{"docstore/data": {"479b311f-2366-4ca1-92f2-c4657a6bb8f9": {"__data__": {"id_": "479b311f-2366-4ca1-92f2-c4657a6bb8f9", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a631f22b-8831-4685-bf31-3eb8baf7bd29", "node_type": "1", "metadata": {}, "hash": "2291549f65d377b1bbf0192d6b118d55a4a43c2eb4cc17a0d6a9e8da86d14c67", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1 D e c . 9 (2): 39-47  Review article  DOI: ttps://www.doi.org/10.54796/njb.v9i2.41914 \n  \n\n \n\u00a9NJB, BSN  39 \n\nEfficacy and Toxicity of Different Forms of Asparaginases Against Acute \nLymphoblastic Leukemia: A Review  \nAdesh Baral, Ritesh Gorkhali, Amit Basnet, Shubham Koirala, Hitesh K. Bhattarai  \n\nDepartment of Biotechnology, Kathmandu University, Dhulikhel, Nepal. \n\nReceived: 15th Jun 2021; Revised: 22nd Dec 2021; Accepted: 25th Dec 2021; Published online: 31st Dec 2021 \n\nAbstract \nAcute lymphoblastic leukemia (ALL) is a form of blood cancer that affects white blood cells and is among the most common \nforms of leukemia with children and adolescents showing the highest number of cases. Most treatment protocols include \nchemotherapy using asparaginase. Asparaginase converts asparagine to aspartic acid and ammonia. Unlike normal, healthy \ncells, cancerous cells depend on asparagine for their growth. When these cells are deprived of asparagine by the action of the \nenzyme, the cancer cells selectively die. As of date, several forms of asparaginases are commercially available and are \nadministered in ALL therapy. But due to limited study, it will be early and inaccurate to predict which forms of the enzymes \nare better. In this review, we aim to compare the efficacy and toxicity of four different asparaginases\u2014native Escherichia coli \nasparaginase, PEG Escherichia coli asparaginase, Erwinia chrysanthemi asparaginase and a recombinant Escherichia coli \nasparaginase\u2014used in ALL therapy in children and adolescents using available clinical trial data. PubMed and Clinical \ntrial.org databases were used to select studies. Asparaginase activity, toxicity, anti-asparaginase antibody level and event-free, \noverall survival was compared for different asparaginases. Seventeen randomized and non-randomized controlled trials were \nincluded. Evidence was insufficient to ascertain which asparaginase is the best. PEG Escherichia coli asparaginase seems to be \nbetter with a high activity among the treated patients but there remains high toxicity for all available asparaginases. This \nstudy highlights a need to discover alternative sources of asparaginase from the organisms, which are evolutionarily distant \nfrom Escherichia coli and Erwinia chrysanthemi with high higher enzyme activity and reduced toxicity. \n\nKeywords: Efficacy, Acute lymphoblastic Leukemia, Asparaginase, Clinical trials. \n\n Corresponding author, email: hitesh321@gmail.com \n\nIntroduction  \nAcute lymphoblastic leukemia or ALL is among the \n\nmost prevalent forms of leukemia and most commonly \n\naffects children. ALL is caused by unchecked and \n\nmalignant proliferation of lymphoid progenitor cells in \n\nblood, bone marrow, and extramedullary sites [1]. ALL \n\npresents itself via three main pathological processes: \n\nfirst is the failure of bone marrow, second is the \n\ninfiltration of other tissues by blasts via malignancy and \n\nthird is the systemic effects arising from cytokines \n\nreleased by the cancerous cells. ALL often shows \n\ncommon signs and symptoms among children which \n\ninclude: anemia, thrombocytopenia, and pronounced \n\nhepatosplenomegaly or lymphadenopathy [1]. \n\nAmerican Cancer Society estimates that 6590 cases were \n\ndiagnosed in 2016 alone with 1400 death [2]. It is \n\nsuspected that a combination of prenatal mutations and \n\nenvironmental factors cause ALL. \n\nL-asparaginase is an enzyme that catalyzes the \n\nformation aspartic acid and ammonia from asparagine. \n\nIt is commonly produced in bacteria, but not in humans.", "start_char_idx": 47, "end_char_idx": 3609, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a631f22b-8831-4685-bf31-3eb8baf7bd29": {"__data__": {"id_": "a631f22b-8831-4685-bf31-3eb8baf7bd29", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "479b311f-2366-4ca1-92f2-c4657a6bb8f9", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "2a0dddb1839eac33d3f2ea1338316c95ac96588271312f37cd9d67cc80513d7a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1d78f89e-a9b7-4d1f-a0b1-2f98c6778fe1", "node_type": "1", "metadata": {}, "hash": "24d012d848b2c54c332ab7b577f3387838f2d2223cbcb9e08be5345d7caee7bb", "class_name": "RelatedNodeInfo"}}, "text": "It is commonly produced in bacteria, but not in humans. \n\nOrganisms produce it in the course of their normal life \n\ncycle and it can be extracted and purified for industrial \n\nand medical purposes. L-asparaginase was introduced \n\nin the early 70s as part of the treatment protocols for \n\nALL in children. Now, L-asparaginase has become a \n\nroutine part of treatment protocols of ALL [3]. \n\nAsparaginase works via depletion of asparagine in the \n\nblood. L-asparagine is an important amino acid used by \n\ncells in protein synthesis. Most normal cells produce L-\n\nasparagine for their growth using L-asparagine \n\nsynthetase enzyme, synthesizing L-asparagine from \n\naspartic acid and glutamic acid. Neoplastic cells like \n\nALL cells are incapable of producing their own L-\n\nasparagine because they lack L-asparaginase synthetase \n\nenzyme. Thus, they are dependent on the extracellular \n\nsupply of asparagine for their existence and \n\nreproduction. Exogenous source can be the serum where \n\nasparagine from diet and normal cells are pooled. \n\nConsequently, L-asparaginase is used as a therapeutic \n\nagent against ALL. L-asparaginase catalyzes the \n\ndegradation of asparagine into ammonia and aspartate \n\nand depletes the asparagine in the blood serum, which \n\nleads to starvation of cancer cells causing cell death [4].  \n\nL-asparaginase is commercially extracted mostly from \n\nErwinia chrysanthemi and Escherichia coli. The enzyme \n\ncan be used in its purified native state or can be \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-7147-1411\nmailto:hitesh321@gmail.com\nmailto:hitesh321@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  40 \n\nconjugated to increase its half-life. PEG Escherichia coli \n\nasparaginase is a variant made by conjugating the \n\nEscherichia coli L-asparaginase with polyethylene glycol. \n\nThere are many commercially available examples of \n\nPEG Escherichia coli asparaginase as well. Usually, PEG-\n\nasparaginase can be used in lower doses compared to \n\nnative state asparaginases due to its greater half-life [5]. \n\nSome people can have allergic reactions to Escherichia \n\ncoli asparaginase. Erwinia chrysanthemi asparaginase is \n\nused as an alternative for these cases [3]. \n\nTherapeutic results of combining L asparaginase with \n\nchemotherapy protocols have been generally very \n\nsuccessful. Many variations have been made to improve \n\nthe results of these therapies, usually centered around \n\nreducing enzyme related side effects that are common in \n\nthese therapies[6]. Though all three forms of L \n\nasparaginase have been extensively studied for their \n\neffectiveness and safety, unintended enzyme-related \n\nside-effects like hypersensitivity and allergic reactions \n\nabound [7]. Till date, there has been limited study to \n\naddress the efficacy and toxicity of different forms of \n\nasparaginases available for therapy because of which \n\nindividuals who are under therapy are at high risk. This \n\ncreates an urgent need to address and minimize the risk \n\nby providing necessary and relevant information on \n\nsafety and fill in the research gap. In this study, we have \n\nattempted to compile and examine a list of studies to \n\nunderstand efficacy, safety and toxicity of PEG \n\nEscherichia coli asparaginase, Erwinia chrysanthemi \n\nasparaginase, and native Escherichia coli asparaginase to \n\ndetermine if there is a need for an alternative source of \n\nasparaginase to reduce these unwanted side effects.", "start_char_idx": 3554, "end_char_idx": 7191, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1d78f89e-a9b7-4d1f-a0b1-2f98c6778fe1": {"__data__": {"id_": "1d78f89e-a9b7-4d1f-a0b1-2f98c6778fe1", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a631f22b-8831-4685-bf31-3eb8baf7bd29", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "290febe7b4c64b79e4a9b6421b80d8f9b8564420242e88fa120d522cebfe1eba", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b40137a7-b7e6-4167-b788-0090505b3a08", "node_type": "1", "metadata": {}, "hash": "5790f57bb1e945129c1a392dc86a54ad4ea1ce75d0bc155232489cdb37a0552e", "class_name": "RelatedNodeInfo"}}, "text": "Asparagine Concentration: \nOgawa et al. reported that the average plasma \n\nasparagine concentration of patients treated with E. \n\nchrysanthemi asparaginase was 0.218 \ud835\udf41M [8]. \n\nConcentration of < 0.5 \ud835\udf41M is considered as complete \n\ndepletion of asparagine from the blood [9]. Van der \n\nSluis et al reported that complete depletion of \n\nasparagine was seen in 97.8% of patients treated with \n\nrecombinant asparaginase and 97.9% of patients treated \n\nwith native E. coli asparaginase [10]. Likewise, Pieters et \n\nal. claimed that the mean asparagine concentration \n\ndropped to 0.5 \u00b5M under both recombinant E. coli \n\nasparaginase and native E. coli asparaginase in 99% of \n\npatients [11]. In another study by van der Sluis et al. the \n\nmean asparagine concentration dropped to <0.5 \ud835\udf41M in \n\nall patients at all time points measured with \n\nrecombinant E. coli asparaginase[12]. \n\nIn a comparison done between intermittent and \n\ncontinuous routes of PEG E. coli asparaginase among \n\n625 children of Europe, it was found that 95% of patients \n\nwere asparagine depleted during treatment [13]. In 2011, \n\na study conducted to demonstrate PEG E. coli \n\nasparaginase as a viable alternative in patients that have \n\nshown allergic reactions to treatments using native E. \n\ncoli asparaginase, it was found that asparagine level \n\ndepleted to 40% and 20% at day 7 and day 14 \n\nrespectively for hypersensitive patients using PEG \n\nasparaginase. Similarly for non-hypersensitive patients, \n\nit was depleted to 50% at day 14 but while using native  \n\nE. coli asparaginase it was depleted to only 86% on day \n\n14 [14]. 26% of patients receiving E. chrysanthemi \n\nasparaginase showed asparagine depletion during re-\n\ninduction. With E. coli asparaginase receiving patients, \n\nasparagine depletion was seen in 60% to 90% during the \n\nre-induction phase. Serum asparagine levels recovered \n\nafter 4 days for patients administered with E. \n\nchrysanthemi asparaginase compared to 11 days for E. \n\ncoli asparaginase [15] (Table 1). \n\nTable 1. Average asparagine Concentration in treated patients \n\nfrom individual trials \n\nClinical Trial Type of Asparaginase \nAverage \n\nConcentration(\ud835\udf41M) \n\n[8] Erwinia chrysanthemi 0.218 \n\n[16] Native Escherichia coli 0.13 \n\n[10] \nNative Escherichia coli \n(asparaginase medac) \n\n<0.5 \n\n[10] \nRecombinant \nEscherichia coli \n\n<0.5 \n\n[17] \nNative Escherichia coli \n(asparaginase medac) \n\n<0.5 \n\n[17] \nRecombinant \nEscherichia coli \n\n<0.5 \n\n[12] \nRecombinant \nEscherichia coli \n\n<0.5 \n\n[13] PEG Escherichia coli <0.5 \n\n[14] Native Escherichia coli <0.5 \n\nAsparaginase Activity: \nMeasurement of asparaginase concentration during the \n\ntherapy has lots of technical limitations, which is why \n\nasparaginase-enzyme activity is generally used to \n\nmonitor asparaginase. As per U.S FDA, effective plasma \n\nlevel of asparaginase was defined as \u2265 0.1 IU/mL and \n\nused for determination of efficacy in the approval \n\nprocess for asparaginase [18]. Ogawa et al.", "start_char_idx": 7196, "end_char_idx": 10153, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b40137a7-b7e6-4167-b788-0090505b3a08": {"__data__": {"id_": "b40137a7-b7e6-4167-b788-0090505b3a08", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1d78f89e-a9b7-4d1f-a0b1-2f98c6778fe1", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4963a54eb0eedf1ae1b11c40377bbb617601acc5c2af5c02f0b37cf1d06c5dc0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6eb4243c-a16e-4f12-b617-5dd3cf2588fb", "node_type": "1", "metadata": {}, "hash": "03b0b111c6719fbb878f761e899d4018abdc3d28b19df20de600c60381ae4f2b", "class_name": "RelatedNodeInfo"}}, "text": "Ogawa et al. reported that \n\nthe average activity of E. chrysanthemi asparaginase in \n\ntreated patients throughout the study was 0.36 IU/mL, \n\nwhich was much higher than the therapeutic level of \n\nasparaginase [8]. All the treated patients in their study \n\nachieved a therapeutic level of asparaginase. Likewise, \n\nVyas et al. noted in patients treated with native E. coli \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  41 \n\nasparaginase and PEG E. coli asparaginase, the activity \n\nof the enzymes were 0.13 IU/mL for native E. coli \n\nasparaginase and 0.30 IU/mL for PEG E. coli \n\nasparaginase with 86% in the native E. coli group and \n\n94% generic PEG E. coli group achieving a therapeutic \n\nlevel of asparaginase [16]. In case of recombinant \n\nEscherichia coli asparaginase, Van der Sluis et al. \n\nreported that the average asparaginase activity was 0.17 \n\nIU/mL with 62.2 % of patients achieving the therapeutic \n\nlevel of asparaginase and in native E. coli asparaginase \n\naverage asparaginase activity was 0.16 IU/mL with \n\n65.9% patients achieving the therapeutic level of \n\nasparaginase [10]. Similarly, Place et al. claimed that \n\nPEG E. coli asparaginase activity was around 0.7 IU/mL \n\nis treated patients and native E. coli asparaginase \n\nactivity was around 0.1-0.2 IU/mL[19]. \n\nIn a phase II study conducted by Dinndorf et al. for the \n\nFDA, the asparaginase activity and the depletion of \n\nasparagine were measured in days after the first dose. \n\nThey found that between the 2nd and 7th day after the \n\nfirst dose both native E. coli asparaginase and the PEG \n\nEscherichia coli asparaginase had activities above 0.03 \n\nIU/mL in 50 patients. This number decreased to below \n\n10 patients for native E. coli asparaginase group while it \n\nreached 20 for the PEG group in the remission induction \n\nphases [20]. \n\nIn a study, the therapeutic PEG E. coli asparaginase \n\nactivity was observed to be 0.234 IU/mL among 86% of \n\nsurviving patients [17]. In another study by Pieters et al. \n\nmedian asparaginase activity for native E. coli \n\nasparaginase was found to be 0.19 IU/mL while \n\nrecombinant asparaginase showed an activity of 0.14   \n\nIU/mL  (Pieters et al. 2008). Moreover, Van der Sluis et \n\nal. reported the serum asparaginase activities of \n\nrecombinant E. coli asparaginase to be >0.10 IU/mL in \n\n74% patients and was 0.13 IU/mL of all measured \n\nsamples respectively [12]. In Rau et al. none of the study \n\npopulation completed the trail and only one patient had \n\ntolerated the PEG E. chrysanthemi asparaginase with \n\nactivity >0.1 IU/mL [21].  Significantly shorter serum \n\nhalf-life of 0.65 days was observed for E. chrysanthemi \n\nasparaginase enzyme compared to 1.24 days for E coli \n\nasparaginase in a study by Duval et al. [15] (Table 2). \n\nAs stated earlier, asparaginase activity in-vivo was \n\nstandardized to be less than 0.1 IU/mL during the \n\ntherapy, but it happens to be between 0.13-0.70 IU/mL \n\n(Table 3).", "start_char_idx": 10141, "end_char_idx": 13120, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6eb4243c-a16e-4f12-b617-5dd3cf2588fb": {"__data__": {"id_": "6eb4243c-a16e-4f12-b617-5dd3cf2588fb", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b40137a7-b7e6-4167-b788-0090505b3a08", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "8c2f512c4a8fc39df86839a88f9e87f85c6ba18ff44f46337fb26b669d2ac245", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ea668546-aa57-4814-aa74-b5cf731919d3", "node_type": "1", "metadata": {}, "hash": "3f8ac72a0ae0cc90c7df0945c009863291f3a9d201c050faa3ae8577a84cb348", "class_name": "RelatedNodeInfo"}}, "text": "This showcases that most of the individuals, \n\nwho were under therapy achieved the threshold.  \n\nToxicity: \n\nIn a therapy involving asparaginase, toxicity is directly  \n\nTable 3. Average range of asparaginase activity in treated \npatients from all the studied trials. \n\nType of asparaginase \nAverage Activity Range \n(IU/mL) from all trials \n\nNative Escherichia coli 0.13-0.19 \n\nPEG Escherichia coli 0.23-0.70 \n\nErwinia chrysanthemi 0.13-0.58 \n\nRecombinant Escherichia coli 0.13-0.17 \n\nrelated to the dose administered. This is why controlled \n\nadministration of quantity is a must. A trial reported \n\nthat E. chrysanthemi asparaginase hypersensitivity \n\nreaction (urticaria) of grade 1-2 were seen in 2(8%) of \n\npatients, pancreatitis of grade 1-3 in 3(12%) of the \n\npatients, and hyperglycemia of grade 1-2 in 5 (20%) of \n\nthe patients [8]. Similarly, hypersensitivity reaction of \n\ngrade 3-4 was seen in 7(12%) patients in native E. coli \n\nasparaginase treated group, 3 (6%) in PEG E. coli \n\nasparaginase treated group, hyperglycemia of grade 3-4 \n\nwas seen in 2(4%) patients in native E. coli asparaginase \n\ntreated group and in 1(2%) patient in PEG E. coli \n\nasparaginase group [16]. Later, Van der Sluis et al \n\nreported that hypersensitivity reaction was seen in \n\n2(2.1%) of the patients in recombinant E. coli \n\nasparaginase group and 5(5%) in native E. coli \n\nasparaginase group, pancreatitis of grade \u2265 2 was seen \n\nin 1 (1%) of patient in native E. coli asparaginase group \n\n[16]. Also the hypersensitivity reaction of grade 1-4 was \n\nseen in 28(12%) of patients receiving PEG E. coli \n\nasparaginase and in 21(9%) in native E. coli asparaginase \n\nreceiving group.  Pancreatitis of grade \u2265 2 was seen in 27 \n\n(12%) of the patients receiving PEG asparaginase and \n\n22(10%) receiving native E. coli asparaginase group [19]. \n\nIn 1999, in a study by Liang et al. 10,000 IU/m2 dose \n\nEscherichia coli asparaginase was used during the \n\nremission induction therapy on 93 children with \n\nTable 2. Average asparaginase activity from individual \ntrials in treated patients \n\nClinical Trial Type of Asparaginase \nAverage Activity \n\n(IU/mL) \n\n[8] Erwinia chrysanthemi 0.36 \n\n[16] Native Escherichia coli 0.13 \n\n[16] PEG Escherichia coli 0.30 \n\n[10] \nNative Escherichia coli \n\n(asparaginase medac) \n0.16 \n\n[10] \nRecombinant Escherichia \n\ncoli \n0.17 \n\n[19] Native Escherichia coli 0.1-<0.2 \n\n[19] PEG Escherichia coli 0.70 \n\n[17] \nNative Escherichia coli \n\n(asparaginase medac) \n0.19 \n\n[17] \nRecombinant  \n\nEscherichia coli \n0.14 \n\n[12] \nRecombinant  \n\nEscherichia coli \n0.13 \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  42 \n\nstandard-risk or SR (Spontaneous Remission) ALL. They \n\nfound that 26.8% or 25 of the participants showed signs \n\nof toxicity.", "start_char_idx": 13121, "end_char_idx": 15914, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ea668546-aa57-4814-aa74-b5cf731919d3": {"__data__": {"id_": "ea668546-aa57-4814-aa74-b5cf731919d3", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6eb4243c-a16e-4f12-b617-5dd3cf2588fb", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "04327ad9ea1274921bd2114f58de369bd1d339255e433676bd6edb290bcb708e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "97d31fbd-76b0-492f-9bb0-328462ae0624", "node_type": "1", "metadata": {}, "hash": "aa505596c015466dca75e9eae7e2decc33fbbddcda1f0b8c22546584059cf151", "class_name": "RelatedNodeInfo"}}, "text": "Of them, 15 or 16% showed signs of sepsis, 2 \n\nor 2% had pneumonia, 6 or 6% showed signs of \n\nhyperglycemia, and 6 or 6% had hemorrhage. During \n\nremission induction, 19 of 93 or 20.4% of patients \n\ndeveloped a severe infection. Death during induction \n\noccurred in 6 patients [22]. A phase II clinical trial was \n\nconducted by Dinndorf et al. for the PEG Escherichia coli \n\n- asparaginase Oncaspa\u00ae by Enzon Pharmaceuticals in \n\n118 children aged 1 to 9 years. It was a comparative \n\nstudy between a native E. coli asparaginase and the PEG \n\nE. coli asparaginase. They concluded, 14 of the 58 \n\npatients in the PEG asparaginase group compared to 18 \n\nof the 59 patients in the native Escherichia coli group \n\nsuffered from toxic effects. Hyperglycemia was much \n\nmore common in the PEG Escherichia coli asparaginase \n\ngroup with 5% or 3 patients suffering from it and \n\nabnormal liver conditions were much more common in \n\nthe native Escherichia coli asparaginase group, 10 \n\npatients with abnormal liver tests compared to 6 in the \n\nPEG Escherichia coli asparaginase group [20].  \n\nAnother study conducted on 144 patients aged below 22 \n\nyears to study the effect of weekly vs. bi-weekly dosing \n\nregimens with PEG E. coli asparaginase and native E. coli \n\nasparaginase in patients with first relapses of ALL in \n\nreinduction therapy, found that out of the 143 patients \n\nwhose data was evaluated 72 or 50% of them suffered \n\nfrom severe infections. 29 of them had \n\nhypoalbuminemia, 32 of them had low fibrinogen and 9 \n\nof them showed weight loss.  \n\nThere were also specific toxicities seen in the group \n\ngiven PEG E. coli asparaginase. Only 6 of the 144 tested \n\nshowed PEG-asparaginase hypersensitivity, 4 of whom \n\nonly showed grade I allergic reaction. The other 2 had \n\ngrade III hypersensitivity and were given alternative E. \n\nchrysanthemi asparaginase instead [23].  \n\nAdditionally, in the E. coli asparaginase 5000 arm 9 (2.7%) \n\ndeaths were observed, whereas in E. coli asparaginase 10000 \n\narm 23 (6.5%) deaths were observed. Pneumonia was seen in \n\nabout 50% of patients and hypersensitivity reaction was \n\nreported in 4.5% (n=31) patients in E. coli asparaginase 10000 \n\narm. In E. coli 5000 arm 1.8% (n=13) patients showed \n\nhypersensitive reaction [24]. Later, 15 out of 16 deaths were \n\nfor patients over 40 years. Sepsis together with \n\nhepatotoxicity occurred in 50% of the dead patients. \n\nAmong surviving people treated with PEG E. coli \n\nasparaginase pancreatitis and hypoalbuminaemia of \n\ngrade 3+ were recorded on 2 patients (3%) [17]. Pieters \n\net al. reported no death during the trail but recorded \n\ndeep venous thrombosis and severe hyperglycemia in \n\ntwo separate patients given with recombinant \n\nasparaginase and similarly, deep venous thrombosis \n\nand severe neutropenia in two different patients treated \n\nwith native E. coli asparaginase [11]. A study conducted \n\nby Van der Sluis et al. found that 12 patients under \n\ntreatment showed hemorrhage, nose bleeding, \n\nthrombosis of the superior vena cava and increased \n\nalanine aminotransferase CTC grade III [12]. Rau et al. \n\nreported complications of chest tightness and facial \n\nerythema with mild swelling and anaphylaxis in three \n\npatients [21].", "start_char_idx": 15915, "end_char_idx": 19153, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "97d31fbd-76b0-492f-9bb0-328462ae0624": {"__data__": {"id_": "97d31fbd-76b0-492f-9bb0-328462ae0624", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ea668546-aa57-4814-aa74-b5cf731919d3", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "b66488208a5537cf9507599eec84ce48fef72515d2fab70822fce79d73866d9a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4c323e45-41fa-44f3-9a98-d14c126c825d", "node_type": "1", "metadata": {}, "hash": "aa5328d3a1776308f66ba26e430577b9534b27d92a5996ecdfb034937009b837", "class_name": "RelatedNodeInfo"}}, "text": "Albertsen et al. in 2019, demonstrated that 60 (9.6%) \n\npatients experienced toxicity during PEG E. coli \n\nasparaginase treatment and 23 (3.7%) after the last dose. \n\nAmong those showing symptoms, hypersensitivity was \n\nseen in 13 (2%), osteonecrosis in 29 (4.6%), pancreatitis \n\nwas seen in 24 (3.84%) and thromboembolisms in 17 \n\n(2.72%).   In a 3-year period of observation, incidence of \n\nany form of toxicity associated with first asparaginase \n\ntreatment after randomization was found to be much \n\nhigher in children of age 10 years or older compared to \n\nchildren younger than 10 years, but did not differ \n\nbetween boys and girls or between patients at \n\nintermediate risk or standard risk [13]. Additionally in \n\nanother study comparing native E. coli asparaginase \n\nwith E. chrysanthemi asparaginase, E. coli asparaginase \n\narm had more instances of coagulation abnormalities \n\n(30.2%) compared to E. chrysanthemi asparaginase arm \n\n(11.8%) [15]. \n\nAnother trial conducted in Spain by Ribera et al \n\nconcluded that during induction therapy, percentage of \n\npatients with detectable infection, hypersensitivity, \n\nthrombosis, hepatic toxicity, pancreatitis and \n\ncoagulopathy (all of them within grade 3-4) for native E. \n\ncoli asparaginase was 56%, 1%, 6%, 21%, 1%, 11%  \n\nrespectively while for PEG E. coli asparaginase was 45, \n\n0, 9, 38, 3, 18 respectively. Similarly during \n\nconsolidation therapy, percentage of patients with \n\ndetectable infection, hypersensitivity, thrombosis, \n\nhepatic toxicity, pancreatitis and coagulopathy (all \n\nwithin grade 3-4) for native E. coli asparaginase \n\nwas 16%, 1%, 0.4%, 3%, 0%, 0.4% respectively while for \n\nPEG E. coli asparaginase was 13%, 1%, 0%, 11%, 0%, 5% \n\nrespectively [25].   \n\nToxicities caused by different forms are asparaginases \n\nare relatively similar in every patient group (Table 4). \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  43 \n\nEvent-Free Survival (EFS): \nFollowing initial treatment for cancer, patients generally \n\nremain free of any complications or symptoms that were \n\nintended to delay or prevent their treatment. This is \n\nknown as an event-free survival. Vyas et al. reported \n\nthat 2-years event-free survival of patients treated with \n\nPEG E. coli was 84% and  treated with native E. coli \n\nasparaginase was 80.7% [16]. Place et al evaluated 5-\n\nyears of event-free survival of patients to be 90% in PEG \n\nE. coli asparaginase treated group and 89% native E. coli \n\nasparaginase treated group[19]. \n\nPession et al. study found that 5 year and 10 year event-\n\nfree survival (EFS) of native E. coli asparaginase group \n\nwas 84.6% and 82.5% for the 494 patients enrolled in the \n\ntrial. 58 cases failed to achieve event-free status with 1 \n\ncase of second malignancy and 22 relapses. The study by \n\nLiang et al. was a comparison between the use of native \n\nE. coli asparaginase and epidoxorubicin in the treatment \n\nof SR ALL in the remission induction therapy. They also \n\nsaw 5 relapses in their asparaginase arm of the study \n\ngroup. They estimated an EFS at 3 years to be 72%[22]. \n\nThe FDA phase II trial study by Dinndorf et al.", "start_char_idx": 19156, "end_char_idx": 22320, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4c323e45-41fa-44f3-9a98-d14c126c825d": {"__data__": {"id_": "4c323e45-41fa-44f3-9a98-d14c126c825d", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "97d31fbd-76b0-492f-9bb0-328462ae0624", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "60748fb6bf8f919560559db0a99cd2334b1cf2891bcb2db82bcfccace6a1dc97", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "78f4e0fb-6f3c-48ea-b035-560d88643f95", "node_type": "1", "metadata": {}, "hash": "8bc09cc164e9f3571b35cccfa541c4f456ac85258d89d7de701bb9d6e2ad6a15", "class_name": "RelatedNodeInfo"}}, "text": "The FDA phase II trial study by Dinndorf et al. did not \n\nattempt to find long-term EFS. They estimated an 80% \n\nEFS for both their Erwinia asparaginase group and  PEG \n\nasparaginase group. Karachunskiy et al. conducted a \n\nstudy on event-free survival rates and found that at 10 \n\nyears the probabilities of event-free survival rates for \n\nEscherichia coli 5000 arm (79 \u00b1 2%) were not significantly \n\ndifferent from Escherichia coli 10000 arm (75 \u00b1 2%) [24]. \n\nWhile comparing native E. coli asparaginase with E. \n\nchrysanthemi asparaginase, event-free survival predicted \n\nwas 6 years and percentage patient survival was 73.4% \n\nversus 59.8% [15] (Table 5). \n\nBy observing years of event free survival ranges, it can \n\nbe discerned that PEG E. coli asparaginase gives low \n\ndegree of disease reoccurrence (Table 6). \n\nFollow up period for event free survival patients were \n\nreported for 10 years for native E. coli (10 years), 6 years \n\nfor PEG E. coli and 6 years for E. chrysanthemi \n\nasparaginases. \n\nOverall Survival: \nA clinical trial reported 2-year event-free survival of \n\npatients at 93% treated with PEG E. coli asparaginase  \n\nTable 5: Time period of Event-Free Survival in treated patients \nfrom individual trials. \n\nClinical \ntrial \n\nType of \nAsparaginase \n\nEvent free \nsurvival \n\nyear \n\n% of \npatients \nsurvived \n\nFollow \nup \n\nperiod \n(Years) \n\n[16] Native \nEscherichia coli \n\n2 80.7 2 \n\nPEG \nEscherichia coli \n\n2 84 \n\n[19] Native \nEscherichia coli \n\n5 89 6 \n\nPEG \nEscherichia coli \n\n5 90 \n\n[26] Native \nEscherichia coli \n\n5 84.6 10 \n\nNative \nEscherichia coli \n\n10 82.5 \n\n[24] Native \nEscherichia coli \n\n10 73-81 10 \n\n[15] Native \nEscherichia coli \n\n6 73.4 6 \n\nErwinia \nchrysanthemi \n\n6 60 \n\n[22] Native \nEscherichia coli \n\n3 72 3 \n\nTable 6: Average year range of Event-Free Survival in treated \npatients from all the studied trials. \n\nType of asparaginase \nAverage year range of Event-\nFree Survival Year (% range) \n\nNative Escherichia coli 5(84%-89%)-10 (75%-82%) \n\nPEG Escherichia coli 5(90%) \n\nErwinia chrysanthemi 6 (59%) \n\nand 84% treated with native E. coli asparaginase [16] and \n\nanother trial evaluated 5-years of overall survival of \n\npatients. The statistics was 96% treated with PEG-\n\nasparaginase and 94% treated with Escherichia coli \n\nasparaginase [19]. Likewise, Karachunskiy et al. \n\nreported that patients of E. coli asparaginase 5000 arm \n\n(86 \u00b1 2% ) had slightly superior probability of overall \n\nsurvival at 10 years  compared to E. coli asparaginase \n\n10000 arm (82 \u00b1 2%) [24]. Overall survival estimated at 5 \n\nand 10 years was 94.4% and 93.7% in the group with \n\nasparaginase versus 89.8% and 88.6% in the group \n\nwithout asparaginase , respectively [26] (Table 7). \n\nMoreover, study conducted in 2002 demonstrated that 6 \n\nyear survival rate using native E. coli asparaginase was \n\ngreater than using E. chrysanthemi asparaginase (83.9% \n\nvs 75.1%) [15]. \n\n  \n\nTable 4. Average range of occurring toxicity in treated patients from all the studies trials.", "start_char_idx": 22273, "end_char_idx": 25270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "78f4e0fb-6f3c-48ea-b035-560d88643f95": {"__data__": {"id_": "78f4e0fb-6f3c-48ea-b035-560d88643f95", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4c323e45-41fa-44f3-9a98-d14c126c825d", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "1d557b32cb39a7bbf738a7b24c75c0e8ca45e46f43eeeed432295d52a3d1f279", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "17e8f834-a42e-47d3-839c-72ead74320db", "node_type": "1", "metadata": {}, "hash": "e748de5c736e49879a7e2887a942092bfbc29bf430ed617cb36298f6a5cb1732", "class_name": "RelatedNodeInfo"}}, "text": "Average range of occurring toxicity in treated patients from all the studies trials. \n\n         Asparaginase                            \nTypes \nToxicity    \n\nEscherichia coli \nAsparaginase (%) \n\nPEG- Escherichia coli \nAsparaginase (%) \n\nErwinia chrysanthemi \nAsparaginase (%) \n\nRecombinant Escherichia \ncoli Asparaginase (%) \n\nHypersensitivity 1.8 \u2013 12 2\u2013 12 1.3 \u2013 12 2.1 \nPancreatitis 1 \u2013 10 3-12 NA NA \nHyperglycemia 4 \u2013 6 2 5 \u2013 20 NA \nThe range of toxicities are of 1-4 grade from all the trials. \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  44 \n\n In terms of overall survival PEG asparaginase from \n\nEscherichia coli showed better result than other forms of \n\nasparaginases (Table 8) \n\nTable 8. Average year range of Overall Survival in treated \npatients from all the studied trials. \n\nType of asparaginase \nAverage year range of Overall \n\nsurvival Year (% range) \n\nNative Escherichia coli 5 (94%) - 10 (93%) \n\nPEG Escherichia coli 5 (96%) \n\nFollow up period of overall survival of patients were \n\nreported for 10 years for native E. coli asparaginase, 6 \n\nyears for PEG E. coli asparaginase and 6 years for E. \n\nchrysanthemi asparaginase. \n\nAnti-Asparaginase Antibody (AAA) \nThe most common adverse reactions of asparaginase in \n\nchildren are produced by anti-asparaginase antibodies. \n\nThese adverse reactions can manifest as mild or severe \n\nallergic reactions. It was reported that 10% (9 in native \n\nE. coli asparaginase and 10 in recombinant Escherichia \n\ncoli asparaginase group) patients were detected positive \n\nfor AAA [10]. The Dinnodorf et al. FDA study found \n\nthat 16 out of 57 or 28% of their patients treated with \n\nnative E. coli asparaginase had anti-aparaginase \n\nantibodies at any given time in of the treatment. Three \n\nsubjects were known to have pre-existing anti-\n\naparaginase antibodies. In the patients treated with the \n\nPEG E. coli asparaginase 11% of the 55 or 6 of them had \n\nasparaginase antibodies. Rau et al. 2018 reported very \n\nlow anti-PEG IgM among three patients. Anti-PEG IgG \n\nwas observed in three patients except one after 5.5-years \n\nof exposure to PEG E. coli asparaginase [21] (Table 9). \n\n \n\nTable 9. individual patients positive with Anti-Asparaginase \nAntibody (AAA) from trials. \n\nClinical Trial \nType of \n\nAsparaginase \n\n% of patients positive \nfor Anti-Asparaginase \n\nAntibody (AAA) \n\n[10] \n\nRecombinant \nEscherichia coli \n\n \n10 \n\nNative Escherichia coli \n(Asparaginase \n\nmedac) \n\n[20] \nNative Escherichia coli 28 \n\nPEG Escherichia coli 11 \n\nPEG asparaginase from Escherichia coli has shown \n\npromising advantages over other forms of \n\nasparaginases. Anti-asparaginase antibodies are found \n\nin lower numbers in PEG asparaginase . (Table 10).   \n\nTable 10. Average % range of patients positive for Anti-\nAsparaginase Antibody (AAA) from all the studies trials.", "start_char_idx": 25186, "end_char_idx": 28044, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "17e8f834-a42e-47d3-839c-72ead74320db": {"__data__": {"id_": "17e8f834-a42e-47d3-839c-72ead74320db", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "78f4e0fb-6f3c-48ea-b035-560d88643f95", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "148a6103d1f704282c50a90f58afafc9d5eabc5ca5e5e24364df31af4136996a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b56c9ae9-03e7-46c1-af27-e9ca1cdec9cd", "node_type": "1", "metadata": {}, "hash": "e0d75b222100da0839914973a8be588796d53fe458c43913bd0d87b8ad8d9597", "class_name": "RelatedNodeInfo"}}, "text": "Type of asparaginase \nAverage % range of patients positive \n\nfor Anti-Asparaginase Antibody (AAA) \n\nNative Escherichia coli 10%-28% \n\nPEG Escherichia coli 11% \n\nDiscussion and Conclusion \nData was obtained from various clinical trials (Table 11) \n\non asparaginase activity, concentration, and toxicity of \n\nthe three major types of asparaginases used for ALL \n\ntherapies: PEG-asparaginase, Erwinia chrysanthemi \n\nasparaginase and native Escherichia coli asparaginase. \n\nPEG E. coli asparaginase activity was seen to be between \n\n0.3-0.7 IU/mL, which is the highest in comparison to \n\nothers (Table 3). In terms of toxicity, all three forms of \n\nasparaginases showed similar results (Table 4). \n\nToxicities like hyperglycemia and pancreatitis were seen \n\nin a significant number of cases leading to a decrease in \n\nthe effectiveness of the enzyme in the treatment of the \n\npatients. In several studies, we can see that incidence of \n\ntoxicity increases with the dose of the enzyme. Another \n\nminor conclusion that we can derive from clinical trials \n\nby Place et al and Vyas et al is that overall survival was \n\nslightly higher for PEG E. coli asparaginase than native \n\nE. coli asparaginase (Table 8). This difference is very \n\nslight and the results stay the same for event free \n\nsurvival making conclusions about the higher efficacy of \n\none type of asparaginase over the other difficult (Table \n\n6).  \n\nThere were number of other studies that did not directly \n\nevaluate the safety and efficacy of asparaginases in \n\nclinical trials for children. Since these studies were also \n\nincluded in this review, we will discuss the findings of \n\nthese studies. In the study by Rau et al. pegcrisantaspase \n\n(Erwinia chrysanthemi pegylated asparaginase) treatment \n\nalso reported hypersensitivity reaction to the patients \n\nTable 7. Time period of Overall Survival in treated patients \nfrom individual trials. \n\nClinical \ntrial \n\nType of \nAsparaginase \n\nOverall \nsurviva\nl year \n\n% of \npatients \nsurvived \n\nFollow \nup period \n\n(Years) \n\n[16] Native Escherichia coli 2 84 2 \n\nPEG Escherichia coli 2 93 \n\n[19] Native Escherichia coli 5 94 6 \n\nPEG Escherichia coli 5 96 \n\n[26] Native Escherichia coli 5 94.4 10 \n\nNative Escherichia coli 10 93.7 \n\n[24] Native Escherichia coli 10 80-88 10 \n\n[15] Native Escherichia coli 6 84 6 \n\nErwinia chrysanthemi 6 75 \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  45 \n\nwith previously showing hypersensitivity reaction to PEG asparaginase treatment.  \nTable 11. General data of selected studies. \n\nAuthor, year Country \nType of Enzyme \n(Asparaginase) \n\nRoute of \nadministration \n\nPeriod of treatment \nof Enzyme \n\nTotal \nNumber of \nParticipant \n\nStudy \ngroup(age) \n\nOgawa et al. 2017 Japan Erwinia chrysanthemi Intramuscular 2weeks 24 2-16 years \n\nVyas et al. 2018 India Native Escherichia coli) Intramuscular 10 weeks 106 Less than \n18 years generic PEG Escherichia \n\ncoli) \nIntravenous \n\nVan der Sluis al, \n2018 \n\nNetherlands Recombinant Escherichia \ncoli \n\nIntravenous 5 weeks 199 Children \n\nNative Escherichia coli \n\nPlace et al.", "start_char_idx": 28047, "end_char_idx": 31149, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b56c9ae9-03e7-46c1-af27-e9ca1cdec9cd": {"__data__": {"id_": "b56c9ae9-03e7-46c1-af27-e9ca1cdec9cd", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "17e8f834-a42e-47d3-839c-72ead74320db", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "47beba88650d458f60d86940a4b6bce3da43652ba56cfbd943233e32cefa4983", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1dcf1813-f8c0-4c55-8546-293016535281", "node_type": "1", "metadata": {}, "hash": "baa9700d356fcc3359d051f8dc33d770b7c2e9894d4c673cdf2ddd4062df8ed0", "class_name": "RelatedNodeInfo"}}, "text": "2015 USA and \nCanada \n\nPEG Escherichia coli) Intravenous 30 weeks 463 1-18 years \n\nEscherichia coli Intramuscular \nPession et al. 2005 Italy 90% of the patients \n\nreceived Erwinia \nchrysanthemi \n\n10%  Escherichia coli \n\nIntramuscular 24 months 490 1-15 years \n\nLiang et al. 1999 Taiwan Escherichia coli Intramuscular 110 weeks 201 1-15 years \n\nDinndorf et al. 2007 USA Escherichia coli[Elspar\u00ae \nfrom Merck] PEG \n\nEscherichia coliOncaspa\u00ae \nBy Enzon Pharmaceuticals, \n\nInc \n\nBoth \nintramuscular \n\n12 Weeks 118 1-9 years \n\nAbshire et al. 2000 USA PEG Escherichia coli \nErwinia  chrysanthemi \n\nIntramuscular 4 weeks 144 Below 22 \nyears of \n\nage \nKarachunskiy et al Moscow\u2013\n\nBerlin \nNative  Escherichia coli Intramuscular 200 days 774 1-19 years \n\nPatel et al. 2017 UK PEG Escherichia coli - 8 weeks 91 25-65 years \nPieters et al. Netherlands Recombinant  Escherichia \n\ncoli asparaginase \nAsparaginase medac \n\nIntravenous 39 days 32 1-14 years \n\nvan der Sluis et al. \n2013 \n\nNetherlands \nand \n\nGermany \n\nRecombinant  Escherichia \ncoli -asparaginase \n\n- 39 days 12 Below 1 \nyears \n\nRau et al. 2018 USA PEG Erwinia chrysanthemi Intravenous 29 days 4 1-20 years \n\nAlbertsen et al. \n2019 \n\nDenmark, \nFinland, \nIceland, \n\nNorway, or \nSweden \n\nPEG- Escherichia coli Intramuscular 30-33 weeks 625 Children \n\nDuval et al. 2002 Belgium, \nFrance and \n\nPortugal \n\nEscherichia coli Intravenously 6 weeks 700 Less than \n18 years Erwinia chrysanthemi \n\nKurtzberg et al. \n2011 \n\nUSA, \nCanada \n\nPEG Escherichia coli \nasparaginase \n\nIntramuscularly 4 weeks 76 Less than \n21 years \n\nNative  Escherichia coli \nRibera et al. 2017 Spain Native  Escherichia coli Intravenously 4 weeks 126 18-60 years \n\nIt is possible that PEG (poly ethylene glycol) can be \n\nimmunogenic and anti-PEG IgG antibodies are formed \n\nduring PEG-asparaginase treatment. The remaining \n\nimmunological memory may mediate hypersensitivity \n\nreaction during pegcrisantaspase treatment. One of the \n\npatients involved in this study had not been exposed to \n\npegaspargase for 5.5 years. He did not experience a \n\npegcrisantaspase hypersensitivity reaction. A lack of a \n\ndurable immunologic memory from anti-PEG-mediated \n\nimmune reactions may be the case for this patient. It is \n\nsuggested that patients who have been recently exposed \n\nto PEG in the formulation of other medicine in any \n\ndisease should not use any PEG asparaginase in any \n\nform. Instead, native Escherichia coli or Erwinia \n\nchrysanthemi asparaginase should be used. \n\nIn another study by Patel et al. it was shown that \n\nasparaginase toxicity can be substantial in older \n\npatients, making it difficult to deliver safely to those \n\naged above 40 years [17]. Similarly, when Ribera et al \n\nused native or PEG asparaginase in adult patients there \n\nno significant difference observed in complete \n\nremission, diseases free survival, and overall survival \n\nwith no influence in patient response and outcome [25]. \n\nBased on numerous previous studies teenagers and \n\nyounger adults typically have better outcome from \n\ninduction and consolidation treatment compared to \n\nadults (aged above 40 years).", "start_char_idx": 31150, "end_char_idx": 34263, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1dcf1813-f8c0-4c55-8546-293016535281": {"__data__": {"id_": "1dcf1813-f8c0-4c55-8546-293016535281", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b56c9ae9-03e7-46c1-af27-e9ca1cdec9cd", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "7d819c8590151025758907d90ffa43264bcca2fd0cc5b8be4be0d6f55b6473b1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e11216de-4e31-42a5-94c4-170a90fcff54", "node_type": "1", "metadata": {}, "hash": "c452ec52a97d27d07265dc75c65727425c80db350cfe0dbb53a0d6441a59b7f3", "class_name": "RelatedNodeInfo"}}, "text": "Careful timing of \n\nadministration and avoidance of overlapping toxicities \n\nare recommended for the older patients. \n\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  46 \n\nIn the Liang et al. study authors state that the increased \n\nmortality was due to immunosuppression via depletion \n\nof blood asparagine by the enzyme. They had increased \n\nthe doses of the Leunase brand asparaginase to match \n\nthat of the dose preparation by Nesbit et al. of \n\nEscherichia coli preparation called Crasnitin. Authors \n\nattribute the severe infection and unexpected mortality \n\nto this dose change [22]. The product sold as Medac, is \n\nalso known to cause excessive toxicity when given at a \n\nhigh dose, leading to a 50% reduction in the dosage [27]. \n\nKarachunskiy et al. have reported that patients treated \n\nwith E. coli asparaginase of dose 10000 U/m2 have \n\nreported the severe hypersensitivity reaction more \n\nfrequently than patients provided with 5000 U/m2 of \n\ndose. Also, death in complete remission occurred \n\nsignificantly more in 10000 U/m2 provided patients [24]. \n\nAn advantage of higher doses was not found in the \n\ngroup.  \n\nThus, it will be advantageous to discover and use a \n\nhigher activity asparaginase, thereby allowing use of \n\nlower dose of enzyme to reduce the incidences of \n\ntoxicity. Enzymes with low Km value will have higher \n\nactivity against L-asparagine. Besides the L-asparagine \n\nactivity of L-asparaginase, there is the secondary activity \n\nof mentioned asparaginase enzymes against L-\n\nglutamine, that has been linked to the different toxic \n\nside effects[28,29]. Also, the role of L-glutamine activity \n\nhas not been seen in anti-cancer activity of the \n\nenzyme[30]. One method of finding new asparaginase is \n\nto extract it from an organism other than Escherichia coli \n\nor Erwinia chrysanthemi. We can expect an organism that \n\nis evolutionarily distant from Escherichia coli or Erwinia \n\nchrysanthemi to have different enzyme activity. Thus, we \n\ncan expect to find better alternatives to commercially \n\navailable asparaginases with higher activity than \n\nEscherichia coli and Erwinia chrysanthemi [31, 32]. \n\nAccording to this study, PEG asparaginase provides \n\nbetter enzyme concentration than E. coli or Erwinia \n\nchrysanthemi asparaginase in various clinical trials. \n\nSimilarly, two studies show that PEG asparaginase has \n\nhigher 2-year overall survival than native E. coli \n\nasparaginase. The difference is very minor to \n\nconclusively say PEG asparaginase is superior. Using \n\nErwinia chrysanthemi asparaginase when E. coli and PEG \n\nasparaginase fail, as is currently done, is recommended \n\nfrom this study as well. Furthermore, alternative source \n\nof asparaginase from the organisms, which are \n\nevolutionarily distant from Escherichia coli and Erwinia \n\nchrysanthemi and with a lower Km value i.e., higher \n\nenzyme activity toward L-asparagine, and low activity \n\ntowards the L-glutamine need to be discovered. Such \n\nnovel asparaginases can be used in lower dose thereby \n\nby reducing toxicity. \n\n \n\nAcknowledgement \nThe authors acknowledge the Department of \n\nBiotechnology, Kathmandu University, Dhulikhel, \n\nNepal for providing all support during the study \n\nperiod.  \n\nCompeting Interests \nThe authors declare no competing interests. \n\nReferences: \n1.  Mitchell C, Hall G, Clarke RT. Acute leukaemia in children: \n\nDiagnosis and management. BMJ. 2009;338(7709):1491\u20135.  \n2.", "start_char_idx": 34264, "end_char_idx": 37721, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e11216de-4e31-42a5-94c4-170a90fcff54": {"__data__": {"id_": "e11216de-4e31-42a5-94c4-170a90fcff54", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1dcf1813-f8c0-4c55-8546-293016535281", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "de31cb410849e69d7d5a3c270d8c45db7e930171e5398eb1d94e61acbc311c3c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a58e8c7e-5aa1-4076-884f-5059587e148a", "node_type": "1", "metadata": {}, "hash": "b4a3b54fef35c79c61d30f8eb6caa84b4f01107c1dd730eb7808824d9c8bd85f", "class_name": "RelatedNodeInfo"}}, "text": "2009;338(7709):1491\u20135.  \n2.  Terwilliger T, Abdul-Hay M. Acute lymphoblastic leukemia: a \n\ncomprehensive review and 2017 update. Blood Cancer J. \n2017;7:577.https://doi.org/10.1038/bcj.2017.53 \n\n3.  Piatkowska-Jakubas B, Krawczyk-Kuli\u015b M, Giebel S, Adamczyk-\nCioch M, Czyz A, Mara\u0144da EL, et al. Use of L-asparaginase in \nacute lymphoblastic leukemia: Recommendations of the Polish \nadult leukemia group. Pol Arch Med Wewn. 2008;118(11):664\u20139. \nhttps://doi.org/10.20452/pamw.518 \n\n4.  Batool T, Makky EA, Jalal M, Yusoff MM. A Comprehensive \nReview on l-Asparaginase and Its Applications. Appl Biochem \nBiotechnol. 2016;178(5):900\u201323. https://doi.org/10.1007/s12010-\n015-1917-3 \n\n5.  Avramis VI, Sencer S, Periclou AP, Sather H, Bostrom BC, Cohen \nLJ, et al. A randomized comparison of native Escherichia coli \nasparaginase and polyethylene glycol conjugated asparaginase \nfor treatment of children with newly diagnosed standard-risk \nacute lymphoblastic leukemia: A Children\u2019s Cancer Group study. \nBlood. 2002 Mar;99 (6): 1986\u201394. https://doi.org /10.1182 \n/blood.V99.6.1986 \n\n6.  M\u00fcller HJ, Boos J. Use of L-asparaginase in childhood ALL. Vol. \n28, Critical Reviews in Oncology/Hematology. Elsevier; 1998. p. \n97\u2013113. https://doi.org/10.1016/S1040-8428(98)00015-8 \n\n7.  Ettinger LJ, Kurtzberg J, Vo\u01d4te PA, J\u00fcrgens H, Halpern SL. An \nopen\u2010label, multicenter study of polyethylene glycol\u2010L\u2010\nasparaginase for the treatment of acute lymphoblastic leukemia. \nCancer. 1995;75(5):1176\u201381. https://doi.org/10.1002/1097-\n0142(19950301)75:5<1176::AID-CNCR2820750519>3.0.CO;2-Y  \n\n8.  Ogawa C, Taguchi F, Goto H, Koh K, Tomizawa D, Ohara A, et \nal. Plasma asparaginase activity, asparagine concentration, and \ntoxicity after administration of Erwinia asparaginase in children \nand young adults with acute lymphoblastic leukemia: Phase I/II \nclinical trial in Japan. Pediatr Blood Cancer. 2017;64(9):1\u20138. \nhttps://doi.org/10.1002/pbc.26475 \n\n9.  Schore RJ, Devidas M, Bleyer A, Reaman GH, Winick N, Loh ML, \net al. Plasma asparaginase activity and asparagine depletion in \nacute lymphoblastic leukemia patients treated with pegaspargase \non Children\u2019s Oncology Group AALL07P4*. Leuk Lymphoma. \n2019; 60(7): 1740\u20138. https://doi.org/ 10.1080/ 10428194. 2018. \n1542146  \n\n10.  van der Sluis IM, de Groot-Kruseman H, te Loo M, Tissing WJE, \nvan den Bos C, Kaspers GJL, et al.", "start_char_idx": 37694, "end_char_idx": 40054, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a58e8c7e-5aa1-4076-884f-5059587e148a": {"__data__": {"id_": "a58e8c7e-5aa1-4076-884f-5059587e148a", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e11216de-4e31-42a5-94c4-170a90fcff54", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "e143173afb2160936029ff0f9a300f9725a8bc604d5538599d52ace89eecb4e9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a46ef0c3-b335-4292-8b84-d4e910b8eea4", "node_type": "1", "metadata": {}, "hash": "8c77b16131369fabccd62250ae0270953faa980bfeb4392cd639ce9dbf913641", "class_name": "RelatedNodeInfo"}}, "text": "Efficacy and safety of \nrecombinant E. coli asparaginase in children with previously \nuntreated acute lymphoblastic leukemia: A randomized \nmulticenter study of the Dutch Childhood Oncology Group. \nPediatr Blood Cancer. 2018;65(8):1\u20138. https://doi.org/10.1002/ \npbc.27083  \n\n11.  Pieters R, Appel I, Kuehnel HJ, Tetzlaff-Fohr I, Pichlmeier U, Van \nDer Vaart I, et al. Pharmacokinetics, pharmacodynamics, efficacy, \nand safety of a new recombinant asparaginase preparation in \nchildren with previously untreated acute lymphoblastic \nleukemia: A randomized phase 2 clinical trial. Blood. 2008 \nDec;112(13):4832\u20138. https://doi.org/10.1182/blood-2008-04-\n\nhttps://doi.org/10.1038/bcj.2017.53\nhttps://doi.org/10.20452/pamw.518\nhttps://doi.org/10.1007/s12010-015-1917-3\nhttps://doi.org/10.1007/s12010-015-1917-3\nhttps://doi.org/10.1016/S1040-8428(98)00015-8\nhttps://doi.org/10.1002/1097-0142(19950301)75:5%3C1176::AID-CNCR2820750519%3E3.0.CO;2-Y\nhttps://doi.org/10.1002/1097-0142(19950301)75:5%3C1176::AID-CNCR2820750519%3E3.0.CO;2-Y\nhttps://doi.org/10.1002/pbc.26475\nhttps://doi.org/10.1002/pbc.27083\nhttps://doi.org/10.1002/pbc.27083\nhttps://doi.org/10.1182/blood-2008-04-149443\n\n\nNepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 39-47     Baral et al. \n \n\n \n\u00a9NJB, BSN  47 \n\n149443 \n12.  van der Sluis I, M\u00f6ricke A, Escherich G, von Stackelberg A, \n\nHolter W, Klingebiel T, et al. Efficacy and safety of recombinant \nE. coli-asparaginase in infants (less than one year of age) with \nacute lymphoblastic leukemia. Haematologica. 2013 \nNov;98(11):1697\u2013701. https://doi.org/10.3324/haematol.2013. \n090563 \n\n13.  Albertsen BK, Grell K, Abrahamsson J, Lund B, Vettenranta K, \nJ\u00f3nsson \u00d3G, et al. Intermittent versus continuous PEG-\nasparaginase to reduce asparaginase-associated toxicities: A \nNOPHO ALL2008 randomized study. J Clin Oncol. \n2019;37(19):1638\u201346. https://doi.org/10.1200/JCO.18.01877 \n\n14.  Kurtzberg J, Asselin B, Bernstein M, Buchanan GR, Pollock BH, \nCamitta BM. Polyethylene glycol-conjugated L-asparaginase \nversus native L-asparaginase in combination with standard \nagents for children with acute lymphoblastic leukemia in second \nbone marrow relapse: A children\u2019s Oncology Group Study (POG \n8866). J Pediatr Hematol Oncol. 2011;33(8):610\u20136. \nhttps://doi.org/10.1097/MPH.0b013e31822d4d4e  \n\n15.  Duval M, Suciu S, Ferster A, Rialland X, Nelken B, Lutz P, et al.", "start_char_idx": 40055, "end_char_idx": 42422, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a46ef0c3-b335-4292-8b84-d4e910b8eea4": {"__data__": {"id_": "a46ef0c3-b335-4292-8b84-d4e910b8eea4", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a58e8c7e-5aa1-4076-884f-5059587e148a", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "87bf5d9313317403eab67d8a81a0f85cdc31f75065e090343a2e4b61f773fb75", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ae49c980-d3fb-483b-af8a-d4ce3ec7319e", "node_type": "1", "metadata": {}, "hash": "cf91fa63c1f090dda98e2cf0acc20e15aa675eed200088eacc11648cfa1587a7", "class_name": "RelatedNodeInfo"}}, "text": "Comparison of Escherichia coli-asparaginase with Erwinia-\nasparaginase in the treatment of childhood lymphoid \nmalignancies: Results of a randomized European Organisation \nfor Research and Treatment of Cancer - Children\u2019s Leukemia \nGroup phase 3 trial. Blood. 2002 Apr;99(8):2734\u20139. \nhttps://doi.org/10.1182/blood.V99.8.2734 \n\n16.  Vyas C, Jain S, Kapoor G, Mehta A, Takkar Chugh P. Experience \nwith generic pegylated L-asparaginase in children with acute \nlymphoblastic leukemia and monitoring of serum asparaginase \nactivity. Pediatr Hematol Oncol. 2018;35(5\u20136):331\u201340. \nhttps://doi.org/10.1080/08880018.2018.1538277  \n\n17.  Patel B, Kirkwood AA, Dey A, Marks DI, McMillan AK, Menne \nTF, et al. Pegylated-asparaginase during induction therapy for \nadult acute lymphoblastic leukaemia: Toxicity data from the \nUKALL14 trial. Leukemia. 2017 Jan;31(1):58\u201364. \nhttps://doi.org/10.1038/leu.2016.219 \n\n18.  Salzer WL, Asselin B, Supko JG, Devidas M, Kaiser NA, Plourde \nP, et al. Erwinia asparaginase achieves therapeutic activity after \npegaspargase allergy: a  report from the Children\u2019s Oncology \nGroup. Blood. 2013 Jul;122(4):507\u201314. \nhttps://doi.org/10.1182/blood-2013-01-480822  \n\n19.  Place AE, Stevenson KE, Vrooman LM, Harris MH, Hunt SK, \nO\u2019Brien JE, et al. Intravenous pegylated asparaginase versus \nintramuscular native Escherichia coli L-asparaginase in newly \ndiagnosed childhood acute lymphoblastic leukaemia (DFCI 05-\n001): A randomised, open-label phase 3 trial. Lancet Oncol. \n2015;16(16):1677\u201390. https://doi.org/10.1016/S1470-2045(15) \n00363-0  \n\n20.  Dinndorf PA, Gootenberg J, Cohen MH, Keegan P, Pazdur R. \nFDA Drug Approval Summary: Pegaspargase (Oncaspar\u00ae) for \nthe First\u2010Line Treatment of Children with Acute Lymphoblastic \nLeukemia (ALL). Oncologist. 2007;12(8):991\u20138. \nhttps://doi.org/10.1634/ theoncologist.12-8-991  \n\n21.  Rau RE, Dreyer ZA, Choi MR, Liang W, Skowronski R, \nAllamneni KP, et al. Outcome of pediatric patients with acute \nlymphoblastic leukemia/lymphoblastic lymphoma with \nhypersensitivity to pegaspargase treated with PEGylated Erwinia \nasparaginase, pegcrisantaspase: A report from the Children\u2019s \nOncology Group. Pediatr Blood Cancer. 2018 Mar;65(3). \nhttps://doi.org/10.1002/pbc.26873 \n\n22.  Liang DC, Hung IJ, Yang CP, Lin KH, Chen JS, Hsiao TC, et al. \nUnexpected mortality from the use of E. coli L-asparaginase \nduring remission induction therapy for childhood acute \nlymphoblastic leukemia: A report from the Taiwan Pediatric \nOncology Group. Leukemia. 1999;13(2):155\u201360. https://doi.org/ \n10.1038/sj.leu.2401260  \n\n23.  Abshire TC, Pollock BH, Billett AL, Bradley P, Buchanan GR.", "start_char_idx": 42424, "end_char_idx": 45056, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ae49c980-d3fb-483b-af8a-d4ce3ec7319e": {"__data__": {"id_": "ae49c980-d3fb-483b-af8a-d4ce3ec7319e", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a46ef0c3-b335-4292-8b84-d4e910b8eea4", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "639355bf257da8eb11ffc2195cc10a2cce89cab04e909128f4f8a4c8b1bfd67d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ebc587c3-d4b2-4536-8db5-abd6a7b33e3d", "node_type": "1", "metadata": {}, "hash": "f46773ed774d519fcc061c4e6eeef5f2cdfeb06158fae7c7e4088e801bdbf00a", "class_name": "RelatedNodeInfo"}}, "text": "Abshire TC, Pollock BH, Billett AL, Bradley P, Buchanan GR. \nWeekly polyethylene glycol conjugated L-asparaginase compared \nwith biweekly dosing produces superior induction remission \nrates in childhood relapsed acute lymphoblastic leukemia: A \npediatric oncology group study. Blood. 2000;96(5):1709\u201315. \nhttps://doi.org/10.1182/blood.V96.5.1709  \n\n24.  Karachunskiy A, Tallen G, Roumiantseva J, Lagoiko S, Chervova \n\nA, von Stackelberg A, et al. Reduced vs. standard dose native E. \ncoli-asparaginase therapy in childhood acute lymphoblastic \nleukemia: long-term results of the randomized trial Moscow\u2013\nBerlin 2002. J Cancer Res Clin Oncol. 2019 Apr;145(4):1001\u201312. \nhttps://doi.org/10.1007/s00432-019-02854-x \n\n25.  Ribera JM, Morgades M, Montesinos P, Martino R, Barba P, Soria \nB, et al. Efficacy and safety of native versus pegylated Escherichia \ncoli asparaginase for treatment of adults with high-risk, \nPhiladelphia chromosome-negative acute lymphoblastic \nleukemia. Leuk Lymphoma. 2018;59(7):1634\u201343. \nhttps://doi.org/10.1080/10428194.2017.1397661  \n\n26.  Pession A, Valsecchi MG, Masera G, Kamps WA, Magyarosy E, \nRizzari C, et al. Long-term results of a randomized trial on \nextended use of high dose L-asparaginase for standard risk \nchildhood acute lymphoblastic leukemia. J Clin Oncol. \n2005;23(28):7161\u20137. https://doi.org/10.1200/JCO.2005.11.411  \n\n27.  Ahlke E, Nowak-G\u00d6TTL U, Schulze-Westhoff P, Werber G, \nB\u00f6rste H, W\u00fcrthwein G, et al. Dose reduction of asparaginase \nunder pharmacokinetic and pharmacodynamic control during \ninduction therapy in children with acute lymphoblastic \nleukaemia. Br J Haematol. 1997;96(4):675\u201381. https://doi.org/ \n10.1046/ j.1365-2141.1997.d01-2089.x  \n\n28.  Parmentier JH, Maggi M, Tarasco E, Scotti C, Avramis VI, \nMittelman SD. Glutaminase activity determines cytotoxicity of L-\nasparaginases on most leukemia  cell lines. Leuk Res. 2015 \nJul;39(7):757\u201362. https://doi.org/10.1016/j.leukres.2015.04.008 \n\n29.  Warrell RPJ, Chou TC, Gordon C, Tan C, Roberts J, Sternberg SS, \net al. Phase I evaluation of succinylated Acinetobacter \nglutaminase-asparaginase in adults. Cancer Res. 1980 \nDec;40(12):4546\u201351.  \n\n30.  Chan WK, Lorenzi PL, Anishkin A, Purwaha P, Rogers DM, \nSukharev S, et al. The glutaminase activity of L-asparaginase is \nnot required for anticancer activity  against ASNS-negative cells. \nBlood. 2014 Jun;123(23):3596\u2013606. https://doi.org/10.1182/ \nblood-2013-10-535112 \n\n31.  Baral A, Gorkhali R, Basnet A, Koirala S, Bhattarai HK. Selection \nof the Optimal L-asparaginase II Against Acute Lymphoblastic \nLeukemia: An In Silico Approach. JMIRx Med.", "start_char_idx": 44997, "end_char_idx": 47613, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ebc587c3-d4b2-4536-8db5-abd6a7b33e3d": {"__data__": {"id_": "ebc587c3-d4b2-4536-8db5-abd6a7b33e3d", "embedding": null, "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-1", "node_type": "4", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "4df5d97114955a2fefa49af84b0af0fce3b455a507c31c3a048f18861c6717b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ae49c980-d3fb-483b-af8a-d4ce3ec7319e", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "1ceda14b70443b8afc5363522e6908f7c97620ddd684d09fb5288fa7fa94e43f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "550a9e9d-670c-45e3-861f-e539e0b2b8f4", "node_type": "1", "metadata": {}, "hash": "4b23752ad923faef0d43afb87f304a627de08bb71accb3f7eb474218d8083b94", "class_name": "RelatedNodeInfo"}}, "text": "JMIRx Med. 2021 \nSep;2(3):e29844. https://doi.org/10.2196/29844  \n\n32.  Thomas J. Kindt, Barbara A. Osborne RAG. Kuby Immunology, \nSixth Edition. 6th ed. W. H. Freeman & Company; 2006. 9\u201311 p.  \n\n \n\nhttps://doi.org/10.1182/blood-2008-04-149443\nhttps://doi.org/10.1200/JCO.18.01877\nhttps://doi.org/10.1097/MPH.0b013e31822d4d4e\nhttps://doi.org/10.1182/blood.V99.8.2734\nhttps://doi.org/10.1080/08880018.2018.1538277\nhttps://doi.org/10.1038/leu.2016.219\nhttps://doi.org/10.1182/blood-2013-01-480822\nhttps://doi.org/10.1016/S1470-2045(15)00363-0\nhttps://doi.org/10.1016/S1470-2045(15)00363-0\nhttps://doi.org/10.1002/pbc.26873\nhttps://doi.org/10.1007/s00432-019-02854-x\nhttps://doi.org/10.1080/10428194.2017.1397661\nhttps://doi.org/10.1200/JCO.2005.11.411\nhttps://doi.org/10.1016/j.leukres.2015.04.008\nhttps://doi.org/10.1182/blood-2013-10-535112\nhttps://doi.org/10.1182/blood-2013-10-535112\nhttps://doi.org/10.2196/29844", "start_char_idx": 47603, "end_char_idx": 48518, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "550a9e9d-670c-45e3-861f-e539e0b2b8f4": {"__data__": {"id_": "550a9e9d-670c-45e3-861f-e539e0b2b8f4", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ebc587c3-d4b2-4536-8db5-abd6a7b33e3d", "node_type": "1", "metadata": {"identifier": "njb-1", "author": "Baral, Adesh; Gorkhali, Ritesh; Basnet, Amit; Koirala, Shubham; Bhattarai, Hitesh K", "title": "Efficacy and Toxicity of Different Forms of Asparaginases Against Acute Lymphoblastic Leukemia: A Review", "date": "2021-12-30", "file": "njb-1.pdf"}, "hash": "83b292a70691ae6140f60d00cf859a29e18fdfc23cd4ab2bf755fa8e544d45b4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e8181b0-dc91-41d3-8ae2-dd3f0c7df2e2", "node_type": "1", "metadata": {}, "hash": "80d0d7c4fd1a52f860e1ca1caa3188f953b6aa01ebfd94e771dba96edb9a03da", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 1-6  Research article  DOI: https://www.doi.org/10.54796/njb.v9i2.41892 \n  \n\n\u00a9NJB, BSN   1 \n\nIsolation, Identification and Screening of Bacillus species with \nAntimicrobial Activity from Different Soil Samples of Kathmandu \nValley \nAishwarya Thapa , Anupa Budhathoki, Anupama Sapkota, Muskan Sainju, Prativa Shrestha, \n Shyam Prasad Pant \nDepartment of Microbiology, St. Xavier\u2019s College, Maitighar, Kathmandu, Nepal \n\nReceived: 21st Oct 2020; Revised: 24th Jun 2021; Accepted: 26th Dec 2021; Published online: 31st Dec 2021 \n\nAbstract \nBacillus species are one of the predominant soil bacteria that are able to produce essential secondary metabolites that have \nantagonistic effects on other microorganisms. They are Gram-positive, endospore-forming, chemoheterotrophic, aerobic or \nfacultative anaerobic rods usually consisting of peritrichous flagella for motility. The major aim of this study was to isolate \nthe antimicrobials producing Bacillus spp. from soil samples of different parts of the Kathmandu Valley, identify them and to \nassess their antimicrobial activity against different pathogenic bacteria. The test organisms used were Staphylococcus aureus \n(ATCC 25923), E. coli (ATCC 25922), Pseudomonas spp., Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA) and \nextended spectrum beta-lactamase (ESBL) producing E. coli. Twenty four isolates from 9 soil samples identified as Bacillus \nspp. showed the zone of inhibition around their growth on Nutrient agar during isolation. These 24 isolates were chosen for \nprimary screening of production of antimicrobial by perpendicular streaking method using four test organisms. . Of these 24 \nisolates, six isolates showing a significant zone of inhibition (\u22651mm) against two or more test organisms from the primary \nscreening were chosen for secondary screening which was further tested with six test organisms including ESBL E.coli and \nMRSA. They were further characterized through different physiological and biochemical tests. All 6 isolates showed \ninhibitory action against MRSA and the largest zone of inhibition (30mm) was shown by isolate U6. Isolate U3 was found to \nhave broad spectrum antimicrobial activity with inhibitory effect against gram negative organisms- Pseudomonas and \nSalmonella and gram positive organism S. aureus (ATCC 25923). Isolate U5 showed a zone of inhibition of about 25mm against \nS. aureus which was comparable to that of erythromycin. Hence, this study determines the soil in Kathmandu Valley as a \npotential source of antimicrobial producing Bacillus spp. and recommends isolation and further characterization of  Bacillus \nisolates as a possible source of novel drug to combat with the emergence of multidrug resistant strains.  \n\nKeywords: Antimicrobials, Kathmandu, Bacillus spp., Staphylococcus aureus, MRSA \n\n Corresponding author, email: aaishu.thapa01@gmail.com \n\nIntroduction \nMembers of the Bacillus genus are the predominant soil \n\nbacteria with resistant-endospore forming ability and \n\nplay a major role in organic matter decomposition, bio-\n\ntransformation, biogas production and nitrogen fixation \n\nby associating with the plant roots which ultimately \n\npromote the growth of the plants [1,2]. They exhibit a \n\nwide range of physiological abilities and produce several \n\nmetabolites with antagonistic effects as a strategy to \n\nsurvive, eliminate competition with other existing \n\norganisms and colonize their natural habitat. Most of the \n\nBacillus species accompany actinomycetes and other \n\nantibiotic producers in the ecosystem.", "start_char_idx": 48, "end_char_idx": 3653, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e8181b0-dc91-41d3-8ae2-dd3f0c7df2e2": {"__data__": {"id_": "9e8181b0-dc91-41d3-8ae2-dd3f0c7df2e2", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "550a9e9d-670c-45e3-861f-e539e0b2b8f4", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "46529af59ba7699ecc23affc64f2d143b2d02aa0e1ca3d3c6054e292964f941c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e63eb483-2ac0-403d-a4b3-4482640e12f2", "node_type": "1", "metadata": {}, "hash": "f0c3a5a33d06969509cf141bde1a4c31067b3e22d9ed87801824244adff35c2f", "class_name": "RelatedNodeInfo"}}, "text": "Therefore, they \n\nmight have acquired resistance to antibiotics from such \n\nsources [3,4]. They produce a wide range of antimicrobial \n\ncompounds that have different chemical structures and \n\nstability through a broad range of pH and temperature \n\nand are resistant to enzyme treatments to some extent, \n\ntherefore, can be used as antibacterial, antifungal and \n\nantiviral agents [5]. A Bacillus strain is singly capable to \n\nproduce different antimicrobial compounds and each \n\ncompound can be active only against the same or closely \n\nrelated species i.e. other gram positive bacteria or may \n\nhave broad spectrum activity, for example, bacteriocins \n\nusually show action against closely related bacteria [4, 6]. \n\nMost of the species from the genus Bacillus are considered \n\nas safe microorganisms and they are easier to handle in \n\nthe lab with a low incubation period i.e. only 24-48hrs \n\nthereby, making Bacillus spp. a preferable microorganism \n\nto investigate antimicrobial properties [6]. \n\nThe multidrug resistant pathogens are emerging at an \n\nalarming rate, and this situation can be linked to \n\ninappropriate usage and shortage on the part of the \n\nmanufacturers causing a steady decline of efficient \n\nantibiotics. Moreover, it has created a consequential issue \n\nin the treatment of infectious disease, so, the exploration \n\non this topic is yet important for the discovery of novel \n\nantibiotics with new metabolites from thus far unscathed \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-2571-1197\nmailto:aaishu.thapa01@gmail.com\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 1-6   Thapa et al. \n\n\u00a9NJB, BSN 2 \n\nhabitats of potential sources that help in controlling the \n\nproblem [7]. \n\nKathmandu Valley lies in an alluvial plain where melting \n\nsnow of four different mountain ranges in the Himalayas \n\nfeeds rivers and streams that flow down from the \n\nmountain. Based on the variation in different locations of \n\nthe valley, soil type and their composition, there is a \n\npossibility of different diversified habitat. Therefore, the \n\nquest of discovering novel microflora and finding \n\nantimicrobial producing Bacillus spp. can be fulfilled [8]. \n\nThus, the project will help in analysis of distribution and \n\nantimicrobial activities of Bacillus spp. collected from the \n\nsampling sites. \n\nMaterials and Methods \nSample collection \nNine soil samples were collected from different areas of \n\nthe Kathmandu Valley (organically cultivated fields, \n\nrhizospheric area and river banks) which were processed \n\nduring the study. The samples were collected from the \n\ndepth of 10-12.5 cm in the sterile polythene bags and \n\ntaken to the laboratory. The sample collection sites were \n\nSundarijal, Balaju, Bhaktapur, Sitapaila and Dillibazar \n\nand the samples were designated as S1, S2, S3, S4, S5, S6, \n\nS7, S8 and S9.", "start_char_idx": 3654, "end_char_idx": 6640, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e63eb483-2ac0-403d-a4b3-4482640e12f2": {"__data__": {"id_": "e63eb483-2ac0-403d-a4b3-4482640e12f2", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e8181b0-dc91-41d3-8ae2-dd3f0c7df2e2", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "fc146d07f87baa529fb2084d54ea4a84683c91c8778c18ef2839a8a87abb7c2f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "22e5014b-115f-4b48-a565-c6b414044372", "node_type": "1", "metadata": {}, "hash": "799494055d4e6aa74c5e57c75d72e8bb6682eaa18807f1d1b3ebbd6ca62779d3", "class_name": "RelatedNodeInfo"}}, "text": "The samples S1 and S8 were from Bhaktapur (Nagarkot \n\nLatitude: 27\u00b04254\u00b0N, Longitude: 85\u00b03114\u00b0E and \n\n27\u00b04015.7\u00b0N, 85\u00b02621.3\u00b0E), S2 from Dillibazar (27.7054\u00b0 \n\nN, 85.3267\u00b0 E), S3 from Sundarijal (27.7909\u00b0 N, 85.4272\u00b0 \n\nE),S4 from Shivapuri (27.8129\u00b0 N, 85.3859\u00b0 E) ,S5 from \n\nGokarneshwor ( 27\u00b0 44' 2.688\" N 85\u00b0 22' 55.38\" E), S6 and \n\nS7 from Balaju (27.73192\u00b0 N, 85.29945\u00b0 E and  27.7309\u00b0 N, \n\n85.2955\u00b0 E) and S9 from Sitapaila (27.7170\u00b0 N, 85.2735\u00b0 E). \n\nIsolation and screening of antimicrobial \nproducing species \nThe bacteria were isolated using a spread plate technique \n\nafter heat treatment of dilutions (10-4 and 10-5) for 10 \n\nminutes in a water bath at 80\u2103 [9].  The zone of inhibition \n\nproducing colonies were observed and selected \n\npreliminarily on Nutrient Agar. Initially, the isolated \n\ncolonies were identified by Gram staining and spore \n\nstaining and then sub-cultured. \n\nPrimary screening was done by \u2018perpendicular streaking \n\nmethod\u2019 in which a vertical line of isolates was streaked \n\non Nutrient Agar (with 2% agar to prevent spreading \n\ncolonies of Bacillus spp.) and incubated at 37\u02daC for 24 hrs. \n\nThe test organisms are  S. aureus,  E. coli, Pseudomonas \n\nspp. and Salmonella spp. (standardized with 0.5 \n\nMcFarland, with cell suspension 108 CFU/ml) were then \n\nstreaked perpendicular to the line of growth of isolate. \n\nThese plates were then incubated overnight at 37\u02daC .The \n\nzone of inhibition was measured respectively after the \n\ncomplete incubation [10]. \n\nCharacterization of antimicrobial producing \nisolates \nCharacterization of Bacillus spp. was done on the basis of \n\nmorphological properties, biochemical tests and growth \n\nat a temperature at 55\u2103 and salt concentration of 6.5% \n\nNaCl according to Bergey's Manual of Determinative \n\nBacteriology [11]. \n\nProduction of crude extract \nSix isolates with high antimicrobial activity through \n\nprimary screening were selected and inoculated a loopful \n\nof sample in about 25 ml of nutrient broth (NB) in a \n\nconical flask and incubated for 48 hrs at 37\u02daC in the \n\nshaker incubator. The broth culture was then transferred \n\nin tubes and centrifuged at 2012 g for 20 min. After the \n\ncentrifugation, the pellets were discarded and the \n\nsupernatant was mixed with ethyl acetate (1:1 ratio), \n\ncollected in screw cap tubes using sterile dropper and \n\nthen centrifuged at 6000 rpm for 20 min. The upper layer \n\nwas collected and transferred to vials. This extract was \n\nlabeled as crude extract [12]. \n\nAgar well diffusion method \nSix test organisms, MRSA, ESBL E. coli (Available at the \n\nresearch laboratory of St. Xavier\u2019s College, Maitighar, \n\nKathmandu, Nepal) and four same test organism as used \n\nin the primary screening and were taken and swabbed on \n\nMuller Hinton agar plates after standardization with 0.5 \n\nMcFarland solution. On the agar plate, 5 wells of 8 mm \n\ndiameter using sterile cork borer and 150 \u00b5l of crude \n\nextract was poured in each well. They were then \n\nincubated at 37oC without inverting for 24 hrs.", "start_char_idx": 6643, "end_char_idx": 9659, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "22e5014b-115f-4b48-a565-c6b414044372": {"__data__": {"id_": "22e5014b-115f-4b48-a565-c6b414044372", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e63eb483-2ac0-403d-a4b3-4482640e12f2", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2c1228d2a637554c3b32b1f46cdd4e59c801d543f687fc6ff6e959760bf8953a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3bfbf594-6939-4e10-a74e-a1a75d7440e2", "node_type": "1", "metadata": {}, "hash": "21da175f1b7846750eef12416120db2de1c69c53d0871398d76de6dfa11d2492", "class_name": "RelatedNodeInfo"}}, "text": "The ethyl \n\nacetate was used as negative control and antibiotic discs \n\nerythromycin (15 mcg), nitrofurantoin (300 mcg), \n\nvancomycin (30 mcg) and gentamicin (30 mcg) (HiMedia \n\nLaboratories) were used as positive control. [12] \n\nResults \n\nIsolation and identification of the Bacillus spp. \nFrom the 9 soil samples collected from different parts of \n\nthe Kathmandu Valley, 24 isolates that produced a zone \n\nof inhibition around their growth on the isolation media \n\ni.e. Nutrient agar selected for identification and \n\nscreening. The zone of inhibition around them is \n\nsuggestive of antimicrobial(s) produced by them that \n\ndiffuse through the media and inhibit growth of other \n\nmicroorganisms around them. These isolates were \n\nidentified as Bacillus spp. on the basis of gram staining \n\n(gram positive rods) and spore staining (sporulating).", "start_char_idx": 9660, "end_char_idx": 10506, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3bfbf594-6939-4e10-a74e-a1a75d7440e2": {"__data__": {"id_": "3bfbf594-6939-4e10-a74e-a1a75d7440e2", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "22e5014b-115f-4b48-a565-c6b414044372", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "c8129aa20e1a0f47a5f4e9461e6127ffcf161c441dbc169b600f036647ab017c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a5092e80-1d5b-43e8-b6da-4695fbd46910", "node_type": "1", "metadata": {}, "hash": "904babd7d1628dd5cb3b2640718f5f4669d2001ec4eef779f8b2c6e1dd550648", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 1-6   Thapa et al. \n\n\u00a9NJB, BSN 3 \n\nPrimary screening of the antimicrobial \nproducing isolates  \nThe 24 isolates of Bacillus spp. were chosen for primary \n\nscreening to detect their ability to produce antimicrobial \n\nsubstances by perpendicular streaking method. All of the \n\nisolates were found to show the zone of inhibition around \n\ntheir growth in primary screening (perpendicular \n\nstreaking method) against four test organisms viz. S. \n\naureus, E. coli, Pseudomonas spp. and Salmonella s pp. \n\nOf these 24 isolates, eight isolates produced a significant \n\nzone of inhibition (\u22651mm) against any one of the test \n\norganisms (Table1). \n\nCharacterization of antimicrobial producing \nisolates \nAmong these eight isolates six isolates showing a \n\nsignificant zone of inhibition (\u22651mm) against two or more \n\ntest organisms during primary screening were chosen for \n\nsecondary screening and were further characterized \n\nthrough different morphological, physiological and \n\nbiochemical tests  (Table 2). \n\nSecondary Screening of the antimicrobial \nproducing isolates  \nThe six isolates showing a significant zone of inhibition \n\n(\u22651mm) against two or more test organisms during \n\nTable 1. Zone of inhibition demonstrated by antimicrobial metabolite producing isolates against test organisms \n\nSample Isolate Number Name of the \n\nisolate \n\nS. aureus E. coli Pseudomonas spp. Salmonella spp. \n\nS2 5 U1 1 mm - - 6 mm \n\nS3 1 U2 22 mm 1 mm - 8 mm \n\nS4 1 U3 11 mm - - - \n\nS5 3 U4 1 mm - 1 mm 2 mm \n\nS6 1 U5 6 mm - - 9 mm \n\nS1 2 U6 15 mm - - - \n\nS3 1 U7 5 mm - - - \n\nS6 2 U8 5 mm - - - \n\n- : not inhibited; U1-8: Bacillus spp. isolates \n\nTable 2. Characterization of the isolates based on morphology and different biochemical tests \n\nCharacteristics U1 U2 U3 U4 U5 U6 \n\nSpore Spore-former Spore-former Spore-former Non Spore-former Spore-former Spore-former \n\nMotility Motile Motile Non-motile Non-motile Motile Motile \n\nCell Diameter >1mm <1mm >1mm <1mm <1mm <1mm \n\nCatalase + + + + + + \n\nMR + + + + - - \n\nVP - + - - + + \n\nCitrate - + + - + + \n\nStarch hydrolysis + + - - + + \n\nNitrate reduction - - - - + + \n\nAcid from glucose  + - - - - + \n\nAcid from mannitol - + + - + - \n\nAcid from  arabinose  - - - - - - \n\nGrowth at 55\u02daC - - - - - - \n\nGrowth in 6.5%NaCl + + + - + + \n\nIdentified as: Bacillus  spp. \n\n+ : positive, - : negative  \n\nTable 3. Zone of inhibition demonstrated by crude antimicrobial metabolite extract against test organisms \n\nTest organisms \n\nIsolates MRSA S. aureus ESBL \nproducing E. \n\ncoli \n\nE. coli Pseudomonas spp. Salmonella spp. \n\nU1 11 mm 12 mm - - - 20 mm \nU2 12 mm 12 mm - - - 15 mm \nU3 12 mm 12 mm - - 15 mm 15 mm \nU4 20 mm 15 mm - - - - \nU5 20 mm 25 mm - - - - \nU6 30 mm - - - - 18 mm \n\n- : not inhibited", "start_char_idx": 10511, "end_char_idx": 13270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a5092e80-1d5b-43e8-b6da-4695fbd46910": {"__data__": {"id_": "a5092e80-1d5b-43e8-b6da-4695fbd46910", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3bfbf594-6939-4e10-a74e-a1a75d7440e2", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "141b60855140f437928d455f699b18e86073fc858f2ee187e8af34472c6c3174", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1edde5d4-535a-46b4-b1b6-b5571fdbd1fa", "node_type": "1", "metadata": {}, "hash": "6d735e4ab752e2e339c79ac5c451316284fbbbe746fd37dd5cc7b42e803c8861", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 1-6               Thapa et al. \n \n\n\u00a9NJB, BSN   4 \n\nprimary screening were chosen for secondary screening \n\nby agar well diffusion method and tested against test \n\norganisms including MRSA and ESBL producing E. coli  \n\nas tabulated in Table 3.  \n\nThe results obtained in this study showed that the \n\nisolates U3, U5 and U6 have the potential for producing \n\nantimicrobial substances inhibiting the Gram positive. \n\nSimilarly, U1, U2 and U3  have inhibited Gram negatives  \n\n Salmonella spp. as well. The most prominent finding of \n\nthe study was isolate U5, which produced a zone of \n\ninhibition of about 25 mm against Staphylococcus aureus \n\n(ATCC 25923) which was comparable to the zone of \n\ninhibition produced by erythromycin against the \n\norganism. \n\nDiscussion \nThe study was carried out to isolate and identify \n\nantimicrobial metabolites producing Bacillus spp. from \n\nvarious sites of Kathmandu valley in Nepal. The initial \n\nselection of the antimicrobial producing strains was \n\nbased on the clear zone around their colony shown by \n\ndifferent bacterial colonies in primary culture on NA \n\nplates (Figure 1). In a similar study done by Rai et al, the \n\ncolonies with a clear halo zone were considered as \n\nantibiotic producers. The competition for the growth of a \n\ntype of organism present in the sample was inhibited by \n\nthe other organisms marking a boundary where no \n\norganism grew giving rise to the halo zone [12].  \n\nThe identification tests performed as per Bergey\u2019s \n\nManual of Determinative Bacteriology showed that the \n\nisolates U1, U3 and U4 were close to those of Bacillus \n\nmacquariensis, B. sphaericus and B. inosolitus respectively \n\nwhereas the results of isolates U2, U5 and U6 were close \n\nto that of B. subtilis (Figure 2). NB used as a culture \n\nmedium during production of crude extract in this study \n\nsupported the production of antimicrobial compounds \n\nwhich was observed in the form of a zone of inhibition \n\nagainst the test organisms (Figure 3). In contrast, a study \n\nshowed that culture of B. subtilis B38 strain in NB resulted \n\nin sufficient growth, but did not exhibit any antibacterial \n\nactivity [6]. Extract from trypticase soy broth (TSB) \n\nshowed better results than NB in another study [13]. \n\nAlthough majority of the antimicrobial compounds do \n\nnot require metal ions to perform their biological \n\nactivities, there are some compounds that need metal \n\nions to maintain their structures and physiological roles \n\nproperly which may not be provided by nutrient broth \n\nand the difference in inhibitory effect of the extracts \n\nmight have differed due to the type of the compound \n\nproduced [14]. \n\n \nFigure 1. Isolates with antimicrobial activity on Nutrient \n\nagar. \n\n \nFigure 2. Grams staining under 100x \n\n \n\n \n \n\nFigure 3. Agar-well diffusion method with test organism \n\nS. aureus (Central well- Negative control).  \n\n \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 1-6               Thapa et al. \n \n\n\u00a9NJB, BSN   5 \n\nFurther, extracts of all 6 isolates demonstrated a zone of \n\ninhibition against MRSA with the highest zone of \n\ninhibition of 30 mm being shown by the isolate U6. It can \n\nbe inferred that the antimicrobials produced by the \n\nisolates have greater potency as a novel remedy for the \n\nalarming rate of antibiotic resistance and in particular, \n\nthe methicillin resistant Staphylococcus aureus. \n\nThe extract from the isolates showed more inhibition \n\nagainst Gram positive bacteria than Gram negative \n\nbacteria, however, E. coli and ESBL producing E. coli were \n\nnot inhibited by any of them. In a similar study, 63.63% \n\ncolonies demonstrated inhibitory effects against gram \n\npositive bacteria and 27.27% colonies were inhibitory to \n\ngram negative bacteria. 9.09% of the colonies had a broad \n\nspectrum activity [12].", "start_char_idx": 13275, "end_char_idx": 17100, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1edde5d4-535a-46b4-b1b6-b5571fdbd1fa": {"__data__": {"id_": "1edde5d4-535a-46b4-b1b6-b5571fdbd1fa", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a5092e80-1d5b-43e8-b6da-4695fbd46910", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "96768a8edd2647e3142713fb18115c34e0fdac630c1887e0b625bce666c2bcde", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8daa0b36-43fe-408c-b85c-6d9b1de56e26", "node_type": "1", "metadata": {}, "hash": "67a0550ef39da0c8806da4ccec80412274bd0e21d7c1395759dd5310e67a5b3e", "class_name": "RelatedNodeInfo"}}, "text": "9.09% of the colonies had a broad \n\nspectrum activity [12]. It has been reported that \n\nantimicrobial properties of genus Bacillus show greater \n\ninhibition to the gram positive bacteria than gram \n\nnegative bacteria in comparison. The resistance of Gram-\n\nnegative test strains to certain antimicrobial metabolites \n\nis due to the barrier created for the hydrophobic \n\ncompounds because of the presence of outer \n\nlipopolysaccharide layer which is less permeable. It is \n\nalso suggested that the produced antimicrobial \n\nsubstances produced by a Gram-positive bacterium are \n\nrestricted to other gram-positive bacteria [15,16]. In \n\ncontrast, the antimicrobials produced by B. subtilis MIR \n\n15 in a study have shown inhibitory action mostly against \n\ngram-negative bacteria including E. coli and P. aeruginosa \n\n[4]. Among all 6 isolates, crude obtained from isolate U3 \n\nwas found to have broad spectrum activity with \n\ninhibitory effect even against gram negative organisms \n\nlike Pseudomonas spp. and Salmonella spp. One of the \n\nimportant findings that this isolate was active against \n\nPseudomonas spp. is of great importance as Gram negative \n\nbacterium such as Pseudomonas spp.  is usually resistant \n\nto a wide range of antibiotics [17]. \n\nThe extracellular secretion of antimicrobial metabolites in \n\nsoluble form are significant from an industrial point of \n\nview as disruption of the bacterial cells and solubilization \n\nprocesses can be avoided during downstream \n\nprocessing. The reason that we could not characterize \n\nBacillus spp. was due to unavailability of molecular tools \n\n(detection of marker genes) in the routine laboratory of \n\nSt. Xavier\u2019s College, Nepal and limited capital (non-\n\nfunded) for outsourcing.  \n\nThe increase in antibiotic resistance has been attributed \n\nto inappropriate use, inadequacies on the part of the \n\nmanufacturers and leads to the steady decline of effective \n\nantibiotics annually worldwide. Antibiotic resistance is \n\npresent in every country. The patients with drug resistant \n\ninfections consuming more healthcare resources are high \n\nrisk to clinical outcome and eventually death than the \n\npatients with nonresistant infections. This situation is a \n\nserious challenge to drug manufacturers, public health \n\npractitioners worldwide [18]. \n\nReducing the spread of resistant pathogens and the rate \n\nof evolution of resistance is complex. Antibiotic \n\nresistance is forcing scientists to search for these \n\nantibiotic-producing bacteria in hopes of finding new \n\nways of killing pathogens. Therefore, this study is an \n\nattempt to identify Bacillus species with potential of \n\nantibiotic production that could be used to stem the \n\nscourge of drug resistance which suggest that the soil of \n\nKathmandu valley is inhabited by different \n\nantimicrobials producing Bacillus spp and its proper \n\nstudy may create a way towards the development of \n\nnovel antibiotics. \n\nConclusion  \nThis study demonstrated that the isolated strains of \n\nantimicrobial producing Bacillus spp. can be used as a \n\npotent source of antibiotics as isolates U3, U5 and U6 \n\nshowed an appreciable zone of inhibition against the test \n\norganisms especially with the Gram positive ones \n\nincluding MRSA, than the gram negative ones. However, \n\nisolates U1, U2 and U3 were effective antimicrobials for \n\nGram negative organisms such as Salmonella spp. and \n\nPseudomonas spp. \n\nAuthors' contributions  \nAT designed and conceptualized the project. AT, AB, AS \n\nMS and PS carried out the experiments, lab works and \n\nparticipated in the data analysis. SP helped to design the \n\nstudy, amended the methodology, managed necessary \n\narrangements during laboratory investigations and \n\nsupervised the complete study. All authors have read, \n\nedited and approved the final manuscript. \n\nCompeting interests  \nNo competing interests were disclosed. \n\nFunding  \nThe authors declared that there were no grants were \n\ninvolved in supporting this work. \n\nAcknowledgements  \nOur sincere gratitude to the Department of Microbiology, \n\nSt. Xavier\u2019s College, Maitighar, Kathmandu, Nepal for \n\nproviding the laboratory facilities and all the support for \n\ncompleting this study. \n\n\n\nNepal J Biotechnol.", "start_char_idx": 17041, "end_char_idx": 21252, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8daa0b36-43fe-408c-b85c-6d9b1de56e26": {"__data__": {"id_": "8daa0b36-43fe-408c-b85c-6d9b1de56e26", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1edde5d4-535a-46b4-b1b6-b5571fdbd1fa", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "df174bfcfda264b152f947e46ee1c28d16a51eb508f88d0845c05a9b1210cf18", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "98603d5f-4b31-4331-a33a-c7f3b137adca", "node_type": "1", "metadata": {}, "hash": "dbf50fcddc177e69ad2bd31347cbb0abbfb2106633ca29976b169ba0d46f9db8", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 1-6               Thapa et al. \n \n\n\u00a9NJB, BSN   6 \n\nEthical approval and consent  \nHumans or human samples were not used in this study. \n\nSo, ethical approval from concerned authority was not \n\nneeded.  \n\nReferences \n1. Mackie TJ, McCartney JE. Mackie, McCartney practical medical \n\nmicrobiology. 1st ed. Singapore: Longman Singapore Publishers \nLtd. 1996. \n\n2. Wafula EN, Kinyua J, Karuiki D, Muigui A, Mwirichia R. Isolation \nand characterization of Bacillus species from soil in Ngere tea \ncatchment area of Murang\u2019A County, Kenya. International Journal \nof Life Sciences Research. 2014;2(3):27-35. \n\n3. Mansour A, Zeinab R, Amanollah ZA. Isolation and identification \nof Bacillus Species from soil and evaluation of their antibacterial \nproperties. Avicenna J Clin Microb Infec. 2015;2(1):e23233, \ndoi: 10.17795/ajcmi-23233 \n\n4. Aslim B, Saglam N, Beyatli Y. Determination of some properties of \nBacillus isolated from soil. Turk J Biol. 2002;26:41\u20138. \n\n5. Baruzzi F, Quintieri L, Morea M, Caputo L. Antimicrobial \ncompounds produced by Bacillus spp. and applications in food. \nScience against microbial pathogens: communicating current \nresearch and technological advances. 2011;1102-1111. \n\n6. Tabbene O, Slimene IB, Bouabdallah F, Mangoni ML, Urdaci MC, \nLimam F. Production of anti-methicillin-resistant Staphylococcus \nactivity from Bacillus subtilis sp. strain B38 newly isolated from \nsoil. Appl Biochem Biotechnol. 2009;157:407\u2013419.  \n\n7. Blomberg B. Antimicrobial resistance in developing countries. \nTidsskr Nor Laegeforen. 2008;128(21):2462-2466. \n\n8. Vinod AS, More SM. Isolation, identification and characterization \nof Bacillus species from Lonar lake for production of cyclodextrin \nglycosyltransferase. Int.J .Curr. Microbiol. App. Sci. 2013;2(12):14-\n23. \n\n9. Sharma M, Manandhar M. Practical Approach to Microbiology. \n3rd ed. Kathmandu: National Book Centre; 2017. 200 p.  \n\n10. Iqbal S, Qasim M,  Begum F,  Rahman H, Sajid I. Screening, \ncharacterization and optimization of antibacterial peptides, \nproduced by Bacillus safensis strain MK-12 isolated from waste \ndump soil KP, Pakistan .Biorxiv. 2018. \ndoi: https://doi.org/10.1101/308205 \n\n11. Goodfellow M, Kampfer P, Dusse HJ, Trujillo ME, Suzuki KI, \nLudwig W, Whitman WB Bergey\u2019s manual of systematic \nbacteriology. 2nd ed. New York: Springer; 2012. \n\n12. Rai M, Aryal S, Parajuli P. Characterization and activity of \nantimicrobial       polypeptide of Bacillus spp from wastelands of \nKathmandu valley. Annals of Applied Bio-Sciences. 2017;4(1):50-\n57. \n\n13. Kumar A, Saini P, Shrivastav JN. Production of peptide antifungal \nantibiotic and biocontrol activity of Bacillus subtilis. Indian Journal \nof Experimental Biology. 2009;47:57-62. \n\n14.", "start_char_idx": 21233, "end_char_idx": 23993, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "98603d5f-4b31-4331-a33a-c7f3b137adca": {"__data__": {"id_": "98603d5f-4b31-4331-a33a-c7f3b137adca", "embedding": null, "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-106", "node_type": "4", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "2cac58b24d1f04cf750d0647f3c035200e003a8965e161eebe3bc591d1858bf1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8daa0b36-43fe-408c-b85c-6d9b1de56e26", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "5b02c30ad47ae6b2a4ebf367a68612d2f930201b26d47920efdd08735ac44929", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c0818a0a-5549-4844-ac41-8b42d1a45859", "node_type": "1", "metadata": {}, "hash": "acaaca0b1c1368d412b5db4608d768456531343829aefa08720161a6c580a377", "class_name": "RelatedNodeInfo"}}, "text": "2009;47:57-62. \n\n14. Jamil B, Hasan F, Hameed A, Ahmed S. Isolation of Bacillus subtilis-\nMH4 and its potential of polypeptide antibiotic production. Pak. J. \nPharm. Sci. 2007;20(1):26-31. \n\n15. Al- Tarawni AH, Al- Zereini WA, Tarawneh KA. Bacillus sp. 1A1 \nas a producer of antibacterial crude extract: taxonomy, cultivation \nand partial purification. Current Research in Bacteriology. \n2015;8:18-25. \n\n16. Motta  AS, Olivera FC, Brandelli A. Screening for \nantimicrobial activity among bacteria isolated from the Amazon \nBasin. Braz. J. Microbiol. 2004;35(4):307-310 \n\n17. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic \nresistance in Pseudomonas aeruginosa: mechanisms and alternative \ntherapeutic strategies. Biotechnology Advances.2019;37(1):177-\n192. \n\n18. Antimicrobial resistance [Inernet]. World Health Organization; \n2020  [ cited 2021 March,  4]. Available from: https://www.who.int \n/news-room/fact-sheets/detail/ antimicrobial-resistance  \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nhttp://dx.doi.org/10.17795/ajcmi-23233", "start_char_idx": 23973, "end_char_idx": 25135, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c0818a0a-5549-4844-ac41-8b42d1a45859": {"__data__": {"id_": "c0818a0a-5549-4844-ac41-8b42d1a45859", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "98603d5f-4b31-4331-a33a-c7f3b137adca", "node_type": "1", "metadata": {"identifier": "njb-106", "author": " Thapa, Aishwarya;  Budhathoki, Anupa;  Sapkota, Anupama; Sainju, Muskan; Shrestha, Prativa; Prasad Pant, Shyam", "title": "Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley", "date": "2021-12-30", "file": "njb-106.pdf"}, "hash": "9849e71ee734d3d5f87965742e6fd08eae1952bdeb1c5e5e6920817662b43ffa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "50cdb48f-ac30-460d-9899-b477de184966", "node_type": "1", "metadata": {}, "hash": "8d9b04548f922879fa4dfd0a0e969c90545c8e9f277be74933f3dda6179798d2", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 93-108 Review article  DOI: https://doi.org/10.3126/njb.v9i1.38671 \nSPECIAL ISSUE: ICBSN 2021 \n\n\u00a9NJB, BSN 93 \n\nIntellectual Property Right on Basmati Rice: Current Scenario and \nEvidences of Origin, Diversity, Cultivation and Use Values of Basmati \nRice in Nepal \nBal Krishna Joshi1 , Krishna Hari Ghimire1, Prakash Raj Bista2, Ram Baran Yadaw3, Ram Krishna Shrestha4, Gaurish \nKrishna Kharel5, Prakash Paneru6, Ram Bahadur KC6 and Deepak Bhandari6 \n\n1National Agriculture Genetic Resources Center, NARC; Khumaltar, Kathmandu, Nepal;  \n2Ministry of Agriculture and Livestock Development, Kathmandu;  \n3Directorate of Agricultural Research, NARC, Province-2, Parwanipur, Bara;  \n4Crop Development and Agro-biodiversity Conservation Center, Department of Agriculture, Lalitpur; \n5Kto Inc, Kathmandu  \n6Nepal Agricultural Research Council, Kathmandu,  \n\nReceived: 0 Mar 2021; Revised: 14 May 2021; Accepted: 22 May 2021; Published online: 31 Jul 2021 \n\nAbstract \nBasmati rice, also called the king/prince of rice landraces has very special values in Nepalese society as well as in other countries \nof Indian Subcontinent. With the objectives of collecting, analyzing and documenting Basmati related information in Nepal, we \nvisited different sites; carried out key informant surveys; organized focus group discussions, online interaction and discussion \nmeetings; requested all relevant offices/ persons/ stakeholders through phone, website, and letter to share information; \norganized high level official meeting, and Basmati rice expert meeting; documented video documentary and did online as well \nas library search. Because of its high market value at global level, many countries and organizations have been attempting to \nget intellectual property rights (mainly patent and geographical indication tag) on Basmati rice. India applied for GI tag to \nBasmati rice in the European Union (EU) in July 2018, and Nepal submitted opposition letter along with proofs and evidences \nof origin, diversity, cultivation and use values of Basmati rice on 9 December 2020. A total 133 Basmati type rice landraces are \ngrown in 60 districts of Nepal. Basmati rice is traditionally grown, sold, and consumed in geographically localized areas of \nNepal since ancient time. International and national scientists have defined lower altitude of Nepal as one of the centers of \norigin of Basmati rice. Many Nepalese basmati rice landraces have been characterized and evaluated using morphological traits, \nisozymes and DNA markers. Four basmati type of rice landraces have been registered in National Seed Board. Many \ncommunity seed banks have maintained different types of Basmati rice landraces. National Agriculture Genetic Resources \nCenter and International genebanks have collected more than 80 and conserved  68 basmati landraces. Basmati rice landraces \nhave geo-linked traits. The historical culture of production, consumption and marketing of native basmati rice in Nepal should \nalways be favored by both national and international rules and regulations. Nepal has ample and valid evidences to get \ngeographical indication (GI) right on Basmati rice. \n\nKeywords: Geographical indication, Basmati rice, origin, diversity, historical literature \n\n Corresponding author, email: joshibalak@yahoo.com \n\nIntroduction  \nRice diversities consisting of 2500 native landraces and \n\n153 improved varieties are being grown in 75 out of 77 \n\ndistricts and within an altitude range from 60 to 3050 m in \n\nNepal [1\u20137].", "start_char_idx": 48, "end_char_idx": 3590, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "50cdb48f-ac30-460d-9899-b477de184966": {"__data__": {"id_": "50cdb48f-ac30-460d-9899-b477de184966", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c0818a0a-5549-4844-ac41-8b42d1a45859", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "6ba38444184c8b8c0b8729b8517d95bafd47c51e185420a7fc34fe797d78103f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c408c375-90e2-4385-a121-4101ef945486", "node_type": "1", "metadata": {}, "hash": "6036266b0f54e6122cefd2602d61173c47e59556628cb76de6a4455d65f8e1d9", "class_name": "RelatedNodeInfo"}}, "text": "Before 1980, Nepal was exporting rice \n\nincluding aromatic rice to India, China, Singapore and \n\nBangladesh [1,8\u201312[. In 1977, a total of 105,000-t rice was \n\nexported [1]. The word \u2018Basmati\u2019 is used as an adjective \n\ndescribing the things having aroma or fragrance. Basmati, \n\nthe prince/king of rice is a valued and expensive cereal. \n\nMany landraces are very localized and possess specific \n\ntraits, for instance, Basmati with aroma [2,13,14]. In \n\ngeneral, Basmati type landraces include all aromatic rice \n\nlandraces. Aromatic rice is grown in 10% of total rice area \n\n(i.e. 150,000 ha out of total 1491,744 ha rice area) with total \n\nproduction of 375,000t in Nepal [15]. Average \n\nproductivity of such landraces is about 2.5 t/ha. Basmati \n\nrice emits aroma which could be a geographical indication \n\n(GI).  \n\nGeographical indication is a sign used on products that \n\nhas a specific geographical origin and possess qualities or \n\nreputation that are due to that origin. It is a very common \n\npractice to provide GI tag to the agricultural products in \n\nthe world to monopolize the marketing. Germany has the \n\nhighest number of GI tagged products with 9,499, but, \n\nNepal does not have any one [7]. A total of 361 GI \n\nproducts have been registered in India as of September \n\n2019. Darjeeling tea was the first GI tagged product in \n\nIndia, registered in 2004. In 2010, Basmati rice also got \n\nregistered as GI product in India. India has also submitted \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttp://orcid.org/0000-0002-7848-5824\nmailto:joshibalak@yahoo.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108 Joshi et al. \n\n\u00a9NJB, BSN 94 \n\napplication to EU for geographical indication tag to \n\nBasmati rice in 2018 [16]. In addition to EU, India has also \n\nsought registration of \u2018Basmati\u2019 in different countries.  \n\nBasmati rice is grown in Indian Subcontinent and many \n\ncountries have their own native Basmati rice [16\u201318]. \n\nMany diverse Basmati types of rice landraces are being \n\ngrown in different parts of Nepal since ancient times \n\n[1,6,14,19,20]. Therefore, the Nepalese farming \n\ncommunities have rights on using Basmati rice. Nepal \n\napplied opposition letter to EU with regards to GI tag to \n\nIndian Basmati rice on 9 December 2020. To be eligible to \n\nget GI tag to Basmati rice, Nepal needs to develop and \n\ngenerate relevant proofs and evidences. The objectives of \n\nthis paper, therefore, were to compile Basmati rice related \n\nproofs and evidences of origin, diversity, cultivation and \n\nuse values in Nepal; to analyze historical cases of Basmati \n\nand aromatic rice landraces and to aware and generate \n\ninformation about GI tag to Basmati rice. \n\nMethodologies \nBasmati rice type (which include all aromatic rice \n\nlandraces) have been grown in different parts of Nepal \n\nsince ancient time. There are key farmers and researchers \n\nwho are well familiar with Basmati type rice landraces. \n\nBoth primary and secondary data were collected, \n\nanalyzed and discussed. Information related to \n\ngeographical indication and Basmati rice were telecasted, \n\npublished in different media and shared widely to make \n\naware and improve understanding of geographical \n\nindication. Nine different methods to generate and \n\ncompile Basmati rice related proofs and evidences were \n\nadopted.", "start_char_idx": 3591, "end_char_idx": 7034, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c408c375-90e2-4385-a121-4101ef945486": {"__data__": {"id_": "c408c375-90e2-4385-a121-4101ef945486", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "50cdb48f-ac30-460d-9899-b477de184966", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "92131d0a4130d92aed20808edee20295fca77c59dd0d1a513937a4de75468c66", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7a985bfa-bd0c-48fe-996e-d06fa33f5c10", "node_type": "1", "metadata": {}, "hash": "934094e33ff3eaa8b5ef0b4c4ad5e75b422d6df178e103eeb87e23764ee889b8", "class_name": "RelatedNodeInfo"}}, "text": "We visited 6 different sites; surveyed 15 key \n\ninformants; organized 5 times focus group discussion; \n\norganized 3 times online interaction and discussion \n\nmeetings; requested call through phone, email, website \n\nand different media to relevant organization across the \n\ncountry; organized two times high-level-official meetings; \n\norganized a Basmati rice expert consultation meeting; \n\ndeveloped three video documentaries, and surveyed \n\nliteratures. Proofs and evidences were grouped and \n\nanalyzed under 10 different areas as shown in Figure 1. \n\nDatabase of Genesys (https://www.genesys-pgr.org/), \n\nNational Genebank of Nepal and community seed banks \n\nwere analyzed. Districts growing aromatic rice landraces \n\nwere mapped in the country map. Total number of \n\naromatic rice landraces was estimated based on their \n\nname given by farmers. Aromatic rice diversities were \n\ngrouped under two: Basmati group (any landraces that \n\ncontain at least the word Basmati in their name) and non-\n\nbasmati aromatic group (any landraces that emits aroma \n\nbut do not have the word Basmati in their name).    \n\nFigure 1. Groups of potential proof and evidences of \norigin, diversity, cultivation and use values of Basmati \nrice  \n\nFindings \nAll relevant stakeholders, farming communities, experts \n\nand high officials attended in various official meetings \n\nagreed that Basmati rice originated in Nepal and different \n\ntypes of Basmati rice are being grown. Basmati rice has \n\nmultidimensional values associated with farming \n\ncommunities, wealthy people and special occasions. \n\nFarmers and researchers are also well familiar with the \n\nhistorical importance, diversity, market value and use of \n\nBasmati rice in Nepal.  \n\nGeographical indication in Nepal \nBecause of climatic variation, Nepal is rich in \n\nagrobiodiversity and some of them are produced in very \n\nspecific areas e.g. Jumli Marshi, Jethobudo, Basmati, Juju \n\nDhau, Pharping pear, etc. More than 100 agricultural \n\nproducts are potential for geographical indication (GI) tag \n\nin Nepal [7,21]. It is well known that if Jethobudo grows \n\nother than Pokhara valley, its quality decreases. None of \n\nthe products are registered as GI in Nepal; however, there \n\nare many products including Basmati, marketed \n\ninformally as GI and getting higher price for assured \n\nbetter quality in different parts of the country. Three traits \n\n(famous, special trait and origin) are very important on GI \n\nsystem. Basmati is very famous, has a very special trait \n\nand originated in Nepal, and therefore, hold capacity to \n\nget GI tag. Legally registration system as GI has not been \n\nexisted in Nepal, but there is a policy provision for GI [7]. \n\nBasmati rice in Nepal and India  \nBasmati rice emits a specific aroma in the field, at \n\nharvesting, in storage, during milling, cooking and eating. \n\nSome landraces may emit aroma in only few stages e.g. at \n\n12\n\n34\n\n5\n\n6\n\n8\n\n7\n\n9\n\n10\n\nDeep rooted evidences for GI\n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108 Joshi et al. \n\n\u00a9NJB, BSN 95 \n\nharvesting, during cooking, etc. In Nepal Basmati type \n\nrice covers all aromatic rice landraces (short grain, \n\nmedium and long grain types) and varieties [22].  It is \n\ngrown in Tarai and Mid Hill agricultural ecozones. This is \n\nhighly reputed rice and cost very high. Therefore, normal \n\nfamily cannot offer such rice all the day. Basmati rice are \n\nsold in many locations by the name of production areas. \n\nMany short and medium grain aromatic landraces are \n\ngrown and consumed locally but they are not known \n\nmuch in the international market.", "start_char_idx": 7035, "end_char_idx": 10627, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7a985bfa-bd0c-48fe-996e-d06fa33f5c10": {"__data__": {"id_": "7a985bfa-bd0c-48fe-996e-d06fa33f5c10", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c408c375-90e2-4385-a121-4101ef945486", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "5c0134652de20a3d5e418d6059cb6b4ad869fa1f6cd352c60aeab818bd31b723", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "361092ca-30ea-4572-b7dc-b4761ec5f68b", "node_type": "1", "metadata": {}, "hash": "a1b7b730a070fb5894eae0ceb5c24f6cb38f59b5ee245271cdc9dc9a299469a9", "class_name": "RelatedNodeInfo"}}, "text": "In India, Basmati type \n\nrice include only long grain rice that emits aroma in most \n\nof the time [16\u201318,23]. Along with Basmati, many short \n\nand medium grain aromatic rice varieties are grown in \n\ndifferent parts of India. Some of them are superior for taste \n\nand aroma as compared to long grain aromatic varieties \n\n[18]. Though Basmati rice includes all aromatic rice in \n\nNepal, we have to standardize the Basmati rice for \n\ninternational trade as per the international standard \n\nincluding in India and Pakisan.  \n\nRelatively traditional grown Basmati rice are better in \n\nquality and aroma than Basmati varieties developed by \n\nbreeder and grown in high input conditions. Tools and \n\ntechniques now are available to check the adulteration of \n\nBasmati that help protect the interest of consumers and \n\nfarmers [18]. Ready-to-use kit along with DNA markers \n\ncould be used for Basmati authentication. An international \n\ncode of practice has been developed for maintaining the \n\nreputation of Basmati rice [18]. \n\nFigure 2. Historical events on intellectual property right \n\n(IPR) over Basmati Rice and its geographical indication \n\n(GI) cases in Nepal \n\nHistorical Events on IPR over Basmati Rice \nBecause of very highly recognized and preferred trait of \n\nBasmati rice, different types of intellectual property rights \n\nare being tried on Basmati rice around the world. For \n\nexample, US-based RiceTec Company patented Basmati \n\nrice in 1997 [24]. India registered Basmati rice under the \n\nregime of geographical indication in 2010. In Nepal IPR \n\nover agricultural products and technologies are very \n\nnegligible. In case of Basmati rice, Nepal applied detail \n\nproofs and evidences of origin, diversity, cultivation and \n\nuse values of Basmati rice in EU in Dec 2020. Historical \n\ndetails of IPR over the Basmati rice and GI cases are given \n\nin Figure 2.  \n\nEvidences of origin, diversity, cultivation and \nuse values of Basmati rice \nBasmati rice possess geo-linked trait i.e. quality and \n\naroma. Same genotype of Basmati rice if grown in other \n\nthan its original home localities, their quality differs and \n\ncould not get same quality products as produced in their \n\nnative localities. This property of Basmati rice is then \n\nprovided as GI tag. Many different types of information \n\nand methodologies are needed to get GI tag [21]. \n\nEvidences on aromatic rice in Nepal are described below.", "start_char_idx": 10628, "end_char_idx": 13034, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "361092ca-30ea-4572-b7dc-b4761ec5f68b": {"__data__": {"id_": "361092ca-30ea-4572-b7dc-b4761ec5f68b", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7a985bfa-bd0c-48fe-996e-d06fa33f5c10", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "45d92585ce3261989c740b2173ba2a64ef9d0590b3f6f67851615f3de0ddb965", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "582d0844-197d-43df-afb2-2ca5155b14e5", "node_type": "1", "metadata": {}, "hash": "772b48c91e6ddc63618ad490c01d82a050edb3c0a811b4712c9f4629bd1b8cd8", "class_name": "RelatedNodeInfo"}}, "text": "Evidences on aromatic rice in Nepal are described below.  \n\n1997 Detail evidences submitted to EU\n\n2 Sept 1997\n\nNepal, member of WIPO\n\nRiceTech, US patented Basmati rice\n\nJune 2000 India opposed US patent on Basmati rice\n\n23 April 2004\n\n2002 RiceTech withdrew basmati rice patent\n\nNepal, member of TRIPS/WTO\n\nMay 2010\n\nJuly 2018\n\n11 Sept 2020\n\n5 Oct 2020\n\n28 Nov 2020\n\n2 Dec 2020\n\n7 Dec 2020\n\n8 Dec 2020\n\n9 Dec 2020\n\n15 Dec 2020\n\n16 Dec 2020\n\n20 Dec 2020\n\n6 Dec 2020\n\n5 Dec 2020\n\n4 Feb 2021\n\nGI tag to Basmati rice in India \n(7 states) by APEDA\n\nIndia applied GI tag to Basmati rice in EU\n\nNotice on GI tag to Basmati rice published in OJ of the EU\n\nPakistan decided to oppose India claim over GI tag in EU \n\nGK Kharel got informed notice of OJ of the EU on GI tag to Basmati rice \nNotice of OJ of the EU on GI tag to \nBasmati rice published in Dekhapadhi\n\nNotice of OJ of the EU on GI tag to \nBasmati rice published in Setopati\n\nBasmati GI tag issue entered in DI and MoICS\nafter application notice by APAA Nepal\n\nBasmati GI tag issue entered \nin NARC and MoALD\n\nProofs and \nevidences collected \n\n1st high level meeting \nand decided to oppose \n\nOppose letter prepared and submitted \nand received acknowledgement from EU \n\nDisseminated widely through media \n\nMeeting with NAPA \n\n2nd High level meeting\n\n29 Dec 2020Panel Discussion on Basmati case, NAPA\n\n1 Jan 2021Committee formed \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108 Joshi et al. \n\n\u00a9NJB, BSN 96 \n\nLinguistic and ancient literature evidences \nIn literal meaning, basmati means aroma or scented \n\n[3,7,9,25]. It is made up of two Sanskrit words, \u2018Vas\u2019 means \n\naroma and \u2018Mati\u2019 means ingrained from the origin. In \n\nNepali language, the equivalent of Vas is Bas (aroma) and \n\ntherefore, aroma related rice landraces and varieties are \n\ncalled Basmati. Almost all Nepali understand the meaning \n\nand importance of Basmati and Basmati rice and \n\ntherefore, this word Basmati has become the very \n\ncommon Nepali word.  \n\nAncient literatures have mentioned Basmati rice as an \n\nimportant food, nutritional and medicinal items [1,26\u201330]. \n\nChandranighantu (250 years old literature) has described \n\nSali Dhan [29,31]. Ayurved has grouped rice diversity in \n\nthree categories and among them shukdhanya category \n\nincludes 15 sali dhanya jaat including Basmati type [29]. \n\nNepali literature of 1960 BS (1903 AD) reported 61 \n\nlandraces including Basmati type [27]. In Lumbini, which \n\nis birth place of Lord Gautam Buddha, there is aromatic \n\nrice landrace called Kalanamak, which was used as holy \n\ngrain during Baudha period (900 BC). Because of its \n\nreligious important, community has started conservation \n\nworks for Kalanamak rice landrace (called Bhaudhakalin \n\nKalanamak Dhan) and reviving the culture [32,33]. The \n\nFather of Lord Budhha is the king Suddhodhan which \n\nmean pure rice with aroma. Nepal has also registered \n\naromatic rice by this name, Suddhodhan Kalanamak. \n\nKalanamak rice also contain high iron and zinc [34].", "start_char_idx": 12978, "end_char_idx": 15974, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "582d0844-197d-43df-afb2-2ca5155b14e5": {"__data__": {"id_": "582d0844-197d-43df-afb2-2ca5155b14e5", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "361092ca-30ea-4572-b7dc-b4761ec5f68b", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "0f036e0ab5b244df3f11e504fb6db0b9a4c0692d7659e3c695103a85f20c84e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5ef865c7-2421-43e6-974c-18ea61ce45a7", "node_type": "1", "metadata": {}, "hash": "dc7339982270e83101aa35fb4d8d4c182f8ba92a35c3640e07f69b6ca4164fad", "class_name": "RelatedNodeInfo"}}, "text": "Kalanamak rice also contain high iron and zinc [34]. Many \n\nresearchers have documented and published about \n\nNepalese basmati rice landraces and many of these are \n\navailable online [1,6,9,19,30,35\u201348]. \n\nFolklore \nBasmati is common word in Nepal and has been used for \n\ngiving name to ladies. Other plant species having aroma \n\nare also named with this word e.g. Basmati sponge gourd, \n\nBasmati Rayo, Basmati banana, etc. We can see some \n\nstories, poems, songs associated with Basmati in Nepal. \n\nFor example, there is Deuda song in Western Nepal, \n\n\u201cBasmati ko dhan pakya garai basai gaya\u201d which mean \n\nduring maturity, Basmati rice emits aroma and any one \n\ncan feel its aroma around the field. Similarly, fair dance \n\nsong called Hathhorha is held in Baisakhi festival. There \n\nis a long song this fair dance and its part (\u201cBasmati \n\nropanya syara O bamja Jhuprai bamja\u201d) also relate \n\nBasmati rice [49]. \n\nTraditions, specialty and reputation  \nBasmati rice is traditionally grown, sold, and consumed in \n\nNepal since ancient time [1,10\u201312,25,33\u201336,44,48,50\u201358]. \n\nHistorically, public mind always considers Basmati rice as \n\na special grain aromatic rice grown and produced in a \n\nparticular geographical area. Basmati rice landraces have \n\ngeo-linked traits and they are grown in different parts of \n\nNepal [4\u20137]. It is highly valued and most important rice \n\nlandraces fetching premium price in the market \n\n(8,9,19,21,25,36,38,38,41,50,57,59\u201362).  \n\nBasmati is a group of rice landraces used on special \n\noccasion [10,51,55]. Farmers\u2019 experiences indicated that \n\norganic Basmati is far better than non-organic in quality \n\nand aroma. The aroma decreases when an aromatic rice is \n\ngrown with chemical fertilizers. The application of home \n\nmade compost (made up from native and local materials \n\nand livestock dungs) is a must if the real aromatic rice is \n\nto be produced. Aroma is higher in recently harvested rice \n\nover old stock. Rice dehulled with local Dhiki is with \n\nhigher aroma as compared to grain milled in a rice huller \n\n[19]. Its straw is very soft and long therefore farmers \n\nprefer to make different home items, Gundri, Chakati, \n\nChataai, etc. from its straw. Basmati rice straw is also used \n\nto make marriage-temple or house and this is very old \n\ntradition in Tarai region. \n\nSociocultural, economical and market value  \nBasmati rice has social and cultural values in Nepalese \n\ncommunities [10\u201312,14,33,52,55,57,58]. Social status is \n\nvery high for those family who consume and grow \n\naromatic rice. Common culture from ancient time is to \n\noffer Basmati rice based food items to guest, relatives, VIP, \n\nin festival, special function, marriage ceremony, etc. \n\n[57,60]. Nepali community consider Basmati rice grain as \n\nholy, pure, chokho, virgin and therefore used during \n\nfasting, offer to Gods and Goddess, and used in different \n\nreligious ceremony (chhat, shraddha, etc.). It is also a \n\ncomponent of axeta and vikxa. There are a lot of socio \n\ncultural evidences particularly in Tarai area. It is used \n\nduring Ram Janaki Bibaha, in general marriage ceremony, \n\nKul deuta pooja, etc. \n\nSome farmers use Basmati rice grain in death ceremony \n\nand Ritual Shraddha. For this, they allocate separate land \n\nfor continued growing of Basmati rice and harvest from \n\nthis land is used during ritual program.", "start_char_idx": 15922, "end_char_idx": 19267, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5ef865c7-2421-43e6-974c-18ea61ce45a7": {"__data__": {"id_": "5ef865c7-2421-43e6-974c-18ea61ce45a7", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "582d0844-197d-43df-afb2-2ca5155b14e5", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "7a45bf57319a6d8bb0821a6775003bc03308859ae9d4642ed02fdfb980bd8e32", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e5671dfc-95ee-41cd-8234-3f168209bb1e", "node_type": "1", "metadata": {}, "hash": "057d38ee47ec233384d9b1cc6f056df42f331faa9037d6dffb889a626dc57dda", "class_name": "RelatedNodeInfo"}}, "text": "On the day of \n\nShraddha, their home use to be full of delicious rice smell. \n\nIn many religious events, there is a function called hom-\n\nhalne where aromatic rice grains are used and one can feel \n\nsmell of rice around during this function. \n\nBasmati rice has a very high economic value [19,44,59,60]. \n\nThe aromatic rice is very popular in both domestic and \n\ninternational markets and fetches premium price. Gin and \n\nShahi [28] reported that Nepal used to export about 200 \n\nmetric tons of fine quality aromatic rice per annum \n\nearning about 4l million rupees in 1977.  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108 Joshi et al. \n\n\u00a9NJB, BSN 97 \n\nTable 1. List of Basmati landraces conserved in National \nGenebank, Nepal \n\nSN Accession Landrace  Collected site \n\n1 \nNGRC01669 Jhinuwa \n\nMasino \nGorakhkali, \nGorkha \n\n2 NGRC01698 Basmati Dhan Kalsil, Bajura \n\n3 NGRC 01811 Basmati Dhan  Mundi, Humla \n\n4 NGRC 01815 Basmati Dhan  Tukche, Mustang \n\n5 NGRC 01825 Basmati Dhan  Makai, Humla \n\n6 NGRC 01835 Sunaulo Dhan Badhu, Bajura \n\n7 \nNGRC 01867 Masino Basmati \n\nDhan \nDhading \n\n8 NGRC 01945 Basmati Dhan Lalitpur \n\n9 \nNGRC 01967 Kalanamak \n\nDhan \nKhungai, \nRupandehi \n\n10 \nNGRC 02022 Hanse Dhan Dandagaon, \n\nSalyan \n11 NGRC 02030 Basmati Dhan Dipayal, Doti \n\n12 NGRC 02036 Sunaulo Dhan  Martadi, Bajura \n\n13 \nNGRC 02066 Hansaraj Dhan Madigaon, \n\nBajhang \n\n14 \nNGRC 02093 Hansaraj Dhan Manara, \n\nDadeldhura \n\n15 \nNGRC 02094 Basmati Dhan Manara, \n\nDadeldhura \n\n16 \nNGRC 02103 Sunaulo Dhan Bhandara, \n\nDadeldhura \n\n17 \nNGRC02821 Jhinuwa \n\nMasino \nGorkha Bazaar, \nGorkha \n\n18 NGRC03016 Kanakjira Udayapur \n\n19 NGRC03023 Sunaulo Ghaiya  Sanagaun -7, Doti \n\n20 NGRC03038 Sunaulo Ghaiya Silgadhi-9, Doti \n\n21 \nNGRC03050 Sunaulo Ghaiya Sallaghari-11, \n\nDadeldhura \n\n22 \nNGRC03051 Danda Basmati Dasharat chand-9, \n\nBaitadi \n\n23 \nNGRC03052 Danda Basmati Dasharat chand-9, \n\nBaitadi \n\n24 \nNGRC03096 Kalo Masino Taranagar \n\n(DADO), Gorkha \n\n25 \nNGRC03249 Jhinuwa \n\nBasmati Dhan \nRaluka, Nuwakot \n\n26 \nNGRC03268 Basmati Dhan Thumpakhar, \n\nSindhupalchok \n\n27 \nNGRC03289 Rato Basmati \n\nDhan \nParsa \n\n28 \nNGRC03291 Rato Basmati \n\nDhan \nBara \n\n29 \nNGRC03293 Basmati Nokhi \n\nDhan \nBara \n\n30 NGRC03326 Kanakjira Dhan Sunsari \n\n31 \nNGRC03364 Basmati Dhan Chhinnamasta,", "start_char_idx": 19268, "end_char_idx": 21526, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e5671dfc-95ee-41cd-8234-3f168209bb1e": {"__data__": {"id_": "e5671dfc-95ee-41cd-8234-3f168209bb1e", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5ef865c7-2421-43e6-974c-18ea61ce45a7", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "509267ee3941f6ef1a6a0a4883ccb82b69937ca918fa1117a330bfb1605b7eb1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "926824b7-6c7a-4997-9820-a78e776a382d", "node_type": "1", "metadata": {}, "hash": "c71888b636031be29af93f8b69e598759fcb929d25565a43bdbe2cd6fbf65515", "class_name": "RelatedNodeInfo"}}, "text": "Saptari \n\n32 \nNGRC03369 Kalanamak \n\nDhan \nKapilvastu \n\n33 NGRC03375 Basmati Dhan Dhanusha \n\n34 \nNGRC03389 Basmati Dhan Beldari, \n\nKanchanpur \n\n35 \nNGRC03415 Basmati Dhan Phulkaha Katti, \n\nSiraha \n36 NGRC04999 Kalo Masino Gaikhur, Gorkha \n\n37 \nNGRC05007 Hansaraj \n\nBasmati Dhan \nChaudhari-6, \nMauri bagar, \nBajhang \n\n38 NGRC05017 Hansaraj Dhan Banjh-8, Bajhang \n\n39 NGRC05018 Shyamjiro Banjh-8, Bajhang \n\n40 NGRC05691 Shyam Jira Gadariya-1, Kailali \n\n41 NGRC07862 Jarneli Dhan Barpak, Gorkha \n\n42 NGRC07869 Begani Ghaiya Saurpani, Gorkha \n\n43 \nNGRC07889 Masino Basmati Bichaur-4, \n\nLamjung \n\n44 \nNGRC07900 Mohanbhog Patharaiya-9, \n\nKailali \n\n45 \nNGRC07915 Kalo Jhinuwa Ghanpokhara, \n\nLamjung \n\n46 \nNGRC07923 Lekali Basmati Ghanpokhara, \n\nLamjung \n\n47 \nNGRC08267 Seto Basmati \n\nDhan \nShivasatasi \nMunicipality, \nJhapa \n\n48 \nNGRC08273 Kalo Tuned \n\nBasmati Dhan \nShivasatasi \nMunicipality, \nJhapa \n\n49 \nNGRC08276 Chhoti Basmati \n\nDhan \nShivasatasi \nMunicipality, \nJhapa \n\n50 \nNGRC08277 Rato Basmati \n\nDhan \nShivasatasi \nMunicipality, \nJhapa \n\n51 \nNGRC08278 Hansaraj Dhan Shivasatasi \n\nMunicipality, \nJhapa \n\n52 \nNGRC08289 Kanakjira \n\nBasmati Dhan \nShivasatasi \nMunicipality, \nJhapa \n\n53 \nNGRC08300 Jorayal Basmati \n\nDhan \nShivasatasi \nMunicipality, \nJhapa \n\n54 \nNGRC08303 Hansaraj Dhan Shivasatasi \n\nMunicipality, \nJhapa \n\n55 \nNGRC08308 Kalo Basmati \n\nDhan \nShivasatasi \nMunicipality, \nJhapa \n\n56 \nNGRC08372 Kalo Basmati \n\nDhan \nKawasoti N.Pa.-\n14, Nawalpur \n\n57 \nNGRC08442 Hansaraj Dhan Satyawti Gaupa, \n\nGulmi \n\n58 \nNGRC08586 Basmati Dhan Betali \u2013 4,\n\nRamechhap \n\n59 \nCo - 10271 \n\nHansaraj Dhan Satyawti Gaupa, \nGulmi \n\n60 \nCo - 10406 \n\nBasnadaar \nLamda Dhan \n\nSayaal -5, Doti \n\n61 \nCo - 10512 \n\nBasmati Dhan Betali - 4, \nRamechhap \n\n62 \nCo - 10669 \n\nKalo Tuned \nBasmati \n\nShiwa satasi N. Pa. \n- 3, Jhapa \n\n63 \nCo - 10681 \n\nShyanmjira Khjura Ga. Pa. - 4, \nBanke \n\n64 \nCo - 10691 \n\nBasmati Dhan Bhadrapur N. Pa. - \n1, Jhapa \n\n65 \nCo - 10692 \n\nChulthe \nBasmati \n\nBhadrapur N. Pa. - \n1, Jhapa \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.", "start_char_idx": 21529, "end_char_idx": 23587, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "926824b7-6c7a-4997-9820-a78e776a382d": {"__data__": {"id_": "926824b7-6c7a-4997-9820-a78e776a382d", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e5671dfc-95ee-41cd-8234-3f168209bb1e", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "11a54c1658eb08dcc5ce1f47e848952ea1898da3f5230f822fde417e49187d79", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "98cae205-6e8f-43a3-a9ad-3949043fdfcd", "node_type": "1", "metadata": {}, "hash": "7442de631060f70807dca28a2752df802b910b2b7fbbb0c6ad0c6350f5d01093", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  98 \n\n66 \nCo - 10751 \n\nBasmati Dhan Bagmati - 6, \nLalitpur \n\n67 \nCo - 10764 \n\nAglo Basmati \nDhan \n\nBagmati - 7, \nLalitpur \n\n68 \nCo - 10765 \n\nHocho Basmati \nDhan \n\nBagmati - 7, \nLalitpur \n\n69 \nCo - 10915 \n\nBasmati Dhan Gokulganga Ga. \nPa. - 4, Ramechhap \n\n70 \nCo - 11257 \n\nKalo Basmati  Ganeshaman N. \nPa. - 7, Dhanusha \n\n71 \n\nCo - 11258 \n\nSunaulo \nSugandha  \n\nBhartapur \nMahangarpalika, \nChitwan \n\n72 \nCo - 11381 \n\nGajiyabad \nBasmati \n\nBharatpur sub - \nMC - 19, Chitwan \n\n73 \nCo - 11382 \n\nPuspa Basmati Bharatpur sub - \nMC - 19, Chitwan \n\n74 \nCo - 11416 \n\nBasnadar Kalo Bharatpur sub - \nMC - 19, Chitwan \n\n75 \nCo - 11427 \n\nThaniya \nBasmati \n\nBharatpur sub - \nMC - 19, Chitwan \n\n76 \nCo - 11454 \n\nKalanamak  Bharatpur sub - \nMC - 19, Chitwan \n\n77 \nCo - 11462 \n\nBasphool Bharatpur sub - \nMC - 19, Chitwan \n\n78 \nCo - 11464 \n\nBaspare  Bharatpur sub - \nMC - 19, Chitwan \n\n79 \nCo - 11468 \n\nBasmati \nPaschimko \n\nBharatpur sub - \nMC - 19, Chitwan \n\n80 \nCo - 11470 \n\nJhinuwa \nBasmati  \n\nBharatpur sub - \nMC - 19, Chitwan \n\nNote: There are other Basmati (aromatic) type rice accessions in \nNational Genebank of Nepal that need to further study and \nverification. \n\nMany local rice millers (around 60 rice factories) are \n\nmarketing basmati rice by different brand names at local \n\nand national levels. Some of native aromatic landraces are \n\nas competitive as modern varieties [8,9]. Many \n\nhouseholds prefer to grow economically valued traits ie \n\naroma [63]. Relatively quality of Nepalese Basmati rice is \n\nbetter than other countries. Three landraces (Basmati, \n\nRato Basmati, and Kalo Nuniya) are very popular \n\naromatic landraces in Nepal and have a high market value \n\nin comparison with other varieties [64]. Basmati comes \n\nunder the group of five-qualities (Pancha Gudiya) product \n\nin Nepal. These five qualities are purity, quality, tasty, \n\nhealthy and nutritious. Some of Basmati landraces are \n\nmedicinally important [29]. Basmati rice landraces milled \n\nin local mill (dhiki) content low glycemic index and \n\ntherefore are useful for diabetes patients.  \n\nDatabases  \nFour basmati type of rice landraces have been improved \n\nand registered in National Seed Board of Government of \n\nNepal. They are Pokhreli Jetho Budho rice registered in \n\n2006, Lalka basmati registered in 2010, Suddhodhan \n\nKalanamak and Kalonuniya registered in 2020. These \n\nregistrations have also been published in Nepal Gazette \n\n(Nepal Rajpatra) on different dates [3,65\u201367]. Many \n\ncommunity seed banks have maintained different types of \n\nbasmati rice landraces in their localities [68\u201370].", "start_char_idx": 23591, "end_char_idx": 26173, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "98cae205-6e8f-43a3-a9ad-3949043fdfcd": {"__data__": {"id_": "98cae205-6e8f-43a3-a9ad-3949043fdfcd", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "926824b7-6c7a-4997-9820-a78e776a382d", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "615e8e4a260ad47b089fa44ae083eea20f1ffa219d61d47b1056a5bee5ebedda", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "912a3a96-17cc-4012-9f3f-94254d3b64f3", "node_type": "1", "metadata": {}, "hash": "e43efb246bb84acd74aae46bf867103bc57a27c019d6e308f4be10ff3484db86", "class_name": "RelatedNodeInfo"}}, "text": "National Agriculture Genetic Resources Center (National \n\nGenebank) under Nepal Agricultural Research Council \n\n(NARC, www.narc.gov.np) has collected and conserved \n\nmore than 80 basmati type rice accessions from different \n\nareas of Nepal (Table 1). There are other landraces which \n\nare Basmati type but recognized as different names. Their \n\nexamples include Hansraj, Jethobudho, Jhinuwa, Kalo \n\nmasino, Tilki, Ghiu puri, Begani, Jarneli, Gauriya, Kalo \n\nnuniya, Kala Namak, Kanak Jira, Kariya Kamod, \n\nKrishnabhog, Sali Dhan, Shyamjira, etc [4\u20136,45,71]. \n\nInternational genebanks (https://www.genesys-\n\npgr.org/) has conserved more than 68 basmati rice \n\naccessions collected from different parts of Nepal (Table \n\n2) and some of them were collected in early 1970s. Some \n\nof basmati type accessions conserved in the International \n\nRice Research Institute (www.irri.org) genebank include \n\nAsamiya Basmati, Basmati, Basmati Anpjhutte, Basmati \n\nDhan, Basmati Gola, Basmati Lamo, Basmati Masino, \n\nBasmati Nokhi, Basmati Pahade, Basmati Red, Basmati \n\nUzarka, Basmati White, Danda Basmati, Kalo Basmati, \n\nMasino Basmati, Rato Basmati, Sete Basmati, Seto \n\nBasmati, etc. Many of these accessions conserved in IRRI \n\nhas already been shared with other countries for research \n\nand utilization [67]. \n\nTable 2. Nepali rice accessions named Basmati available \nfrom Genesys \n\nSN Accession  Acquisition \nDate \n\nLocal Name  \n\n1 PI 549247 1984/12/13 Basmati mutant \n2 IRGC 16213 1972/06/30 Masino Basmati \n3 IRGC 23861 1972/04/05 Dhera Dun \n\nBasmati \n4 IRGC 23787 1972/04/05 Basmati Dhan \n\n5 IRGC 58881 1981/08/31 Basmati Lamo \n\n6 IRGC 58882 1981/08/31 Basmati Masino \n(Purple Tip) \n\n7 IRGC 58886 1981/08/31 Basmati Red \n\n8 IRGC 58884 1981/08/31 Basmati Nokhi \n\n9 IRGC 58883 1981/08/31 Basmati Masino \n\n10 IRGC 59054 1981/08/31 Kalo Basmati \n\n11 IRGC 58880 1981/08/31 Basmati Gola \n\n12 IRGC 58885 1981/08/31 Basmati Pahade \n\n13 IRGC 16136 1972/06/30 Asamiya Basmati \n\n14 IRGC 83309 1994/09/22 Basmati \n\n15 IRGC 83679 1994/09/22 Seto Basmati \n\n16 IRGC 83317 1994/09/22 Basmati Mutant \n\n17 IRGC 83316 1994/09/22 Basmati Mixed 2 \n\n18 IRGC 83650 1994/09/22 Rato Basmati \n\n19 IRGC 83310 1994/09/22 Basmati Dhan \n20 IRGC 83314 1994/09/22 Basmati Dhan \n\nhttp://www.narc.gov.np/\nhttps://www.genesys-pgr.org/\nhttps://www.genesys-pgr.org/\nhttp://www.irri.org/\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB,", "start_char_idx": 26177, "end_char_idx": 28601, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "912a3a96-17cc-4012-9f3f-94254d3b64f3": {"__data__": {"id_": "912a3a96-17cc-4012-9f3f-94254d3b64f3", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "98cae205-6e8f-43a3-a9ad-3949043fdfcd", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "d587349f45d5cbf19ee44ba72f83c0b71bf1d1853484c9b43fd0943af93195df", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f62eeabd-d40c-45b5-a97f-b1c5a60009de", "node_type": "1", "metadata": {}, "hash": "c2b46a7a2f4942e712e1bb52c0a6cc5460d211ba5335b5c2c6bba7bca48a53b2", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  99 \n\n21 IRGC 83784 1994/09/22 Basmati \n\n22 IRGC 86925 1996/10/24 Basmati Dhan \n\n23 IRGC 83313 1994/09/22 Basmati Dhan \n\n24 IRGC 83312 1994/09/22 Basmati Dhan \n\n25 IRGC 83661 1994/09/22 Red Basmati \n\n26 IRGC 83308 1994/09/22 Basmati \n27 IRGC 83315 1994/09/22 Basmati Dhan \n\n28 IRGC 88761 1995/06/02 Basmati Mixed 1 \n\n29 IRGC 83678 1994/09/22 Seto Basmati \n\n30 IRGC 83676 1994/09/22 Sete Basmati \n\n31 IRGC 83311 1994/09/22 Basmati Dhan \n\n32 IRGC 16130 1972/06/30 Basmati \n\n33 IRGC 58887 1981/08/31 Basmati White \n\n34 IRGC 58879 1981/08/31 Basmati \nAnpjhutte \n\n35 IRGC 59205 1981/08/31 Rato Basmati \n\n36 IRGC 58888 1981/08/31 Basmati Uzarka \n\n37 IRGC 110313 1996/10/24 Danda Basmati \n\n38 IRGC 62000 1982/04/20 Masino Basmati \n\n39 IRGC 117438 2008/12/15 Basmati \nLamo::IRGC \n58881-1 \n\n40 IRGC 127766 2011/05/01 Rato \nBasmati::IRGC \n59205-1 \n\n41 IRGC 132324 2013/11/01 Basmati \nRed::IRGC 58886-\n2 \n\n42 IRGC 133780 2011/11/01 Basmati \nDhan::IRGC \n83313-1 \n\n43 IRGC 133781 2011/11/01 Basmati \nDhan::IRGC \n83315-1 \n\n44 IRGC 133779 2011/11/01 Basmati \nDhan::IRGC \n23814-1 \n\n45 IRGC 133785 2011/11/01 Basmati \nGola::IRGC \n58880-1 \n\n46 IRGC 133805 2011/11/01 Basmati \nMasino::IRGC \n58883-1 \n\n47 IRGC 133810 2011/11/01 Basmati \nMutant::IRGC \n83317-1 \n\n48 IRGC 133824 2011/11/01 Basmati \nWhite::IRGC \n58887-1 \n\n49 IRGC 133809 2011/11/01 Basmati Mixed \n1::IRGC 88761-1 \n\n50 IRGC 133806 2011/11/01 Basmati Masino \n(Purple \nTip)::IRGC \n58882-1 \n\n51 IRGC 133815 2011/11/01 Basmati \nNokhi::IRGC \n58884-1 \n\n52 IRGC 133962 2011/11/01 Danda \nBasmati::IRGC \n110313-1 \n\n53 IRGC 134149 2011/11/01 Kalo \nBasmati::IRGC \n59054-1 \n\n54 IRGC 134335 2011/11/01 Rato \nBasmati::IRGC \n83650-1 \n\n55 IRGC 134357 2011/11/01 Red \nBasmati::IRGC \n83661-1 \n\n56 IRGC 134410 2011/11/01 Sete \nBasmati::IRGC \n83676-1 \n\n57 IRGC 134411 2011/11/01 Seto \nBasmati::IRGC \n83679-1 \n\n58 IRGC 134645 2011/11/01", "start_char_idx": 28596, "end_char_idx": 30463, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f62eeabd-d40c-45b5-a97f-b1c5a60009de": {"__data__": {"id_": "f62eeabd-d40c-45b5-a97f-b1c5a60009de", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "912a3a96-17cc-4012-9f3f-94254d3b64f3", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "b702599d788665a75588fdcf3da721bcd2a3465523d7d3320355328d2c3d8d03", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1303ccbd-120a-482f-9ea6-ec333b88dc59", "node_type": "1", "metadata": {}, "hash": "d43614da59e7c5c8b393a0297374f1396e1b76efa84007d1e314c64baf1a9cdb", "class_name": "RelatedNodeInfo"}}, "text": "83679-1 \n\n58 IRGC 134645 2011/11/01 Dhera Dun \nBasmati::IRGC \n23861-1 \n\n59 IRGC 134791 2012/11/01 Basmati \nDhan::IRGC \n83310-2 \n\n60 IRGC 134792 2012/11/01 Basmati \nDhan::IRGC \n83312-2 \n\n61 IRGC 134836 2012/11/01 Masino \nBasmati::IRGC \n62000-2 \n\n62 IRGC 134880 2011/11/01 Basmati \nDhan::IRGC \n86925-1 \n\n63 IRGC 134948 2013/11/01 Basmati \nDhan::IRGC \n83311-2 \n\n64 IRGC 135656 2011/11/01 Basmati Mixed \n2::IRGC 83316-1 \n\n65 IRGC 135657 2011/11/01 Basmati \nPahade::IRGC \n58885-1 \n\n66 IRGC 135712 2014/05/01 Basmati \nDhan::IRGC \n83314-2 \n\n67 IRGC 136202 2015/05/01 Basmati \nUzarka::IRGC \n58888-2 \n\n68 IRGC 140367 2018/01/05 Seto \nBasmati::IRGC \n83678-1 \n\nNote: There are many other Basmati (aromatic) type rice \naccessions (with other than Basmati name) collected from Nepal \nin this Genesys database.  Source: [67], https://www.genesys-\npgr.org/ \n\n Center of diversity \nBasmati rice has been originated in Indian Subcontinent \n\n[16\u201318,72\u201374]. The center of diversity of aromatic rice are \n\nthe foothills of Himalayas in the Indian states of Uttar \n\nPradesh (UP) and Bihar, and Tarai region of Nepal \n\n[17,38,54,75] and produced in geographically localized \n\nareas of Nepal. The center of diversity and dispersal route \n\nare indicated in Figure 3. International and national \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  100 \n\nscientists have defined lower altitude of Nepal as one of \n\nthe centers of origin of basmati rice [38,56,72,76,77]. The \n\nTarai belt of Nepal was once considered as the bowel of \n\naromatic rice landraces [38]. Choi et al [76] reported three \n\nmajor geographically structured genetic groups of \n\naromatic rice and they are Bhutan and Nepal which is \n\nadmixture of cluster 2 and 3; Bangladesh, India and \n\nMyanmar which made distinct cluster, and Iran and \n\nPakistan which also made distinct cluster. \n\n \nFigure 3. Center of diversity and dispersal route of aromatic rice \nin Asia.Source: [17] \n\nHistorical evidences and research  \nAncient documents (Ayurved, Chandraniganthu), old \n\nscientific literatures [1,26,27,29,32,33] and culture of Lord \n\nGautam Bauddha [33] have mentioned different features \n\nand uses of aromatic rice. Cultures and values associated \n\nwith Basmati rice have been passed from generation to \n\ngeneration. Basmati rice is the most preferred by all \n\nNepali people. Basmati rice was used by King family, very \n\nrich people, in special occasion, festival, very special \n\nfunction, etc. It is common culture in Nepal to offer \n\nBasmati rice to VIP and guest. Family having Basmati rice \n\nalso got respected by the communities. It has been used as \n\nindicator of rich people and neighbors easily know \n\ncooking variety of rice by smelling aroma.", "start_char_idx": 30428, "end_char_idx": 33154, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1303ccbd-120a-482f-9ea6-ec333b88dc59": {"__data__": {"id_": "1303ccbd-120a-482f-9ea6-ec333b88dc59", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f62eeabd-d40c-45b5-a97f-b1c5a60009de", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "470077bff79e66c7d58c968ca933165a9f90f618b27081bd584c9afc990626d7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "74849343-ac88-438e-8a30-2f799185a38d", "node_type": "1", "metadata": {}, "hash": "1b8732c1c2b00a1906fb08fd6c9044411d44e32f5506ac24c744e0f667ac8f49", "class_name": "RelatedNodeInfo"}}, "text": "Research on basmati rice started in 1951 with the \n\ncollection of 930 rice germplasm (including aromatic \n\nlandraces) from across 54 districts and their evaluation at \n\nParwanipur and Khumaltar [1,26] in Nepal. NARC and \n\nother organizations have been working for developing \n\naromatic rice varieties using local basmati rice landraces \n\nsince early 1960s [8,9,40,67,78\u201382].  \n\nNepali students (BSc Ag) in India have also experiences of \n\ntaking Basmati rice including wild rice from Nepal to \n\nIndia with expecting good amount of money. They \n\nremembered; teacher taught about the importance of \n\nBasmati rice and possibility of patenting this rice (Prabeen \n\nDahal, 2020 personal communication). \n\nBasmati rice diversity and production areas  \nThere are many different forms of basmati rice landraces \n\nin Nepal, grown in different districts [1,4\u2013\n\n9,19,27,30,35,38,41,50,60,61,71,83\u201385]. We found total 133 \n\naromatic rice landraces by name. Among them, 43 \n\nlandraces contain the words in association with Basmati \n\nin their name (Table 1 and 2) and 90 landraces were \n\nnamed by the word other than Basmati (Box 1). Farmers \n\nin particular area may give their own name to aromatic \n\nrice landraces introduced from other areas. Four aromatic \n\nlandraces have been improved and registered in National \n\nSeed Board of Government of Nepal and two exotic \n\naromatic varieties (Sunaulo Sugandha and Sugandhit \n\nDhan-1) have been released for general cultivation. In \n\nIRRI Genebank, there are about 86 landraces described by \n\nthe name Basmati irrespective of grain dimensions and \n\nintensity of aroma in IRRI [23].  \n\nMaximum variation was observed in Nepal, followed by \n\nIndia and Bangladesh in aromatic germplasm [23]. Very \n\nhigh diversity at both phenotypic and genotypic levels in \n\nBasmati rice have been reported in Nepal \n\n[6,7,38,45,71,78,86]. Intra landrace diversity was also \n\nfound commonly in many aromatic landraces [79,80]. \n\nThese aromatic landraces possess very different traits and \n\nbased on 12 bases, types of aromatic rice landraces and \n\nvarieties along with meaning and examples are given in \n\nTable 3. Some are with awn and some are awnless with \n\nred, white and black grain. Based on grain size, there are \n\nthree types of Basmati rice, namely short, medium and \n\nlong grain Basmati rice [30,70,72,87]. Aromatic rice does \n\nnot exist for deep water condition in Nepal. Rahmani and \n\nBox 1.", "start_char_idx": 33158, "end_char_idx": 35573, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "74849343-ac88-438e-8a30-2f799185a38d": {"__data__": {"id_": "74849343-ac88-438e-8a30-2f799185a38d", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1303ccbd-120a-482f-9ea6-ec333b88dc59", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "41cf41302ba048479b7b8ae68cecbdaff6fc62f3669256fc44869cca4f9d1e9d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4d3e8927-2254-4080-aa86-4ee7f376cd14", "node_type": "1", "metadata": {}, "hash": "1f553dacc8d57549fda3b1767e2970fcb98ab79c2478a9efe9979a89bcce0ee3", "class_name": "RelatedNodeInfo"}}, "text": "Rahmani and \n\nBox 1. List of basmati (aromatic) type landraces (not \nnamed by the word basmati) in Nepal (total= 90) \nAachame Masino, Anadi Basnadar,  Anjana, Bagane, \nBagari, Bahani, Baharni, Barambhusi, Basnadaar Lamda \nDhan, Basnadar Kalo, Baspare, Basphool, Batisara, \nBayarni, Begani, Belguthi, Biramphool, Chengul, \nChiniyapuri, Chirankhe, Dudhe Marsi, Gauria (Gaure), \nGhaiya Rato, Ghyu Puri, Ghyu Kumari, Gude Kalo, Gude \nSeto, Gudgudo, Gudura, Gurdi Kalo, Gurdi Seto, \nHansaraj, Hanse Dhan, Hapsa, Hapsa Rato, Indrabeli, \nJaran Dhan (Kalo), Jarneli, Jaswa, Jethobudho, Jhinuwa, \nJhinuwa Ghaiya, Jirasari, Jogini, Kalanamak, Kalo Bayarni, \nKalo Jhinuwa, Kalo Masino, Kalo Nuniya, Kalo Nuniya \nThulo, Kalo Jhinuwa, Kanak Jira, Kariya Kamod, Kasturi, \nKhairo Anadi, Khalte Kholo, Koili, Krishna Bhog, Krishna \nCharcha, Lajee, Lalbachchhi, Madhukar, Mahabhog, \nMahajogani, Malbhog, Masino Jhinuwa, Motisor, Pahenle, \nPakhe Jhinuwa, Pakhe Tunde, Pokhreli Masino, Pran \nPeuri, Rahumanuwa, Rajbhog, Ram Tulsi, Ramjoin, Sali \nDhan, Samundraphinj, Seto Bayarni, Seto Jhinuwa, \nShyamjira, Sisuwapanheli, Sunaulo Dhan, Sunaulo \nGhaiya, Suwawat, Thapachini, Tilki, Tulsi Prasad, \nTulsiphool, Tunde. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108 Joshi et al. \n\n\u00a9NJB, BSN 101 \n\nHarinkher can also grow in shade area. Similarly Koili is \n\nshade loving aromatic landrace. Pranpyuri is very soft \n\nbasmati rice landrace.  \n\nTable 3.", "start_char_idx": 35553, "end_char_idx": 36985, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4d3e8927-2254-4080-aa86-4ee7f376cd14": {"__data__": {"id_": "4d3e8927-2254-4080-aa86-4ee7f376cd14", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "74849343-ac88-438e-8a30-2f799185a38d", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "915a85d6285870135973e3046d2873e0b0df7b5634baf3ec69a277decb987c22", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c013f254-381d-4386-bea9-21e0709cd972", "node_type": "1", "metadata": {}, "hash": "373f7eadc752c449bd5dd9058ba6fc96b22447a6e66e287dc1b2895b9d38e74c", "class_name": "RelatedNodeInfo"}}, "text": "Table 3. Grouping of Nepalese rice landraces based on different \n\ncriteria \n\nSN Basis Type Meaning Example \n\n1 \nPlanting \n\nseason \n\nChaite \n\naromatic \n\nDhan \n\nSpring rice, \n\ntransplanting \n\nin Chaitra \n\nTauli \n\nBhadayia \n\naromatic \n\nDhan \n\nEarly type \nBhadaiya \n\nBasmati \n\nBarkhe or \n\nAgahani \n\naromatic \n\nDhan \n\nNormal rice \n\nBasmati, \n\nKasturi, \n\nJhinuwa, \n\nJaswa, \n\nChananchur\n\n, Ujrka \n\nBasmati, \n\nLalka \n\nBasmati, \n\nTulsi \n\nPrasad, \n\nGopalbhog \n\nHiunde \n\naromatic \n\nDhan \n\nWinter or \n\nboro rice \n\nPakhe \n\nMasino \n\n2 Maturity \n\nEarly \n\naromatic \n\nrice \n\nEarly \n\nmaturity \n\nBhadaiya \n\nBasmati, \n\nGyu Puri \n\nMedium \n\naromatic \n\nrice \n\nMedium \n\nmaturity \n\nThapachini, \n\nAnadi \n\nBasnadar \n\nLate \n\naromatic \n\nrice \n\nLate maturity \n\nGurdi, \n\nKoili, \n\nBasmati, \n\nKasturi, \n\nJhinuwa, \n\nJaswa, \n\nChananchur\n\n, Ujrka \n\nBasmati, \n\nLalka \n\nBasmati, \n\nTulsi \n\nPrasad, \n\nGopalbhog, \n\nTilki \n\n3 Grain size \n\nShort \n\ngrain \n\naromatic \n\nrice \n\nSmall size \n\ngrain with \n\naroma \n\nJethobudho, \n\nPanhele, \n\nMotisar \n\nMedium \n\ngrain \n\naromatic \n\nrice \n\nMedium size \n\ngrain with \n\naroma \n\nMahajogani \n\nLong \n\ngrain \n\naromatic \n\nrice \n\nLong size \n\ngrain with \n\naroma \n\nAnadi \n\nBasnadhar \n\n4 \n\nEcosystem \n\n(production \n\nenvironment) \n\nRainfed \n\nupland \n\naromatic \n\nrice \n\nUnbunded \n\ncondition \n\nSuwawat, \n\nBegani \n\nGhaiya \n\nRainfed \n\nlowland \n\naromatic \n\nrice \n\nBunded \n\ncondition \n\nHansaraj, \n\nSali Dhan \n\nIrrigated \n\naromatic \n\nrice \n\nBunded \n\ncondition \n\nTilki, Kalo \n\nMasino \n\n6 Cultivation \n\nIntroduce\n\nd \n\naromatic \n\nFrom abroad \n\nSunaulo \n\nSugandha, \n\nSugandhit \n\nDhan-1 \n\nImproved \n\naromatic \n\nDeveloped by \n\nbreeder \n\nLalka \n\nBasmati, \n\nSudodhan \n\nKalanamak \n\nLandrace \n\naromatic \n\nMaintained, \n\ndeveloped by \n\nfarmers \n\nKariya \n\nKamod, \n\nKrishna \n\nBhog \n\n7 Morphotype \n\nTall \n\naromatic \n\nrice \n\nTall height \n\nKalo \n\nMasino, \n\nAnadi \n\nMedium \n\naromatic \n\nrice \n\nMedium \n\nheight \n\nHamsaraj, \n\nThapachini\n\nya \n\nDwarf \n\naromatic \n\nrice \n\nShort height \nTulsi \n\nKathey \n\n8 Grain color \n\nWhite \n\naromatic \n\nrice \n\nGrain with \n\nwhite husk \n\nTauli, \n\nHansaraj \n\nBlack \n\naromatic \n\nrice \n\nGrain with \n\nblack husk \n\nShyam Jira, \n\nKalanamak, \n\nKalo \n\nNuniya \n\nRed \n\naromatic \n\nrice \n\nGrain with \n\nred husk \n\nBegani \n\nGhaiya, Sali \n\nDhan \n\n9 \nPhotoperiod \n\nresponse \n\nAus \n\n(Saro, \n\nGaddar \n\nor \n\nGhaiya) \n\naromatic \n\nrice \n\nMature \n\nwithin a \n\ncertain \n\nperiod, \n\nphotoperiod \n\nnon sensitive \n\nGhiu Puri, \n\nBegani \n\nGhaiya \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.", "start_char_idx": 36977, "end_char_idx": 39447, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c013f254-381d-4386-bea9-21e0709cd972": {"__data__": {"id_": "c013f254-381d-4386-bea9-21e0709cd972", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4d3e8927-2254-4080-aa86-4ee7f376cd14", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "986b9f64a7e2f254fb609d5c1d58f529129045e97e61cd17abe8dfcb64d37a39", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "84fb4d1a-8c47-4051-ad9e-30f569892c8d", "node_type": "1", "metadata": {}, "hash": "4c595778c34d230053ab50f66cd09b8df9b48dea6fbe27369e8efd10451f3417", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  102 \n\n \n\nAman \n\n(Agahani \n\nor \n\nSarihan) \n\naromatic \n\nrice \n\nMature at \n\nparticular \n\ntime, photo \n\nperiod \n\nsensitive \n\nKalanamak, \n\nTilki, \n\nGauriya, \n\nUjaraka \n\nBasmati, \n\nTulsi \n\nPrasad, \n\nKanakjira \n\n10 Awn \n\nAwnless \n\naromatic \n\nrice \n\nGrain without \n\nawn \n\nPokhreli \n\nMasino \n\nShort \n\nawn \n\naromatic \n\nrice \n\nGrain with \n\nvery short \n\nawn \n\nRato \n\nBasmati, \n\nLalka \n\nBasmati \n\nLong awn \n\naromatic \n\nrice \n\nGrain with \n\nlong awn \n\nSeto \n\nBasmati, \n\nHansaraj \n\n11 Aroma \n\nComplete \n\n(universal\n\n) aromatic \n\nrice \n\nEmits aroma \n\nin all stages \n\n(field, \n\nharvesting, \n\nstorage, \n\nmilling, \n\ncooking and \n\neating) \n\nHansaraj, \n\nSali Dhan, \n\nKalo \n\nMasino, \n\nJhinuwa, \n\nJethobudho, \n\nKalanamak, \n\nKalo \n\nNuniya \n\nPartial \n\naromatic \n\nrice \n\nEmits aroma \n\nin only few \n\nstages \n\n(cooking and \n\neating) \n\nLalka \n\nBasmati, \n\nJorayal \n\nBasmati, \n\nAnadi, \n\nChananchur\n\n, Jaswa, \n\nGauriya, \n\nUjarka \n\nBasmati \n\n12 Name \n\nBasmati \n\nnamed \n\nrice \n\nName has at \n\nleast basmati \n\nword \n\nBasmati, \n\nUjarka \n\nBasmati \n\nNon \n\nbasmati \n\naromatic \n\nrice \n\nName has \n\nother than \n\nbasmati word \n\nKasturi, \n\nJhinuwa \n\nSource: [1,7,19,22,36,50,52,57,61,70,86,88,89] \n\nSource: [1,2,6,7,14,19,20,30,35,36,41,47,48, 50\u201353, 57,61,65,68,70,78, 81\u201383, \n\n85\u201391] \n\nAromatic rice is produced in many Asian countries [18]. \n\nAromatic rice landraces mainly Basmati, Kalanamak, \n\nKariyakamod, Kalonuniya, Hansraj, Jethobudho, \n\nJhinuwa, Syamjira, Tilki, etc. are being grown in more \n\nthan 30,000 hectares of 41 districts in Nepal [4\u20136,57,65]. \n\nAromatic rice is cultivated from East Mechi to west \n\nMahakali. There are many Ghaiya aromatic landraces as \n\nwell in Doti and Achham districts. Aromatic rice Gauri, \n\nParanpyuri, Kalo Gude and Jhinuwa are still cultivated in \n\nsmall areas in Surkhet valley. Survey and literatures have \n\nshown that aromatic rices are grown in 60 districts out of \n\n77 in Nepal (Figure 4). The Tarai belt was once considered \n\nas the bowel of aromatic rice landraces [38]. Altitudinal \n\ndistribution of aromatic rice landraces is given in Table 4. \n\nAromatic rices are grown from 60 to 1800 m altitude in \n\nNepal.  \n\n \nFigure 4. Districts (marked by star sign) showing cultivation of \n\nnative Basmati (aromatic) type rice landraces in Nepal \n\nSource: [1,6,7,14,19,20,51,52,65,69,70,78] \n\nAgro-morphological and nutritional based \nevidences   \nCollection, characterization and evaluation of native \n\naromatic rice landraces have been started since 1951 in \n\nNepal [1,26].", "start_char_idx": 39451, "end_char_idx": 41935, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "84fb4d1a-8c47-4051-ad9e-30f569892c8d": {"__data__": {"id_": "84fb4d1a-8c47-4051-ad9e-30f569892c8d", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c013f254-381d-4386-bea9-21e0709cd972", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "b86aefcd72c3d50d52aa9a7fafdc2753549d5d2c307bfa7072fd497210adb9cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4172474c-c784-4d4f-bc67-fa321a59ce66", "node_type": "1", "metadata": {}, "hash": "f565110dbd8006fc6c93de56dea6bf607488aebe683de7fe35da3a67397e65a0", "class_name": "RelatedNodeInfo"}}, "text": "A large number of landraces and introduced \n\naromatic rice varieties were characterized and evaluated \n\nboth at on-farm and on-station by many Nepalese \n\nresearchers using agro-morphological traits \n\n[1,7,9,21,28,30,39,43\u201345,51\u201353,58,70,71,78\u201380,82,84,86\u2013\n\n88,92]. Cooking and eating qualities are the main \n\nimportant features in Basmati rice. These qualities are \n\nassociated with different factors. Nutritional analyses \n\nhave been done in some of native and improved Basmati \n\ntype rice varieties and landraces [21,22,48]. The protein \n\ncontent was maximum in Red Basmati (7.74%) and \n\nminimum in Black Basmati (6.51%) among the four \n\nBasmati varieties [22]. These nutritional profiles indicates \n\nthat there is variation within and among different types of \n\naromatic rice landraces grown in Nepal.  \n\nIsozymes and DNA based evidences  \nMany Nepalese aromatic rice landraces have been \n\nexplored through isozymes and DNA markers \n\n[18,38,45,76\u201378,92\u201394]. DNA profile and fingerprints of \n\nsome aromatic rice landraces using SSR markers are given \n\nin Figure 5. National and foreign scientists have used \n\nNepalese aromatic rice genotypes for genetics study, \n\nmolecular breeding and diversity study, and reported \n\nvariation within and among landraces collected from \n\ndifferent parts of Nepal.   \n\nBardiya\n\nBajhang\nDarchula\n\nMustang\n\nDolpa\n\nMugu\n\nHumla\n\nAchham\n\nKailali\n\nDoti\n\nBaitadi Bajura\n\nJumla\n\nJajarkot\nDailekh\n\nDang\n\nSalyan\n\nSurkhet\n\nBanke\n\nMyagdi\n\nRolpa\n\nRukum\n\nKalikot\n\nTanahu\n\nLamjung\nKaski\n\nManang\n\nGulmi\n\nPalpa\n\nSiraha\n\nM\na\n\nh\no\n\ntt\na\n\nri\n\nBara\n\nParsa\n\nChitwan\n\nGorkha\n\nLTP\n\nNuwakot\n\nRasuwa\n\nDolakha\n\nDhankuta\n\nTaplejung\n\nIlam\n\nJhapa\n\nM\no\n\nra\nn\n\ng\n\nSaptari\n\nB\nKTM\n\nSindhuli\n\nKavre\n\nK\nh\n\no\nta\n\nn\ng\n\nCHINA\n\nINDIA\n\nN\n\nSyangja\n\nAromatic rice growing district, Total: 52\n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  103 \n\nTable 4. Distribution of Basmati type rice landraces in Nepal \n\nSN Rice Cultivar Districts Altitude, m \n\n Achhame Masino Chitwan, Jhapa, Makawanpur, Morang 200-800 \n Asamiya Basmati Morang  <600 \n Badiya Basmati Bara, Rautahat, Parsa <600 \n Bagari Chitawan, Siraha 60-300 \n Baharni Bara, Parsa, Saptari, Siraha <500 \n Basmati Bara, Bajura, Dadeldhura, Darchula, Dhanusha, Doti, Humla, \n\nJhapa, Kapilvastu, Kanchanpur, Kathmandu, Lalitpur, Mahottari, \nMorang, Parsa, Pyuthan, Ramechhap Rautahat, Rupandehi, \nSarlahi, Siraha, Sindhupalchok, Taplejung, Udayapur \n\n200-1000 \n\n Basmati Anadi Bara <300 \n Basmati Anpjhutte Dolakha <800 \n Basmati Nokhi Bara  <300 \n Belguthi Jhapa, Morang, Sunsari, Sankhuwasabha,", "start_char_idx": 41936, "end_char_idx": 44517, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4172474c-c784-4d4f-bc67-fa321a59ce66": {"__data__": {"id_": "4172474c-c784-4d4f-bc67-fa321a59ce66", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "84fb4d1a-8c47-4051-ad9e-30f569892c8d", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "a641b6e8d2d93055397eaf89c122975a6bd9134f3f364fc9fda1bb81b663c010", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c5286e49-5f71-466a-8c6b-fa583ad14e0f", "node_type": "1", "metadata": {}, "hash": "cfd3bbdc9ca5c4517265e91601d338c6c1f631be1a2b86d1656250913a75f58f", "class_name": "RelatedNodeInfo"}}, "text": "Morang, Sunsari, Sankhuwasabha, Panchthar, Ilam, Jhapa, \n\nTerhathum \n<800 \n\n Biramphool Dhading, Jhapa, Kathmandu, Kaski, Lamjung, Morang, Parbat, \nSiraha, Sunsari, Udayapur \n\n400-800 \n\n Charan Basmati Bajura  1000 \n Chengul Bara, Parsa, Sunsari <500 \n Chhoti Basmati Jhapa, Morang, Sunsari <300 \n Chirankhe Bhojpur, Dhankuta, Illam, Okhaldhunga, Panchthar, Terhathum <1800 \n Chulthe Jhapa, Sunsari 60-300 \n Danda Basmati Dadeldhura 1530 \n Deradun Basmati Bake  300 \n Gauria Arghakhanchi, Baglung, Kapilvastu, Lamjung, Myagdi, \n\nNawalparasi, Ramechhap, Rupandehi, Sankhuwasabha, Sunsari, \nTerhathum  \n\n300-1400 \n\n Ghyu Kumari Bara, Parsa, Sarlahi, Sindhuli <500 \n Gola Basmati Sunsari <500 \n Gude (Seto, Kalo) Dailekh <1100 \n Gudgudo Gulmi <1100 \n Hansraj Bajhang, Baitadi, Darchula, Dadeldhura, Jhapa,  Kanchanpur, \n\nMorang, Palpa, Pyuthan, Salyan, Sunsari, Surkhet, Syangja \n60-1100  \n\n Hapsa Jhapa <300 \n Hapsa Rato Jhapa 60-300 \n Indrabeli Dhading, Dhankuta, Gorkha, Lamjung 800-1400 \n Jaran Dhan (Kalo) Arghakhanchi, Bajhang, Dang, Gulmi, Jajarkot, Kaski, Parbat, \n\nRukum, Salyan and Surkhet  \n800-1400 \n\n Jaswa Dhanusha, Mahottari, Morang, Rautahat, Saptari, Siraha, Sunsari 60-300 \n Jethobudho Kaski, Myagdi, Parbat, Sunsari, Syagnja, Tanahun 600-1250 \n Jhinuwa Baglung, Doti, Gorkha, Kailali, Kanchanpur, Kaski, Kathmandu, \n\nLamjung, Myagdi, Nuwakot, Parbat, Shankhuwasaba, \nSindhupalchok, Sunsari, Syangja, Tanahun \n\n300-1300 \n\n Jirasari Jhapa, Morang, Panchthar, Ramechhap, Sunsari <600 \n Jogini Chitwan, Ramechhap 500 \n Jorayal Basmati Doti <800 \n Jorpal Basmati Jhapa, Morang, Sunsari <1200 \n Kalo Basmati Dhankuta, Jhapa, Kathmandu, Morang, Sunsari <1200 \n Kalo Jhuse Basmati Jhapa, Morang, Sunsari <300 \n Kalo Nuniya Jhapa, Morang, Sunsari 60-300 \n Kalo Nuniya Thulo Jhapa, Morang, Sunsari 60-300 \n Kalo/Kala Nimak Bardiya, Chitwan, Nawalparasi, Rupandehi 100-400 \n Kalotunde Basmati Jhapa, Morang, Sunsari <300 \n Kanak Jira Bara, Bardiya, Chitawan, Jhapa, Kailali, Kanchanpur, Kapilbastu, \n\nMorang, Salyan, Sunsari, Syanja \n<600 \n\n Kariya Kamod Dhanusa, Morang, Saptari, Siraha 200-400 \n Kasturi Bara,", "start_char_idx": 44486, "end_char_idx": 46602, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c5286e49-5f71-466a-8c6b-fa583ad14e0f": {"__data__": {"id_": "c5286e49-5f71-466a-8c6b-fa583ad14e0f", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4172474c-c784-4d4f-bc67-fa321a59ce66", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "85e71aa2688958b51b4be7dedd7b4d361d00d850078101a352bb3a6582c25e18", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d776c656-fda4-4a88-9e36-5741f408821c", "node_type": "1", "metadata": {}, "hash": "c900522369148dd11439f2caba1f8f67a27bcd7e11afc9c0e3ef01f0a6d44cb6", "class_name": "RelatedNodeInfo"}}, "text": "Morang, Saptari, Siraha 200-400 \n Kasturi Bara, Kailali, Parsa 500-1400 \n Krishna Bhog Achham, Dhankuta, Kanchanpur, Ramechhap  <1400 \n Krishna Charcha Bajura <1400 \n Lalbachchhi Jhapa, Morang, Sunsari 60-300 \n Lalka Basmati Bara, Dhanusha, Parsa, Rautahat 60-300 \n Lanjhi Bara, Parsa <500 \n Lekali Basmati  Lamjung 1500 \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  104 \n\n Mahabhog Kailali, Dhading, Rasuwa, Bara, Parsa 200-600 \n Mahajogini Bara, Parsa <300 \n Masino Basmati Dhading, Khotang <900 \n Motisar Bara, Parsa <300 \n Pahade Basmati Ilam <1000 \n Pahenle Bajhang, Bardiya, Gorkha, Ilam, Kaski, Lamjung, Myagdi, Palpa, \n\nParbat, Sinduplanchok, Syanja  \n600-800 \n\n Pokhreli Masino Solukhumbu, Sankhuwasabha 600-800 \n Pran Peuri Sallyan, Surkhet 1200-1400 \n Rajbhog Dhading, Kailali, Kanchanpur <600 \n Ram Tulsi Panchthar, Terhathum 800-1100 \n Ramjawain Bara, Parsa <600 \n Rato Basmati Jhapa, Morang, Sunsari 60-300 \n Rato Basmati Sano Bara, Jhapa, Mahottari, Morang, Parsa, Siraha, Sunsari <300 \n Ratotunde Basmati Jhapa, Morang, Sunsari <300 \n Sali Dhan Baitadi, Dadeldhura, Gorkha <1200 \n Samundrabakhi \n\nPhim \nDhading, Nuwakot <600 \n\n Samundraphinj Dhading, Kaski, Makawanpur, Nuwakot 200-600 \n Seto Basmati Bara, Jhapa, Morang, Parsa, Sunsari 60-300 \n Shyamjira Banke, Doti, Jhapa, Kailali, Kanchanpur, Morang, Sunsari 60-300 \n Thapachini Achham, Bajhang, Bajura, Dadeldhura, Kailali, Lamjung, \n\nTerhathum  \n200- 1400 \n\n Tulsi Prasad Nawalparasi, Parsa, Dhanusa 200-1400 \n Tulsiphool Dhanusha, Jhapa, Mahottari, Morang, Saptari, Sindhuli, Siraha, \n\nSunsari, Udayapur \n60-300 \n\n Ujarka Basmati Bara, Parsa, Rautahat 60-300 \nSource: [2,4\u20136,14,19,63,68,90,91] \n\nFigure 5. DNA profile of aromatic and non aromatic rice \nlandraces (upper figure) and DNA fingerprint (down figure) of \nBasmati type rice landraces using SSR markers. Source: [78] \n\nGaps and policy implication  \nResearch and mechanism for implication of GI is urgently \n\nneeded in Nepal as this sector is neglected and \n\nunderutilized. Research, development and education \n\nsystem should focus on native crop diversity and \n\ntraditional knowledge along with traditional products \n\nand process. Documentation of different kinds of \n\ninformation e.g. traditional, folklore, scientific \n\ninformation etc. should be done for all types of native \n\n\n\nNepal J Biotechnol.", "start_char_idx": 46555, "end_char_idx": 48927, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d776c656-fda4-4a88-9e36-5741f408821c": {"__data__": {"id_": "d776c656-fda4-4a88-9e36-5741f408821c", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c5286e49-5f71-466a-8c6b-fa583ad14e0f", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "6ecc6cece80e51df8697ba5abb28d30031a9fef443d49907aac66f9d8e631ae3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d3be94fe-2662-4d8a-b688-7a034aff46ce", "node_type": "1", "metadata": {}, "hash": "506eda6991f732544421a3db5ebfe027b8e6610b053471d97b735598b295e360", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  105 \n\nagricultural genetic resources. Accelerated research is \n\nnecessary to identify the geo-linked traits and products, \n\nand geographical indicator of genetic resources. Simple \n\nmechanism should be in place to register native \n\nagricultural products as GI at Provincial and National \n\nlevels.  \n\nConclusion  \nThere are strong proofs and evidences of the origin, \n\ndiversity, cultivation and use values of Basmati rice in \n\nNepal based on survey, on-farm and on-station trials, lab \n\nresearch, and information from local, regional, national \n\nand global levels. Basmati type rice possesses quality trait \n\nfor getting geographical indication tag and it includes all \n\ntypes of aromatic landraces i.e. short, medium and long \n\ngrain Basmati rice in Nepal. Nepalese farming \n\ncommunities in many districts are maintaining, growing, \n\nusing, marketing and sharing Basmati type rice landraces \n\nsince unknown time period. The rights of Nepalese \n\ncommunities, therefore, should not be prohibited for \n\nproduction, consumption and marketing of their basmati \n\nrice landraces. Legal system for GI tag should be \n\nimmediately established in Nepal and agricultural \n\nproducts must be registered. Harmonized system (HS) on \n\ntrade should also be established separately for aromatic \n\nrice in Nepal.  \n\nCompeting Interests \nNo competing interests. \n\nFunding \nNepal Agricultural Research Council funded this \n\nresearch.  \n\nEthical Approval and Consent \nNot applicable \n\nReferences  \n1.  Mallick RN. Rice in Nepal. Kathmandu: Kala Prakanshan; 1981.  \n2.  Upadhyay MP, Joshi BK. Plant Genetic Resources in SAARC \n\nCountries: Their Conservation and Management: Nepal Chapter \n[Internet]. SAARC Agriculture Information Center; 2003. 297\u2013422 \np. Available from: \nhttps://www.researchgate.net/publication/333641164_Plant_G\nenetic_Resources_in_SAARC_Countries_Their_Conservation_an\nd_Management_Nepal_Chapter \n\n3.  Joshi BK, Bhatta MR, Ghimire KH, Khanal M, Gurung SB, Dhakal \nR, et al. Released and Promising Crop Varieties of Mountain \nAgriculture in Nepal (1959-2016 [Internet]. Pokhara, Nepal: \nNAGRC, LI-BIRD and Bioversity International; 2017. Available \nfrom: \nhttps://www.bioversityinternational.org/fileadmin/user_uplo\nad/Released_and_promising_crop_varieties.pdf \n\n4.  Joshi BK. Rice gene pool for mid and high hills and its \nconservation in Nepal. In: Joshi BK, Joshi SL, Paudyal KP, editors. \nAgricultural Research for Enhancing Livelihood of Nepalese \nPeople [Internet]. Kathmandu: SAS-Nepal and NARC; 2004. p. \n252\u201364. Available from: \nhttps://www.researchgate.net/publication/333654995_Rice_Ge\n\nne_Pool_for_Mid_and_High_Hills_and_its_Conservation_in_Ne\npal \n\n5.  Joshi BK. Rice gene pool for Tarai and Inner Tarai areas of Nepal. \nNepal Agric Res J. 2005;6:10\u201323.  \n\n6.  Joshi BK. Rice and wheat gene pools in Nepal (1959-2002) \n[Internet]. Kathmandu Nepal: NAGRC, NARC; 2015. Available \nfrom: \nhttps://www.researchgate.net/publication/321309500_Rice_an\nd_wheat_gene_pools_in_Nepal_1959-2002 \n\n7.", "start_char_idx": 48908, "end_char_idx": 51985, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d3be94fe-2662-4d8a-b688-7a034aff46ce": {"__data__": {"id_": "d3be94fe-2662-4d8a-b688-7a034aff46ce", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d776c656-fda4-4a88-9e36-5741f408821c", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "3ef2d901e495269738f27a88491b59187a7c5f384cecfed861eefcfe0ba73a7c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5b3b7f69-5592-47be-8d84-aa8c6c7d2259", "node_type": "1", "metadata": {}, "hash": "69dfa7de5025aa18187e723b2c30745fabba1c54ddfb0274084f549ed0cee484", "class_name": "RelatedNodeInfo"}}, "text": "Joshi BK, Acharya AK, Gauchan D, Singh D, Ghimire KH, Sthapit \nBR. Geographical indication: A tool for supporting on-farm \nconservation of crop landraces and for rural development. In: \nJoshi BK, KC HB, Acharya AK, eds), editors. Conservation and \nUtilization of Agricultural Plant Genetic Resources in Nepal \n[Internet]. Dhulikhel, Kathmandu, Nepal: NAGRC, FDD, DoA \nand MoAD; 2017. p. 50\u201362. Available from: \nhttps://www.researchgate.net/publication/321670539_Geogra\nphical_indication_A_tool_for_supporting_on-\nfarm_conservation_of_crop_landraces_and_for_rural_developm\nent \n\n8.  Gauchan D, Rijal D, Mudwari A, Shrestha K, Joshi M, S G, et al. \nBenefits from on-farm conservation of crop diversity: experience \nof Nepal\u2019s in situ agrobiodiversity conservation project. In: \nGauchan, D., Sthapit BR, Jarvis DI, editors. Agrobiodiversity \nconservation on-farm: Nepal\u2019s contribution to a scientific basis for \nnational policy recommendations [Internet]. Rome, Italy: IPGRI; \n2003. p. 32\u20136. Available from: \nhttps://www.bioversityinternational.org/fileadmin/_migrated\n/uploads/tx_news/Agrobiodiversity_conservation_on-\nfarm.Nepal_s__10_February_2002__Kathmandu__Nepal.pdf \n\n9.  Gauchan D, Chaudhary P, Smale M, Sthapit BR, Upadhaya MP, \nJarvis DI. A participatory approach to analysing market based \nincentives for rice landraces: A case study of Bara, Central Terai, \nNepal. In: On-farm management of agricultural biodiversity in \nNepal Proceedings of a National Workshop [Internet]. Lumle, \nNepal: NARC; 2003. p. 184\u201393. Available from: \nhttps://www.researchgate.net/publication/273144828_On-\nfarm_management_of_agricultural_biodiversity_in_Nepal_Proc\needings_of_a_National_Workshop_24-\n26_April_2001_Lumle_Nepal_NARC_LI-BIRD_and_IPGRI \n\n10.  N.A.R.C. Basmati rice in Tharu community, JamuhaniGau, \nRupandehi, Nepal. Video documentary (in Tharu language \n[Internet]. Kathmandu, Nepal: Nepal Agricultural Research \nCouncil; 2020. Available from: \nhttps://www.youtube.com/watch?v=oacmV_T9KfU&t=108s \n\n11.  N.A.R.C. Tharu farmer\u2019s view on Basmati rice in BagahiGau, \nRupandehi, Nepal [Internet]. Kathmandu, Nepal; 2020. Available \nfrom: https://www.youtube.com/watch?v=TjRSF_0kvBI&t=17s \n\n12.  N.A.R.C. Tharu farmer\u2019s view on Basmati rice in BagahiGau, \nRupandehi, Nepal. Part II [Internet]. Kathmandu, Nepal; 2020. \nAvailable from: \nhttps://www.youtube.com/watch?v=Q6plhX_FWFQ \n\n13.  Joshi BK. Local germplasm of rice in Nepal: Diversity, characters \nand uses. In: Paudel MN, Bhandari DR, Khanal MP, Joshi BK, \nAcharya P, Ghimire KH, editors. Rice Science and Technology in \nNepal [Internet]. Khumaltar, Nepal: Crop Development \nDirectorate (CDD), and Agronomy Society of Nepal (ASoN); \n2017. p. 158\u201378.", "start_char_idx": 51987, "end_char_idx": 54688, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5b3b7f69-5592-47be-8d84-aa8c6c7d2259": {"__data__": {"id_": "5b3b7f69-5592-47be-8d84-aa8c6c7d2259", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d3be94fe-2662-4d8a-b688-7a034aff46ce", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "cfc2bc5bd808e88d413048c642134a84742da2b822351ae37af4a566a32a12bc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5fedbfd6-de3d-4514-b6d5-5d63ae2aa5a3", "node_type": "1", "metadata": {}, "hash": "5a341bd903d25d285e782b38f5dde0beab28d88ebc6323fd4c5f0b86c0973a65", "class_name": "RelatedNodeInfo"}}, "text": "158\u201378. Available from: \nhttps://www.researchgate.net/publication/321329622_Local_g\nermplasm_of_rice_in_Nepa_Diversity_characters_and_uses \n\n14.  Upreti HK. Distribution patterns of rice landraces in different \nagro-ecology zones of Nepal. In: Bhandari DR, Khanal MP, Joshi \nBK, Acharya P, Ghimire KH, Paudel MN, editors. Rice Science \nand Technology in Nepal [Internet]. Khumaltar, Kathmandu: \nCDD and Agronomy Society of Nepal (ASoN); 2017. p. 152\u20137. \nAvailable from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n15.  MoALD. Statistical information on Nepalese agriculture 2075/76. \nKathmandu: MoALD; 2020.  \n\n16.  E.C. \u2018BASMATI\u2019  EU No: PGI-IN-02425 \u2013 18.7.2018. Eur Comm \nOff J Eur Union. 2020;63(C301):16\u201320.  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  106 \n\n17.  Khush GS. Taxonomy and Origin of Rice. In: rices A, Singh RK, \nSingh, US, Khush GS, editors. Aromatic rices [Internet]. New \nDelhi: Oxford & IBH Publishing Co. Pvt. Ltd; 2000. p. 5\u201313. \nAvailable from: http://books.irri.org/8120414209_content.pdf \n\n18.  Pachauri V, Singh MK, Singh AK, Singh S, Shakeel NA, Singh VP, \net al. Origin and Genetic Diversity of Aromatic Rice Varieties, \nMolecular Breeding and Chemical and Genetic Basis of Rice \nAroma. J Plant Biochem Biotechnol. 2010;19(2):127\u201343.  \n\n19.  Rijal DK, Kadayat KB, Joshi KD, Sthapit BR. Inventory of \nindigenous rainfed and aromatic rice landraces in Seti river valley \nPokhara, Nepal [Internet]. Pokhara, Nepal: LI-BIRD; 1998. \nAvailable from: https://idl-bnc-\nidrc.dspacedirect.org/bitstream/handle/10625/23370/113255.p\ndf?sequence=1 \n\n20.  Joshi KD, Subedi M, Kadayat KB, Sthapit BR. Factors and \nprocesses behind the erosion of crop genetic diversity in Nepal. \nIn: Pratap T, Sthapit B, editors. Managing agrobiodiversity \n[Internet]. ICIMOD and IPGRI; 1998. p. 183\u201397. Available from: \nhttps://www.researchgate.net/publication/282059405_Factors_\nand_Processes_behind_the_Erosion_of_Crop_Genetic_Diversity\n_in_Nepal \n\n21.  Joshi BK, Ojha P, Gauchan D, Ghimire KH, Bhandari B, HB KC. \nNutritionally unique native crop landraces from mountain Nepal \nfor geographical indication right. In: Traditional Crop \nBiodiversity for Mountain Food and Nutrition Security in Nepal. \nIn: Gauchan D, Joshi BK, Bhandari B, Manandhar HK, Jarvis D, \neds), editors. Tools and Research Results of the UNEP GEF Local \nCrop Project, Nepal NAGRC, LI-BIRD and the Alliance of \nBioversity International and CIAT [Internet]. Kathmandu, Nepal: \nNAGRC, LI-BIRD and Bioversity International; 2020. p. 87\u201399.", "start_char_idx": 54681, "end_char_idx": 57276, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5fedbfd6-de3d-4514-b6d5-5d63ae2aa5a3": {"__data__": {"id_": "5fedbfd6-de3d-4514-b6d5-5d63ae2aa5a3", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5b3b7f69-5592-47be-8d84-aa8c6c7d2259", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "b5e31682c6862e6a638a6c457b4a9c050e4c2e78329f027f70165f2cc0fddf42", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ca76a321-a49e-4179-ad3a-7d9fe3758b11", "node_type": "1", "metadata": {}, "hash": "8fc1cf3705a5cbd9e049350f99c70e4f0cc044553254c90686077a12975562db", "class_name": "RelatedNodeInfo"}}, "text": "87\u201399. \nAvailable from: \nhttps://www.researchgate.net/publication/342145047_Nutritio\nnally_Unique_Native_Crop_Landraces_from_Mountain_Nepal_\nfor_Geographical_Indication_Right \n\n22.  Ojha P, Chaudhary O, Subedi U, Karki R, Dongol D. Milling, \nNutritional, Physical and Cooking Properties of Four Basmati \nRice Varieties. J Nepal Agric Res Counc. 2018;4(1):18\u201324.  \n\n23.  Singh VP. The Basmati Rice of India. In: Singh RK, Singh US, \nKhush GS, editors. Aromatic rices [Internet]. New Delhi: Oxford \n& IBH Publishing Co. Pvt. Ltd; 2000. p. 135\u201351. Available from: \nhttp://books.irri.org/8120414209_content.pdf \n\n24.  Subbiah S. Reaping What They Sow: The Basmati Rice \nControversy and Strategies for Protecting Traditional \nKnowledge. Boston Coll Int Comp Law Rev. 2004;27:529.  \n\n25.  Islam MA, Anik TR, Hossain MM, Uddin MI, Ahmed MS. Genetic \ndiversity analysis of some Bangladeshi aromatic rice (Oryza \nsativa L.) using simple sequence repeat markers (SSRM). Arch \nAgric Environ Sci. 2018;3(3):297-303,.  \n\n26.  Bhattarai AN. A review of rice improvement in Kathmandu \nvalley. Nepal J Agric. 1969;4:47\u201357.  \n\n27.  Chataut RDP. Kata haraye ye dhaankajaatharu. In: Bhandari DR, \nKhanal MP, Joshi BK, Acharya P, Ghimire KH, Paudel MN, \neditors. Rice Science and Technology in Nepal [Internet]. \nKhumaltar, Nepal: Hariharbhawan and Agronomy Society of \nNepal (ASoN; p. 41. Available from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n28.  Gin TP, Shahi BB. Performance of Aromatic Rice Varieties in \nNepal. Proceeding of the 5th Rice Improvement Workshop. Bara, \nNepal: NRIP; 1978.  \n\n29.  Subedi KD, Paudel SS. Ayurvedma dhaanko mahatow. In: Paudel \nMN, Bhandari DR, Khanal MP, Joshi BK, Acharya P, Ghimire KH, \neditors. Rice Science and Technology in Nepal [Internet]. \nKathmandu Nepal: CDD and Agronomy Society of Nepal \n(ASoN); 2017. p. 34\u20137. Available from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n30.  Tripathi MP, Sthapit BR, Subedi LP, Sah SK, Gyawali S. Agro-\nmorphological variation in Jhinuwa rice landraces (Oryza sativa \nL.) of Nepal. Genet Resour Crop Evol. 2013;60:2261\u201371.  \n\n31.  Joshi B, Bhatta M. Joshi BK, MR Bhatta and KH Ghimire. 2013. \nShali Dhan: Elite line of rice from Far West Nepal developed \n\nunder the pre-breeding program in Genebank, Khumaltar. \nNARC Newsletter 20(4):5.  \n\n32.  Tripathi AM. Kapilbastu jillaka dhanka purana sthaniya dhanko \njaatharu.", "start_char_idx": 57270, "end_char_idx": 59737, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ca76a321-a49e-4179-ad3a-7d9fe3758b11": {"__data__": {"id_": "ca76a321-a49e-4179-ad3a-7d9fe3758b11", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5fedbfd6-de3d-4514-b6d5-5d63ae2aa5a3", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "82632f90a8e8fb2e18154fcc95952857ce966b30e08aa0a0eff4e47087398e25", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "49271fb8-d5ea-4634-a430-130d281be5c8", "node_type": "1", "metadata": {}, "hash": "d7e860aaee2d25720407b29b820f2a01198c95a5c755b3e175ae0d058f721f67", "class_name": "RelatedNodeInfo"}}, "text": "In: Bhandari DR, Khanal MP, Joshi BK, Acharya P, \nGhimire KH, editors. Nepali language) In: Rice Science and \nTechnology in Nepal [Internet]. Khumaltar, Kathmandu, Nepal: \nCDD and Agronomy Society of Nepal (ASoN); 2017. p. 819. \nAvailable from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n33.  Chauhan BK. Lopanmukh bhaudhakaalin kalanamak dhaan \nbaali samraxan me jhod. Gorkhapatra. 2020;2077(9/15):9.  \n\n34.  Kumar S, Mishra SB, Chaudhary RC. Breeding bauna kalanamak \n102 as new aromatic variety of heritage rice from Uttar Pradesh. \nInt J Sci Environ Technol. 2018;7(5):1690\u20139.  \n\n35.  Joshi KD, Sthapit BR, Vaidya A, Kadayat KB, Tuladhar JK, Subedi \nKD, et al. Report on Aromatic Rice Survey and RRA in Pokhara \nValley of Nepal: Issues for Rice Research. LARC Work Pap. \n1997;(95).  \n\n36.  Rijal DK, Rana RB, Sherchand KK, Sthapit BR, Pandey YR, \nAdhikari N, et al. Findings of site selection in Kaski, Nepal. \nNepal: NARC, LIBIRD and IPGRI; 1998.  \n\n37.  Sherchand KK, Adhikari NP, Khatiwada SP, Shrivastav AC, \nBajracharya J, Joshi KD, et al. Strengthening the scientific basis for \nin-situ conservation of agrobiodiversity: Findings of site selection \nin Bara. Nepal: NARC, LIBIRD and IPGRI; 1998.  \n\n38.  Singh RK, Singh US, Khush GS, Rashmi Rohilla JPS, Singh G, \nShekhar KS. Small and Medium Grained Aromatic Rices of India. \nIn: Singh RK, Singh, US, Khush GS, editors. New Delhi: Oxford & \nIBH Publishing Co. Pvt. Ltd; 2000. p. 155\u201377. Available from: \nhttp://books.irri.org/8120414209_content.pdf \n\n39.  Tiwari RK, Baniya BK, Bajracharya J, Joshi BK, Joshi KD, \nKhatiwada SP, et al. On-farm characterization of rice landraces \nand population structure study of Jhinuwa in Begnas, Kaski. In: \nSubedi A, Joshi BK, Sthapit BR, Upadhaya MP, Baniya BK, \neditors. On-farm Management of Agricultural Biodiversity in \nNepal [Internet]. Lumle Nepal, NARC: LIBIRD and IPGRI; 2003. \np. 139\u201348. Available from: \nhttps://www.researchgate.net/publication/348591146_On_far\nm_characterization_of_rice_landraces_and_population_structur\ne_of_Jhinuwa_in_Begnas_Kaski \n\n40.  Chaudhary B, Gyawali S, Joshi KD, Khatiwada SP, Shrestha K, \nMudwodi A, et al. Participatory plant breeding in rice: Experience \nof National Rice Research Program, Hardinath, Nepal. In: Joshi \nBK, Joshi SL, Paudyal KP, eds), editors. Agricultural Research for \nEnhancing Livelihood of Nepalese People [Internet]. Kathmandu, \nNepal: SAS-Nepal and NARC; 2004. p.", "start_char_idx": 59738, "end_char_idx": 62208, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "49271fb8-d5ea-4634-a430-130d281be5c8": {"__data__": {"id_": "49271fb8-d5ea-4634-a430-130d281be5c8", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ca76a321-a49e-4179-ad3a-7d9fe3758b11", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "6af8e0f3da72b3fa4ad0beac4d71285e646b09a73274dc6c419b5388c1333f3f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "858ee5fb-194f-4247-9922-153d073732dc", "node_type": "1", "metadata": {}, "hash": "4a36112218aa89a41291a32b0572443135f87a30a27fa0886f7c20902d9454d3", "class_name": "RelatedNodeInfo"}}, "text": "222\u20136. Available from: \nhttps://www.researchgate.net/publication/333640904_AGRIC\nULTURAL_RESEARCH_FOR_ENHANCING_LIVELIHOOD_O\nF_NEPALESE_PEOPLE \n\n41.  F.A.O. Country Report on The State of the Nepal\u2019s Plant Genetic \nResources for Food and Agriculture: Nepal [Internet]. \nKathmandu: ABTRACO; 2008. Available from: \nhttp://www.fao.org/3/i1500e/Nepal.pdf \n\n42.  BK SB, Koirala KB, Ghimire KH, Prasai HK, Poudel RP, Poudel \nHP, et al. Consumers\u2019 preferences on different fine and aromatic \nrice varieties. In: Amgain RB, Manandhar S, Ghimire K, eds), \nAdhikari NP, Giri N, editors. Summer Crops Research in Nepal \n[Internet]. Nepal: NARC; 2012. p. 60\u20132. Available from: \nhttps://www.researchgate.net/publication/325653929_Consum\ners%27_Preferences_on_Different_Fine_and_Aromatic_Rice_Var\nieties \n\n43.  Koirala KB, Ghimire KH, Karki TB, SB BK, Bhattarai EM, Poudel \nRP, et al. Response of fine and aromatic rice varieties under \ndifferent fertilizer levels. In: Giri YP, Manandhar HK, Khatiwada \nSP, Bajracharya J, Bhatta MR, Rai SK, et al., editors. Summer \nCrops Research in Nepal [Internet]. Nepal: NARC; 2012. p. 300\u2013\n6. Available from: \nhttps://www.researchgate.net/publication/328335418_Respons\ne_of_Fine_and_Aromatic_Rice_Varieties_under_Different_Fertil\nizer_Levels \n\n44.  Koirala KB, Ghimire KH, SB BK, Bhattarai EM, Karki TB, Prasai \nHK, et al. Evaluation of fine and aromatic rice varieties under \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  107 \n\ndifferent irrigation regimes. In: Koirala KB, Ghimire KH, \nBishwokarma SB, editors. Promotion of fine and aromatic rice \ncultivation in western region of Nepal Final Technical Report \n[Internet]. Lumle, Kaski, Nepal: RARS, NARC; 2008. p. 26\u201341. \nAvailable from: \nhttps://www.researchgate.net/publication/328410356_Promoti\non_of_Fine_and_Aromatic_Rice_Cultivation_in_Western_Regio\nn_of_Nepal \n\n45.  Joshi BK, Bimb HP, Kansakar D, Ghimire E. Genetic relationship \namong Nepalese rice landraces and cultivars based on RAPD \nmarkers. Nepal J Biotechnol. 2012;2:16\u201325.  \n\n46.  Adhikari SK. Purbeepahadi jillaharuma prachalanma rahyeka \ndhan Balika sthaniya jaatharu. In: Bhandari DR, Khanal MP, Joshi \nBK, Acharya P, Ghimire KH, Directorate CD, et al., editors. Rice \nScience and Technology in Nepal [Internet]. Khumaltar, Nepal: \nHariharbhawan and Agronomy Society of Nepal (ASoN; 2017. p. \n807. Available from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n47.", "start_char_idx": 62209, "end_char_idx": 64699, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "858ee5fb-194f-4247-9922-153d073732dc": {"__data__": {"id_": "858ee5fb-194f-4247-9922-153d073732dc", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "49271fb8-d5ea-4634-a430-130d281be5c8", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "d8058cccea4660b722319acb7a5042b6a60e1f7c606f656d54b3d86ab177c31f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1cc56b1d-ce2a-4559-b127-efcbd73f2a0b", "node_type": "1", "metadata": {}, "hash": "df67554e2f308a6151928275686d4ec09a8734f332e863b025a8cd3f992adb35", "class_name": "RelatedNodeInfo"}}, "text": "Bhatta MR, Joshi BK, Ghimire KH, Khanal M, Pokhrel T. Diversity \nand utilization status of cereals and pseudo-cereals in Nepal. In: \nJoshi BK, KC HB, Acharya AK, eds), editors. Conservation and \nUtilization of Agricultural Plant Genetic Resources in Nepal \n[Internet]. Dhulikhel, Kathmandu, Nepal: NAGRC, FDD, DoA \nand MoAD; 2017. p. 195\u2013214. Available from: \nhttps://www.researchgate.net/publication/348049968_Conserv\nation_and_Utilization_of_Agricultural_Plant_Genetic_Resources\n_in_Nepal_Proceedings_of_2nd_National_Workshop \n\n48.  Dhungana S. Grain quality evaluation of traditional aromatic rice \nvarieties of Nepal. Eur Acad Res. 2017;IV(11):9864\u201373.  \n\n49.  Ahuja SC, Ahuja U, Ahuja S. History and folklore of basmati rice. \nJ Cereal Res. 2019;11(3):206\u201314.  \n\n50.  Rana RB, Rijal DK, Gauchan D, Sthapit BR, Subedi A, Upadhyay \nMP, et al. In-situ crop conservation: Findings of agro -ecological, \ncrop diversity and socio-economic baseline survey of Begnas \necosite. Kaski, Nepal: NARC, LIBIRD and IPGRI; 2000.  \n\n51.  Shrestha P, Subedi A, Yadav RN, Yadav MN. Endangered rice \nlocal landraces in Central Tarai region of Nepal. Pokhara, Nepal: \nADCS, LI-BIRD and USC; 2006.  \n\n52.  Amatya G. Agrobiodiversity related farmer\u2019s based traditional \nknowledge register. Pokhara, Nepal: LI-BIRD; 2013.  \n\n53.  Subodh K, Badal M. Characterization of available rice varieties \nthrough diversity block in Makwanpur and Sarlahi districts, \nNepal. EC Agric. 2015;2(2):307\u201316.  \n\n54.  Prodhan MZH. Effects of temperature on morphoagronomic \nperformance, aroma gene expression and volatile profile of \naromatic rice [Internet] [Doctoral dissertation]. [Malaysia]: \nUniversity of Malaya, Kuala Lumpur; 2016. Available from: \nhttp://studentsrepo.um.edu.my/9519/5/zakaria.pdf \n\n55.  Ryan ML. Khanakhanubhayo (Have you eaten)? Traditional rice \nvarieties, land tenure, and social structure in Nepal\u2019s midhills\" \n[Internet]. [Ames, Iowa]: Iowa State University; 2018. Available \nfrom: https://lib.dr.iastate.edu/etd/16663 \n\n56.  Ahuja SC, Ahuja U, Ahuja S. History and folklore of basmati rice. \nJ Cereal Res. 2019;11(3):206\u201314.  \n\n57.  Mandal A. A case study report on socio-religious and commercial \nvalues of basmati rice in Nepal. Rampur, Chitwan, Nepal; 2021.  \n\n58.  N.A.P.A. Press Release on the Issue Related to Evidence on the \nOrigin of Basmati Rice in Nepal. USA; 2021.  \n\n59.  Gauchan D, Shrestha KH, Shrestha RN, Pandey N. Options for \nmarket linked rice research in Nepal. In: Proceedings of the 3rd \nNational Workshop on Outreach Research [Internet]. Nepal: \nORD, NARC; 1996. p. 250\u20135. Available from: \nhttp://elibrary.narc.gov.np/pages/view.php?ref=1295&k= \n\n60.", "start_char_idx": 64701, "end_char_idx": 67368, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1cc56b1d-ce2a-4559-b127-efcbd73f2a0b": {"__data__": {"id_": "1cc56b1d-ce2a-4559-b127-efcbd73f2a0b", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "858ee5fb-194f-4247-9922-153d073732dc", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "ab386f537a51d8fc322a8f1cbc812caf9f583038f885425748aa999d47da2db7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "54e16e00-17ac-4b0b-a45f-4ec338ccfe70", "node_type": "1", "metadata": {}, "hash": "f1fd654b7498a8dce6bd2f09a38606a54c97751164558ac7aa7c12cb816fd278", "class_name": "RelatedNodeInfo"}}, "text": "Gauchan D, Smale M, Chaudhary P. Market-based incentives for \nconserving diversity on farms: the case of rice landraces in \nCentral Tarai, Nepal. Genet Resour Crop Evol. 2005;52:293\u2013303.  \n\n61.  Rana RB, Chaudhary P, Gauchan D, Khatiwada SP, Sthapit BR, \nSubedi A, et al. In-situ crop conservation: Findings of agro-\necological, crop diversity and socio-economic baseline survey of \nKachorwa ecosite. Bara, Nepal: NARC, LIBIRD and IPGRI; 2000.  \n\n62.  Joshi BK, Gorkhali NA, Pradhan N, Ghimire KH, Gotame TP, KC \nP, et al. Agrobiodiversity and its Conservation in Nepal. J Nepal \nAgric Res Counc. 2020;6:14\u201333.  \n\n63.  Rana R, Gauchan D, Rijal D, Subedi A, Upadhyaya M, Sthapit B, \net al. Factors influencing farmers\u2019 decisions on management of \nlocal diversity on-farm and their policy implications. In: Gauchan \nD, Sthapit BR, Jarvis DI, IPGRI, editors. Agrobiodiversity \nconservation on-farm: Nepal\u2019s contribution to a scientific basis for \nnational policy recommendations [Internet]. Rome, Italy: IPGRI; \n2003. p. 27\u201331. Available from: \nhttps://www.bioversityinternational.org/fileadmin/_migrated\n/uploads/tx_news/Agrobiodiversity_conservation_on-\nfarm.Nepal_s__10_February_2002__Kathmandu__Nepal.pdf \n\n64.  Joshi KD, Upadhyay S, Chaudhary P, Shrestha S, Bhattarai K, \nTripathi BP. The Rice Processing Industry in Nepal: Constraints \nand Opportunities. Agric Sci. 2020;11:1060\u201380.  \n\n65.  C.D.D. Rice varietal mapping in Nepal: Implication for \ndevelopment and adoption [Internet]. Hariharbhawan, Lalitpur: \nCDD, DoA; 2015. Available from: \nhttp://doacrop.gov.np/downloadfile/Rice_Varietal_Mapping_\n1470895701_1512106555.pdf \n\n66.  S.Q.C.C. Notified and Denotified varieties of different crops till \n2076-4-27 [Internet]. Kathmandu, Nepal: SQCC, MoALD; 2020. \nAvailable from: \nhttp://sqcc.gov.np/images/category/Notified_and_Denotified\n_Varieties_till_2076_04_07.pdf) \n\n67.  Joshi BK, Hay F, Sapkota S, Ebana K. Nepalese rice around the \nWorld. In: Bhandari DR, Khanal MP, Joshi BK, Acharya P, \nGhimire KH, Paudel MN, editors. Rice Science and Technology \nin Nepal [Internet]. Khumaltar, Nepal: CDD and Agronomy \nSociety of Nepal (ASoN); 2017. p. 221\u201340. Available from: \nhttps://www.researchgate.net/publication/321329311_Nepales\ne_rice_around_the_World \n\n68.  Joshi BK, Shrestha P, Gauchan D, Vernooy R, editors. Community \nSeed Banks in Nepal. In: 2nd National Workshop Proceedings, 3-\n5 May 2018 [Internet]. Kathmandu Nepal; NAGRC: LI-BIRD and \nBioversity International; 2018. p. 154\u2013208.", "start_char_idx": 67370, "end_char_idx": 69867, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "54e16e00-17ac-4b0b-a45f-4ec338ccfe70": {"__data__": {"id_": "54e16e00-17ac-4b0b-a45f-4ec338ccfe70", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1cc56b1d-ce2a-4559-b127-efcbd73f2a0b", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "05b86698c2d32ca7c25c7d202109ccb7e5583ab6b28e39f2a9af099c3a45efa0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8a8bd86b-c6f1-41af-b65e-55ca7c476e55", "node_type": "1", "metadata": {}, "hash": "2b80e238ed7de2c4e054dec457ad8c3b4cf1b454e8b7fe5dc91a79ffe96ac68a", "class_name": "RelatedNodeInfo"}}, "text": "154\u2013208. Available from: \nhttp://himalayancrops.org/project/community-seed-banks-in-\nnepal-2nd-national-workshop-proceedings/ \n\n69.  Badal M, Chaudhary SN, Subedi B, Tharu CS. Community Seed \nBank Implementation Approach of Action Aid Nepal. In: Joshi \nBK, Shrestha P, Gauchan D, Vernooy R, editors. Community Seed \nBanks in Nepal 2nd National Workshop Proceedings [Internet]. \nKathmandu Nepal: NAGRC, LI-BIRD and Bioversity \nInternational; 2018. p. 95\u2013105. Available from: \nhttp://himalayancrops.org/project/community-seed-banks-in-\nnepal-2nd-national-workshop-proceedings/ \n\n70.  Shrestha DK, Paudel IP, Dhakal R, Rasaili S. Conserved local rice \nlandraces in Community Seed Bank, Kachorwa, Bara [Internet]. \nPokhara, Nepal: LI-BIRD; 2017. Available from: \nhttp://www.libird.org/app/publication/view.aspx?record_id=\n254&origin=results&QS=QS&sortfld_221=Date&reversesearch=t\nrue&top_parent=221 \n\n71.  Ghimire KH, Sah RP, Bhandari HS, Thapa B. Collection and \ncharacterization of local and exotic fine-aromatic germplasm. In: \nGautam AK, Akhtar T, Chaudhary B, Gaire J, Bhatta KR, editors. \nRice Research in Nepal Proc 24th Summer Crops Workshop \n[Internet]. Kathmandu, Nepal: NARC; 2004. p. 408\u201311. Available \nfrom: \nhttp://elibrary.narc.gov.np/pages/view.php?ref=1130&k= \n\n72.  Ahuja U, Ahuja SC, Thakrar R, Rani NS. Scented rices of India. \nAsian Agri-Hist. 2008;12(4):267\u201383.  \n\n73.  Bisne R, Sarawgi AK. Agro-morphological and quality \ncharacterization of badshah bhog group from aromatic rice \ngermplasm of Chhattisgarh. Bangladesh J Agric Res. \n2008;33(3):479\u201392.  \n\n74.  Siddiq EA, Vemireddy LR, Nagaraju J. Basmati rices: Genetics, \nbreeding and trade. Agric Res. 2012;1:25\u201336.  \n\n75.  Ashfaq M, Haider MS, Saleem I, Ali M, Ali A, Chohan SA. \nBasmati \u2013 Rice a class apart (A review). J Rice Res. 2015;3:156.  \n\n76.  Choi JY, Lye ZN, Groen SC. Nanopore sequencing-based genome \nassembly and evolutionary genomics of circum-basmati rice. \nGenome Biol. 2020;21:21.  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):93-108     Joshi et al.  \n\n\u00a9NJB, BSN  108 \n\n77.  Civ\u00e1\u0148 P, Ali S, Batista-Navarro R, Drosou K, Ihejieto C, \nChakraborty D, et al. Origin of the aromatic group of cultivated \nrice (Oryza sativa L.) traced to the Indian Subcontinent. Genome \nBiol Evol. 2019;11(3):832\u201343.  \n\n78.  Joshi BK, Shrestha N, Pokhrel R, Chaudhary R. Molecular \ncharacterization, DNA fingerprinting and genetic diversity \nanalysis of Nepalese rice landraces using SSR markers.", "start_char_idx": 69859, "end_char_idx": 72326, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8a8bd86b-c6f1-41af-b65e-55ca7c476e55": {"__data__": {"id_": "8a8bd86b-c6f1-41af-b65e-55ca7c476e55", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "54e16e00-17ac-4b0b-a45f-4ec338ccfe70", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "c80272ce63aa113bc256638d4d0ed499887548ad680af1cb799b394ca3cf5182", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ab1832e2-3ea4-4339-8701-337018b68898", "node_type": "1", "metadata": {}, "hash": "02d6dd1840c0aba985c461e05168f16b6504215789fc316c0a7a8c53cb2d73b2", "class_name": "RelatedNodeInfo"}}, "text": "NARC, \nKhumaltar, Kathmandu, Nepal; 2020.  \n\n79.  Joshi KD, Tiwari RK, Sthapit BR. Diversity assessment of Jetho \nBudho rice landrace Kaski. In: On-farm management of \nagricultural biodiversity in Nepal Proceedings of a National \nWorkshop [Internet]. Lumle, Nepal: NARC; 2003. p. 262\u20135. \nAvailable from: \nhttps://www.researchgate.net/publication/273144828_On-\nfarm_management_of_agricultural_biodiversity_in_Nepal_Proc\needings_of_a_National_Workshop_24-\n26_April_2001_Lumle_Nepal_NARC_LI-BIRD_and_IPGRI \n\n80.  Joshi KD, Chaudhary P, Yadav RB, Sthapit BR. Diversity \nassessment for the enhancement of selected rice landraces Kariya \nKamodh and Lalka Basmati at Kachorwa, Bara. In: On-farm \nmanagement of agricultural biodiversity in Nepal Proceedings of \na National Workshop [Internet]. Nepal. NARC: Lumle; 2003. p. \n254\u201361. Available from: \nhttps://www.researchgate.net/publication/273144828_On-\nfarm_management_of_agricultural_biodiversity_in_Nepal_Proc\needings_of_a_National_Workshop_24-\n26_April_2001_Lumle_Nepal_NARC_LI-BIRD_and_IPGRI \n\n81.  Sah RP, Akhtar T, Bhandari HS, Thapa B, Ghimire KH. Diallel \nanalysis for estimation of combining ability and gene action in \nfine-aromatic rice. In: Gautam AK, Akhtar T, Chaudhary B, Gaire \nJ, Bhatta KR, editors. Rice Research in Nepal [Internet]. \nKathmandu: NARC; 2004. p. 105\u201313. Available from: \nhttp://elibrary.narc.gov.np/pages/view.php?ref=356&k= \n\n82.  Thakur GC, Sah SN, Akhtar T, Ghimire KH, Khatiwada SP, \nHamal G, et al. Varietal investigation activities for fine grain and \naromatic rice of Nepal. In: Rai SK, Paneru RB, Joshi BK, Ghimire \nKH, Amgain RB, Manandhar S, et al., editors. Summer Crops \nResearch in Nepal [Internet]. Kathmandu, Nepal: NARC; 2012. p. \n144\u201351. Available from: \nhttp://elibrary.narc.gov.np/pages/view.php?ref=3209&k= \n\n83.  Joshi BK, HB KC, Tiwari RK, Shrestha P, Amagain R, Upadhyay \nMP. Varietal richness of agricultural crop species and farmers\u2019 \npreferred traits over space and time in Nepal. Bot Oreintalis J \nPlant Sci. 2005;5:69\u201374.  \n\n84.  Bajracharya J, Rana RB, Gauchan D, Sthapit BR, Jarvis DI, \nWitcombe JR. Rice landrace diversity in Nepal. Socio-economic \nand ecological factors determining rice landrace diversity in three \nagro-ecozones of Nepal based on farm surveys. Genet Resour \nCrop Evol. 2010;57(7):1013\u201322.  \n\n85.  Paudel B, Maharjan S. K, B RR, Shrestha A, Shrestha P, Basnet A, \net al. Findings of Baseline Survey on Socio-economic and \nAgricultural Biodiversity of Western Terai Landscape Project of \nNepal [Internet]. Pokhara, Nepal: Local Initiatives for \nBiodiversity, Research and Development; 2008.", "start_char_idx": 72327, "end_char_idx": 74942, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ab1832e2-3ea4-4339-8701-337018b68898": {"__data__": {"id_": "ab1832e2-3ea4-4339-8701-337018b68898", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8a8bd86b-c6f1-41af-b65e-55ca7c476e55", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "98e1fd154a5b3c700bc1281d02569547a09044fdedb03c6bfed5b7c3c2043013", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "023bca50-91c0-49dc-9479-2c9bab30ae0e", "node_type": "1", "metadata": {}, "hash": "9b5a3426e7b2d7eec6f4723d24b18d3041377316531f57121bb2007feeba99c1", "class_name": "RelatedNodeInfo"}}, "text": "Available from: \n\nhttps://www.researchgate.net/publication/274700362_Finding\ns_of_Baseline_survey_on_socioeconomic_and_agricultural_biod\niversity \n\n86.  Ghimire KH, Koirala KB, SB BK, Prasai HK, Poudel R, Poudel HP. \nCharacterization of local fine and aromatic rice varieties and \nstatus of prevailing rice varieties in the project sites. In: Koirala \nKB, Ghimire KH, Bishwokarma SB, editors. Promotion of fine and \naromatic rice cultivation in western region of Nepal Final \nTechnical Report [Internet]. Lumle, Kaski, Nepal; 2008. p. 4\u20137. \nAvailable from: DOI: \nhttps://www.researchgate.net/publication/328410356_Promoti\non_of_Fine_and_Aromatic_Rice_Cultivation_in_Western_Regio\nn_of_Nepal \n\n87.  Gurung R, Dhakal R, Pudasaini N, Paneru PB, Pant S, Adhikari \nAR, et al. Catalog of Traditional Crop Landraces of Mountain \nAgriculture in Nepal [Internet]. Pokhara; Nepal: NARC, LI-BIRD, \nBioversity International; 2019. Available from: \nhttp://himalayancrops.org/project/catalogue-of-traditional-\nmountain-crop-landraces-in-nepal/ \n\n88.  Yadaw R, Chaudhary P, Bajracharya J, Rijal D, Khatiwada S, \nTiwari RK, et al. Agro-morphological diversity in rice landraces \nof Bara and Kaski eco-sites. In: Subedi A, Joshi BK, Sthapit BR, \nUpadhaya MP, Baniya BK, editors. On-farm Management of \nAgricultural Biodiversity in Nepal [Internet]. Lumle, Kaski, \nNepal: NARC, LIBIRD and IPGRI; 2003. p. 42\u20137. Available from: \nhttps://www.researchgate.net/publication/348116250_On-\nfarm_management_of_agricultural_biodiversity_in_Nepal \n\n89.  Yadav H. Madhya tarai xetrama prachalit dhanka sthaniya \njaatharu tatha tinka jaatiya biseshta. In: Bhandari DR, Khanal MP, \nJoshi BK, Acharya P, Ghimire KH, editors. Rice Science and \nTechnology in Nepal [Internet]. Kathmandu, Nepal: CDD and \nAgronomy Society of Nepal (ASoN); 2017. p. 808. Available from: \nhttp://www.doacrop.gov.np/downloadfile/Rice_science_and_\ntechnology_1512106674.pdf \n\n90.  Joshi BK, Ghimire KH, Shrestha SK, editors. AFACI Pan Asia \nProject (IMPGR): Exploration, regeneration and conservation of \nendangered cereals, grain legumes from Central Mid and High \nHills of Nepal. Khumaltar, Kathmandu, Nepal: NAGRC and \nAFACI; 2014.  \n\n91.  Joshi BK, Bhatta MR, Ghimire KH. Shali Dhan: Elite line of rice \nfrom Far West Nepal developed under the pre-breeding program \nin Genebank, Khumaltar. NARC Newsl. 2013;20(4).  \n\n92.  Bimb HP, Sah RP, Karn NL. Isozyme variations in fine and \naromatic rice genotypes. Nepal Agric Res J. 2004;5:59\u201364.  \n\n93.  Glaszmann JC. Isozymes and classification of Asian rice varieties. \nTheor Appl Genet. 1987;74(1):21\u201330.  \n\n94.  Kishor DS, Seo J, Chin JH, Koh H-J.", "start_char_idx": 74943, "end_char_idx": 77584, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "023bca50-91c0-49dc-9479-2c9bab30ae0e": {"__data__": {"id_": "023bca50-91c0-49dc-9479-2c9bab30ae0e", "embedding": null, "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-108", "node_type": "4", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "fb9800cb77a5607178bffdcf981c87febbec1ed0076af9adcb274cda7e41ec5c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ab1832e2-3ea4-4339-8701-337018b68898", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "f6b8361c4a7fbcc5e566d84e573789475a0b55bddd3da4f0579064af3ef89480", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "80a06d36-c69d-402f-a241-9ffb8ba49236", "node_type": "1", "metadata": {}, "hash": "37986dad074bbef3eb60186ff90c827f31393fd17efb64add8ec97e4e1a83311", "class_name": "RelatedNodeInfo"}}, "text": "Kishor DS, Seo J, Chin JH, Koh H-J. Evaluation of whole-genome \nsequence, genetic diversity, and agronomic traits of basmati rice \n(Oryza sativa L. Front Genet [Internet]. 2020;11(86). Available \nfrom: \nhttps://www.frontiersin.org/articles/10.3389/fgene.2020.00086\n/full", "start_char_idx": 77549, "end_char_idx": 77819, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "80a06d36-c69d-402f-a241-9ffb8ba49236": {"__data__": {"id_": "80a06d36-c69d-402f-a241-9ffb8ba49236", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "023bca50-91c0-49dc-9479-2c9bab30ae0e", "node_type": "1", "metadata": {"identifier": "njb-108", "author": "Joshi, Bal Krishna; Ghimire, Krishna Hari; Bista, Prakash Raj; Yadaw, Ram Baran; Shrestha, Ram Krishna; Kharel, Gaurish Krishna; Paneru, Prakash; KC, Ram Bahadur; Bhandari, Deepak", "title": "Intellectual Property Right on Basmati Rice: Current Scenario and Evidences of Origin, Diversity, Cultivation and Use Values of Basmati Rice in Nepal", "date": "2021-07-31", "file": "njb-108.pdf"}, "hash": "9516b81c47b34ccf25d4e41c7386972f376d57985594db81ea4d52c4872c7f2c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "dc72815e-e29a-44f6-947b-eb04beec440a", "node_type": "1", "metadata": {}, "hash": "df432fc2b6bfc0527e8e11a66451df96df576bb25a18c686b1b66cf4d1f482ab", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 75-78 Research article DOI: https://doi.org/10.3126/njb.v9i1.38670 \n\n\u00a9NJB, BSN 75 \n\nThyroid Function and Thyroglobulin Level in Iodine-Deficient \nChildren of Eastern Nepal \nSaroj Kunwar1, Saroj Khatiwada2 , Basanta Gelal3, Saroj Thapa3, Gaurishankar Shah4, Nirmal Baral3, Madhab \n\nLamsal3 \n1Department of Biochemistry, Modern Technical College, Lalitpur, Nepal \n*2School of Medical Sciences, University of New South Wales Sydney, Australia\n3Department of Biochemistry, B P Koirala Institute of Health Sciences, Ghopa, Dharan, Nepal\n4Department of Obstetrics and Gynaecology, B P Koirala Institute of Health Sciences, Ghopa, Dharan, Nepal\n\nReceived: 13 Oct 2020; Revised: 04 Apr 2021; Accepted: 16 Apr 2021; Published online: 31 Jul 2021\n\nAbstract \nIodine deficiency during childhood affects physical and mental development. Iodine deficiency or excess both can \nnegatively impact thyroid function. We conducted this study to assess iodine nutrition and thyroid function in children \nwith insufficient urinary iodine concentration. A community-based cross-sectional study was conducted among the selected \nschools of Udayapur district. Urinary iodine concentration (UIC) was measured in 1012 school children (6-14 years). Based \non UIC data, 83 blood samples were collected to measure serum thyroglobulin (Tg), thyroid-stimulating hormone (TSH), \nfree triiodothyronine (fT3), and free thyroxine (fT4). UIC was measured by ammonium persulfate digestion method, and \nTg, TSH, fT4, and fT3 were measured using ELISA kits. The median UIE was 236 \u00b5g/L, and 11.1% of the children had \ninsufficient UIC. The mean fT3, fT4, and TSH in children with insufficient UIC were 2.55\u00b10.43 pg/mL, 0.96\u00b10.28 ng/dL, and \n3.60\u00b11.44 mIU/L respectively. Among children with low UIC levels, the median Tg was 17.5 ng/mL. Overt hypothyroidism \nwas seen in 6%, and subclinical hypothyroidism in 3.6%. The children had sufficient iodine nutrition, and the frequency of \nthyroid dysfunction was low among the children with insufficient UIC. \n\nKeywords: Children; Iodine deficiency; Nepal; Thyroid dysfunction; Urinary iodine concentration (UIC) \n\n Corresponding author, email: khatiwadasaroj22@gmail.com \n\nIntroduction \nIodine is an essential micronutrient required for the \n\nproduction of thyroid hormones [1, 2]. Iodine deficiency \n\naffects 2 billion people, with 36.4% being school-age \n\nchildren [3]. Iodine deficiency during pregnancy and \n\nchildhood has severe consequences, including \n\ndevelopmental disorders, poor cognition, and \n\nintellectual disability [4]. Supplementation of iodine in \n\nfood or salt has led to a significant reduction in iodine \n\ndeficiency disorders worldwide [5]. \n\nNepal has been continuously improving in iodine \n\nnutrition after starting the universal salt iodization \n\nprogram [6-8]. Several studies, particularly in eastern \n\nNepal show promising results in the improvement of \n\niodine deficiency as revealed by an increase in median \n\nurinary iodine concentration (UIC) of school children [6-\n\n8]. However, there are also concerns about excess iodine \n\nintake in such a population [9]. Iodine deficiency or \n\nexcess both affects thyroid function; therefore, it is \n\nimportant to maintain thyroid function in such a \n\npopulation [1].", "start_char_idx": 48, "end_char_idx": 3334, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "dc72815e-e29a-44f6-947b-eb04beec440a": {"__data__": {"id_": "dc72815e-e29a-44f6-947b-eb04beec440a", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "80a06d36-c69d-402f-a241-9ffb8ba49236", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "e4690b6ec3e33846e2664ee34cd5a5b71682e7ba6f2ba2983683bfd857b21c40", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bc32d1ea-ce74-4bf2-8da1-e2934a62bb44", "node_type": "1", "metadata": {}, "hash": "94b70c8dba12df8eb8b092b175c56c753a9710d8b129672c5e416c78dd3e0d21", "class_name": "RelatedNodeInfo"}}, "text": "Currently, the impact of iodine \n\ndeficiency on thyroid function and thyroglobulin level \n\nin children living in the eastern part of Nepal is \n\nunknown. Therefore, in this study, we specifically \n\naimed to find the recent iodine status in a large cohort of \n\nschool children, and then measure thyroid function in \n\nthe children with low UIC. \n\nMaterials and Methods \nStudy design  \nThis community-based study was conducted among \n\nschool children from the Udayapur district of eastern \n\nNepal. The study was conducted by the department of \n\nbiochemistry in collaboration with the department of \n\npediatrics of B. P. Koirala Institute of Health Sciences \n\n(BPKIHS), Dharan, Nepal. A multistage random \n\nsampling technique was used to enroll school children \n\naged 6 to 14 years in the study. Out of 46 village \n\ndevelopment committees (VDC) in the Udayapur \n\ndistrict, four VDCs (Beltar, Basaha, Rampur, and \n\nChaudandi) were randomly selected for study purposes. \n\nIn those four VDCs, six schools (four schools from 2 \n\nVDCs and two schools from the other 2 VDCs) were \n\nchosen for sample collection. All the students from those \n\nschools in the age range, and fitting into the inclusion \n\ncriteria were enrolled. A total of 1012 children \n\nparticipated, and in the first stage, 1012 urine samples  \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nmailto:sarojkunwar00@gmail.com\nmailto:gelalbasanta@gmail.com\nmailto:drsarojthapa@gmail.com\nmailto:gaurishankarshah@live.com\nmailto:nirmalbaral@hotmail.com\nmailto:madhablamsal@yahoo.co.uk\nmailto:khatiwadasaroj22@gmail.com\nmailto:khatiwadasaroj22@gmail.com\nmailto:khatiwadasaroj22@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 5 - 7 8      Kunwar et al.  \n\n\u00a9NJB, BSN  76 \n\nwere collected from the participants. After UIC \n\nmeasurement, only those children with insufficient UIC \n\n(UIC<100 \u00b5g/L) were followed up for blood sampling. \n\nBlood samples were taken from 83 children out of 112 \n\nchildren with insufficient UIC (UIC<100 \u00b5g/L), as 29 \n\nchildren dropped out for blood sampling. Blood \n\nsamples were collected to assess thyroid function and \n\nthyroglobulin level. This study was approved by the \n\nInstitutional Review Committee of BPKIHS and \n\nconducted under the Helsinki declaration of 1975. \n\nSample collection and analysis \n Written informed consent was obtained from the \n\nguardian of children prior to participation in the study. \n\nAll the stakeholders, including participants, were \n\ninformed about the purpose of the study. All healthy \n\nschool-going children (aged 6-14 years of age) were \n\nincluded. Children taking drugs that interfere with \n\nthyroid function, taking iodine supplements, or with \n\nany severe illness were excluded. In the first stage of the \n\nstudy, anthropometric measurements (height and \n\nweight) were taken, and spot urine samples were \n\ncollected from all 1012 participants. Urine samples were \n\ncollected in a clean, tightly screw-capped plastic vial \n\nand transported to the biochemistry laboratory for \n\nanalysis. In the second phase of the study, blood \n\nsamples (3ml) were collected in the plain vial by \n\nvenipuncture following the standard protocol. Clotted \n\nblood was centrifuged at 3000 rpm, and serum was \n\nseparated. Serum and urine samples were refrigerated \n\nat -20 \u00baC until analysis. Urinary iodine concentration \n\nwas estimated by ammonium persulfate digestion \n\nmicroplate (APDM) method using Sandell-Kolthoff\u2019s \n\nreaction [10].", "start_char_idx": 3335, "end_char_idx": 6906, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bc32d1ea-ce74-4bf2-8da1-e2934a62bb44": {"__data__": {"id_": "bc32d1ea-ce74-4bf2-8da1-e2934a62bb44", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "dc72815e-e29a-44f6-947b-eb04beec440a", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "75bcdc916c763524132c2758fc26bdc849048446c10b6032aebbf25faace3a94", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5a74875f-81a2-432c-baea-750c20be47f1", "node_type": "1", "metadata": {}, "hash": "0ebb0a0975b1d9a4129439378c4b6d6db28d472b6fb9595452f996c09d0c4065", "class_name": "RelatedNodeInfo"}}, "text": "Serum-free triiodothyronine (T3), free \n\ntetraiodothyronine (T4), TSH, and thyroglobulin (Tg) \n\nwere measured by the ELISA method using commercial \n\nkits from Diametra Company.  \n\n Based on the reference ranges of thyroid hormones (fT3, \n\nfT4, and TSH), thyroid function was classified as normal \n\nthyroid function (fT3, fT4, and TSH within the reference \n\nrange), overt hypothyroidism (TSH above the reference \n\nrange and fT3 and fT4 below the reference range) and \n\nsubclinical hypothyroidism (TSH above the reference \n\nrange, and fT3 and fT4 within the reference range). The \n\nreference range followed for fT3, fT4 and TSH were 1.2-\n\n4.2 pg/ml, 0.8-2.2 ng/dL, and 0.39-6.16 mIU/L \n\nrespectively. \n\nStatistical analysis  \nThe data was analyzed using SPSS version 20.0. The \n\ndata are presented in the form of mean \u00b1 SD (for \n\nnormally distributed variables), median with inter \n\nquartiles (IQR) for non-normally distributed variables \n\nand frequency (percentage). Age, height, weight, serum \n\nfT3, serum fT4 and serum TSH concentration showed \n\nnormal distribution, so they were expressed as \n\nmean\u00b1SD. The UIC and serum thyroglobulin levels were \n\nexpressed as median with IQR as they were not normally \n\ndistributed. Chi-square test was applied to assess \n\nstatistical difference among categorical variables \n\n(gender versus iodine status, gender versus thyroid \n\nfunction). Spearman\u2019s analysis was used to find a \n\nrelationship between quantitative variables (UIC with \n\nthyroid hormones and Tg, Tg with thyroid hormones). \n\nA \u2018p\u2019 value of \u22640.05 was considered statistically \n\nsignificant at a 95% confidence interval. \n\nResults \nA total of 1012 school children (482 males and 530 \n\nfemales) from 4 VDC of the Udayapur district \n\nparticipated in the study. The mean\u00b1SD age, weight, \n\nand height were 10.81\u00b12.32 years, 29.04\u00b18.79 kg, and \n\n131.64\u00b114.61 cm respectively. The median UIC with IQR \n\nwas 236 \u00b5g/L (156, 331) indicating adequate iodine \n\nintake. The median UIC (IQR) among male and female \n\nchildren were 219 \u00b5g/L (149, 335) and 248 \u00b5g/L (165, \n\n330) respectively. \n\nTable 1 shows the classification of iodine nutrition \n\nstatus in the study participants according to modified \n\nWHO assessment criteria with adequate and more than \n\nadequate group merged as sufficient category [3]. The \n\noverall prevalence of iodine deficiency based on \n\nindividual UIC cutoffs was 11.1% (severe, moderate, \n\nand mild deficiency). About one-third, (33.9%) of the \n\nchildren had excessive UIC (UIC>300 \u00b5g/L). \n\nThe mean fT3, fT4, TSH, and median Tg (IQR) in \n\nchildren with insufficient UIC were 2.55\u00b10.43 pg/mL, \n\n0.96\u00b10.28 ng/dL, 3.60\u00b11.44 mIU/L, and 17.5 (12, 29.4) \n\nng/mL respectively. Out of 83 participants with low \n\nUIC, 75 (90.3%) had normal thyroid function, 5 (6.0%) \n\nTable 1. Iodine status according to gender. The data are expressed as frequency (percentage). A Chi-square test was used \nbetween gender and iodine status at a 95% confidence interval.", "start_char_idx": 6907, "end_char_idx": 9871, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5a74875f-81a2-432c-baea-750c20be47f1": {"__data__": {"id_": "5a74875f-81a2-432c-baea-750c20be47f1", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bc32d1ea-ce74-4bf2-8da1-e2934a62bb44", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "8bebcb3f0826854e8f61ff11fff53759079e6a4e7ccbebda6fa40f65ac5b12f5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "112cacad-8e90-4366-a265-643d31860478", "node_type": "1", "metadata": {}, "hash": "c013528680209a1bb1d252b1e6e4849a35a3b81bc720a52735a748dfa7cdb5a6", "class_name": "RelatedNodeInfo"}}, "text": "ID= Iodine deficiency \n\n  Severe ID \n\n(<20 \u00b5g/L) \n\nModerate ID \n\n(20-49 \u00b5g/L) \n\nMild ID (50-99 \n\n\u00b5g/L) \n\nSufficient \n\n(100-299 \u00b5g/L) \n\nExcessive \n\n(>300 \u00b5g/L) \nP value \n\n \n\nGender  \n\nMale (n=482) 5 (0.5%) 13 (1.2%) 40 (3.9%) 266 (26.3%) 158 (15.6%)  \n\n \n\n0.5 \nFemale (n=530) 4 (0.4%) 19 (1.8%) 31 (3.0%) 291 (28.7%) 185 (18.3%) \n\n Total  (n=1012) 9 (0.9%) 32 (3.2%) 71 (7.0%) 557 (55.0%) 343 (33.9%)  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 5 - 7 8      Kunwar et al.  \n\n\u00a9NJB, BSN  77 \n\nhad overt hypothyroidism, and 3 (3.6%) had subclinical \n\nhypothyroidism. Among males with low UIC (n=40), 36 \n\n(90%) had normal thyroid function, 2 (5%) had overt \n\nhypothyroidism, and 2 (5%) had subclinical \n\nhypothyroidism. Among females with low UIC (n=43), \n\nnormal thyroid function, overt hypothyroidism, and \n\nsubclinical hypothyroidism were seen in 39 (91%), 3 \n\n(7%), and 1 (2%) children respectively. No significant \n\ndifferences (p=0.761) in thyroid function was seen \n\nbetween male and female with insufficient UIC.  \n\nIn the children with insufficient UIC, Tg has positive \n\ncorrelation with fT3 (r=0.273, p=0.013). However, Tg \n\nhad no significant correlation with fT4 (r=0.012, \n\np=0.916) and TSH (r=0.056, p=0.615). In the same group, \n\nthere was no significant correlation of UIC with fT3 (r=-\n\n0.075, p=0.503), fT4 (r=-0.139, p=0.212), TSH (r=0.175, \n\np=0.114) and Tg (r=-0.075, p=0.503). \n\nDiscussion \nIodine deficiency has remained the most common cause \n\nof preventable brain damage in children worldwide [11]. \n\nSupplementation of iodine in salt through universal salt \n\niodization has become effective in Nepal as shown by \n\nimproving median UIC in the previous studies from \n\neastern Nepal [6-8]. Median UIC indicates recent iodine \n\nintake and is used as a marker of iodine status in the \n\ncommunity settings. In the present study, the median \n\nUIC was 236 \u00b5g/L, which indicates sufficient iodine \n\nnutrition among children of this district. The previous \n\nreport showed a median UIC of 268 \u00b5g/L among \n\nchildren of this district [7]. \n\nIn this study, 11.1% of the children had UIC< 100 \u00b5g/L, \n\nwhich is considered iodine deficiency. In the previous \n\nstudy, iodine deficiency in this district was 12.7% [7]. \n\nNational surveys conducted in Nepal in the years 1998, \n\n2005, and 2007 depicted iodine deficiency in 43.6%, \n\n27.4%, and 19.4% school-age children respectively [12]. \n\nIn another study in eastern regions, Dhankuta and \n\nDharan, iodine deficiency was 26.6% and 15.6% \n\nrespectively among school children [13, 14]. \n\nThyroglobulin, a thyroid-specific protein, and precursor \n\nin the synthesis of thyroid hormones is considered a \n\nsensitive marker of the iodine status than UIC [15].", "start_char_idx": 9872, "end_char_idx": 12585, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "112cacad-8e90-4366-a265-643d31860478": {"__data__": {"id_": "112cacad-8e90-4366-a265-643d31860478", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5a74875f-81a2-432c-baea-750c20be47f1", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "4c08dc3b37fbb1ad709e055ef6e37e1e9f1f88161de9bbdfd8c2ea73055e31dc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bc744b66-5ddc-449c-b92e-4124ba2e9f7a", "node_type": "1", "metadata": {}, "hash": "f4835e3e0f9df6d07ca414ce9685be6b34a64dc2e1393901056a67ac5173aeac", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nmedian thyroglobulin level in the children with \n\ninsufficient UIC was 17.5 ng/mL. Our previous study \n\namong pregnant women of eastern Nepal reported a \n\nmedian Tg of 6.5 ng/mL [16]. Thyroglobulin increment \n\nin plasma is also seen with a thyroid mass, \n\ninflammation, and hyperactivity of TSH [17]. \n\nWe observed normal thyroid function in most of the \n\nchildren with insufficient UIC. Out of 83 children with \n\ninsufficient UIC, 6% (n=5) had overt hypothyroidism, \n\nand 3.6% (n=3) had subclinical hypothyroidism. \n\nPrevious studies by Shakya et al., Chaudhari et al., and \n\nKhatiwada et al. found subclinical hypothyroidism in \n\n19.5% and 16.7 % (in Morang and Sunsari respectively), \n\n31.8% and 25.59% (in Sunsari and Dhankuta \n\nrespectively) and 17.6 % respectively (in hilly regions) \n\n[18, 14, 19]. Low prevalence of thyroid dysfunction in \n\nthe current study than previous studies may be due to \n\ndifferences in the geographic location, genetic factor, \n\nand small sample size of the current study. In the \n\ncurrent study, one-third (33.9%) of the children had \n\nexcessive UIC, which suggests the risk of iodine-\n\ninduced hyperthyroidism and autoimmune thyroid \n\ndisease in those children. Future studies need to \n\ninvestigate the effect of excessive iodine consumption \n\non thyroid function. Furthermore, the salt iodine \n\nfortification level may need to be readjusted to lower \n\niodine intake in the community. Interestingly, our \n\nfinding of a low rate of thyroid abnormalities in children \n\nwith insufficient UIC suggests that the thyroid gland \n\ncan adapt to low iodine intake. Moreover, UIC is an \n\nindicator of recent iodine intake, and it does not reflect \n\nlong-term iodine nutrition in those children with \n\ninsufficient UIC. In addition, the coexistence of \n\nautoimmune thyroiditis also contributes to abnormal \n\nthyroid function in the community. Overall, thyroid \n\nfunction can be impaired both in the condition of iodine \n\ndeficiency and excess, and therefore, maintaining \n\noptimal iodine intake by continuous monitoring of \n\ndietary iodine intake and UIC is crucial [1]. \n\nWe observed a non-significant negative correlation of \n\nUIC with Tg level and thyroid hormones in children \n\nwith insufficient UIC, and a positive correlation between \n\nthyroglobulin and fT3 (r= 0.273, p=0.013). In iodine-\n\ndeficient cases, thyroglobulin tends to rise due to \n\nhyperactivity of the thyroid gland and TSH; however, \n\nthe thyroid gland may be able to adapt to short-term \n\niodine deficiency maintaining normal thyroid hormones \n\n[1]. The present study has several limitations. Individual \n\nUIC was used as a marker of iodine deficiency, and \n\nthyroid function was not assessed in all the participants. \n\nThyroglobulin was estimated in the children with low \n\nUIC, and other common causes of thyroid dysfunction \n\nsuch as autoimmune thyroiditis were not investigated. \n\nFuture studies need to consider several of these factors. \n\nConclusion  \nSufficient iodine nutrition occurs among the school \n\nchildren of the Udayapur district, with one-third of the \n\nchildren having excessive UIC, and a small proportion \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 5 - 7 8      Kunwar et al.  \n\n\u00a9NJB, BSN  78 \n\nhaving insufficient UIC. The incidence of thyroid \n\ndysfunction is low in children with insufficient UIC. \n\nFuture studies need to be undertaken to measure \n\nthyroid function in children with excessive UIC, and \n\nother potential causes of hypothyroidism in children \n\nwith insufficient UIC need to be investigated.", "start_char_idx": 12586, "end_char_idx": 16122, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bc744b66-5ddc-449c-b92e-4124ba2e9f7a": {"__data__": {"id_": "bc744b66-5ddc-449c-b92e-4124ba2e9f7a", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "112cacad-8e90-4366-a265-643d31860478", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "7dacbfd127ad3069447bab787dae075faa996286b8e5e73009a1683f18ab8f23", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6ed52d5b-0c5c-4095-a1ac-5b8d26097bec", "node_type": "1", "metadata": {}, "hash": "d23c3593a3f15ae360955fcc112568c9b7af09f7ad55352c144ebced692e79ab", "class_name": "RelatedNodeInfo"}}, "text": "Abbreviations \nAPDM: Ammonium persulphate digestion method \n\nFree T4: Free thyroxine \n\nFree T3: Free triiodothyronine \n\nTg: Thyroglobulin \n\nTSH: Thyroid-stimulating hormone \n\nUIC: Urinary iodine concentration  \n\nAuthor contributions \nSK1, SK2, BG, NB, and ML designed the study. SK1 and \n\nST collected the samples. SK1 analyzed the samples. SK2 \n\nperformed data analysis and drafted the manuscript. \n\nSK1 helped in drafting the manuscript. All authors read \n\nand approved the final version of the manuscript. \n\nCompeting interests \nNone \n\nFunding \nNone \n\nAcknowledgments \nWe kindly acknowledge all the participants of the study. \n\nWe kindly acknowledge the department of biochemistry \n\nof B P Koirala Institute of Health Sciences, Dharan for \n\nsupporting the study. \n\nEthics approval and consent  \nThe study was approved by the Institutional Ethical \n\nReview Committee (IRC) of BPKIHS (Code No: \n\nIRC/422/014). Written consent was taken from \n\nchildren\u2019s parents before participation. \n\nAvailability of data and material \nData are available on reasonable request. \n\nReferences  \n1. Chung HR. Iodine and thyroid function. Ann Pediatr \n\nEndocrinol Metab. 2014;19(1):8-12. doi: \n10.6065/apem.2014.19.1.8. \n\n2. Mullur R, Liu YY and Brent GA. Thyroid hormone \nregulation of metabolism. Physiol Rev. 2014;94(2):355-82. \ndoi: 10.1152/physrev.00030.2013. \n\n3. Zimmermann MB, Jooste PL, Pandav CS. Iodine-deficiency \ndisorders. Lancet. 2008;372(9645):1251-62. doi: \n10.1016/S0140-6736(08)61005-3. \n\n4. Zimmermann MB. The effects of iodine deficiency in \npregnancy and infancy. Paediatr Perinat Epidemiol. \n2012;26(1):108-17. doi: 10.1111/j.1365-3016.2012.01275.x. \n\n5. Ershow AG, Skeaff SA, Merkel JM, Pehrsson PR. \nDevelopment of Databases on Iodine in Foods and Dietary \nSupplements. Nutrients. 2018;10(1). pii: E100. doi: \n10.3390/nu10010100. \n\n6. Khatiwada S, Gelal B, Gautam S, Lamsal M, Baral N. \nIodine Status among School Children of remote Hilly \nregions of Nepal. Indian Pediatr. 2015;52(5):436-7. \n\n7. Khatiwada S, Lamsal M, Gelal B, Gautam S, Nepal AK, \nBrodie D, et al. Anemia, Iron Deficiency and Iodine \nDeficiency among Nepalese School Children. Indian J \nPediatr. 2016;83(7):617-21. doi: 10.1007/s12098-015-1924-y. \n\n8. Khatiwada S, Gelal B, Shakya PR, Lamsal M, Baral N. \nUrinary Iodine Excretion among Nepalese School Children \nin Terai Region. Indian J Pediatr. 2016;83(1):15-7. doi: \n10.1007/s12098-015-1755-x. \n\n9. Shakya PR, Gelal B, Baral N. High iodine intakes in school \nchildren in Eastern Nepal. IDD Newsletter. 2011;39:10-3. \n\n10. Ohashi T, Yamaki M, Pandav CS, Karmarkar MG, Irie M. \nSimple microplate method for determination of urinary \niodine. Clin Chem. 2000;46(4):529-36. \n\n11.", "start_char_idx": 16125, "end_char_idx": 18828, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6ed52d5b-0c5c-4095-a1ac-5b8d26097bec": {"__data__": {"id_": "6ed52d5b-0c5c-4095-a1ac-5b8d26097bec", "embedding": null, "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-109", "node_type": "4", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "23be5f492979719cc80c75e1e3cc4bcc45594d6ed5c576a31507015eabee166a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bc744b66-5ddc-449c-b92e-4124ba2e9f7a", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "f68091fbba9787d4a85c1c005662f042eb10867a1bf29753d77d392ed2ce224c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3504ed8c-faac-461f-a10b-9474cf398230", "node_type": "1", "metadata": {}, "hash": "5b9774e00c4b4134fe8a5e26f924ea041e138698475230365081b8f1fb355f2e", "class_name": "RelatedNodeInfo"}}, "text": "Clin Chem. 2000;46(4):529-36. \n\n11. Vincenzo T, Emilio T, Angelo VG, Carlo S, Francesco R, \nBrunella L et al. Role of Iodine, Selenium and Other \nMicronutrients in Thyroid Function and Disorders. Endocr \nMetab and Immune Disord Drug Targets. 2009;9(3):277-94. \n\n12. Ministry of Health and Population, Department of Health \nServices, Government of India and Alliance Nepal. \nNational Survey and Impact Study for Iodine Deficiency \nDisorders (IDD) and availability of iodized salt in Nepal; \n2007. \n\n13. Gelal B, Chaudhari RK, Nepal AK, Sah GS, Lamsal M, \nBrodie DA, et al. Iodine deficiency disorders among \nprimary school children in eastern Nepal. Indian J Pediatr. \n2011;78(1):45-8. doi: 10.1007/s12098-010-0239-2. \n\n14. Chaudhari RK, Gelal B, Brodie D, and Baral N. Thyroid \nfunction and urinary iodine status in primary school age \nchildren of the hills and plains of Eastern Nepal. Indian \nPediatr. 2012;49:332-333. \n\n15. Ma ZF, Skeaff SA. Thyroglobulin as a biomarker of iodine \ndeficiency: a review. Thyroid. 2014;24(8):1195-209. doi: \n10.1089/thy.2014.0052. \n\n16. Chaudhary LN, Khatiwada S, Gelal B, Gautam S, Lamsal \nM, Pokharel H, et al. Iodine and Thyroid Function Status, \nand Anti-thyroid Peroxidase Antibody among Pregnant \nWomen in Eastern Nepal. J Nepal Health Res Counc. \n2017;15(2):114-119. \n\n17. Vejbjerg P, Knudsen N, Perrild H, Laurberg P, Carl\u00e9 A, \nPedersen IB, et al. Thyroglobulin as a marker of iodine \nnutrition status in the general population. Eur J \nEndocrinol. 2009;161(3):475-81. doi: 10.1530/EJE-09-0262. \n\n18. Shakya PR, Gelal B, Das BKL, Lamsal M, Pokharel P K, \nNepal AK, et al. Urinary iodine excretion and thyroid \nfunction status in school age children of hilly and plain \nregions of Eastern Nepal. BMC Res Notes. 2015; 8: 374. \n\n19. Khatiwada S, Gelal B, Baral N, Lamsal M. Association \nbetween iron status and thyroid function in Nepalese \nchildren. Thyroid Res. 2016;9:2. DOI: 10.1186/s13044-016-\n0031-0.", "start_char_idx": 18793, "end_char_idx": 20738, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3504ed8c-faac-461f-a10b-9474cf398230": {"__data__": {"id_": "3504ed8c-faac-461f-a10b-9474cf398230", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6ed52d5b-0c5c-4095-a1ac-5b8d26097bec", "node_type": "1", "metadata": {"identifier": "njb-109", "author": "Kunwar, Saroj; Khatiwada, Saroj; Gelal, Basanta; Thapa, Saroj; Shah, Gaurishankar; Baral, Nirmal; Lamsal, Madhab", "title": "Thyroid Function and Thyroglobulin Level in Iodine-Deficient Children of Eastern Nepal", "date": "2021-07-31", "file": "njb-109.pdf"}, "hash": "702334c082000ddf139b3849e69322a4da7e55caf784c8cbef49edf09d265eaa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1b47f7bb-2300-4c9c-a8f3-b63c2d747f9b", "node_type": "1", "metadata": {}, "hash": "54dbd5cc28067e21490eebaee149b0f2548e45d016f78d799e8e1c92a0de9bb6", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9  Research article DOI: https://doi.org/10.3126/njb.v9i1.38648\n\n\u00a9NJB, BSN 42 \n\nComparative Study of Nutritional Profile of Rice Varieties in Nepal \nEvance Pakuwal , Prakash Manandhar \n\nDepartment of Microbiology, St. Xavier\u2019s College, Kathmandu, Nepal \n\nReceived: 16 Sep 2020; Revised: 04 May 2021; Accepted: 15 May 2021; Published online: 31 Jul 2021 \n\nAbstract\nThe purpose of this study was to evaluate and compare the nutritional quality of different rice varieties (Taichung-176, Khumal-\n4 rice, and Black rice) with Jumli Marsi rice. The highest nutritional factors and phytochemical components were found in the \nMarsi rice (RR) and Black rice (BR). The highest amount of antioxidant property and phenolic content was found in Black rice \nwhich was 61.58 \u00b1 0.02% and 22.75 \u00b1 0.02GAE/100g respectively. The reducing sugar was found to be highest in the TR rice \nvariety, which was 2.74\u00b10.01%. The results also highlight the cooking and physicochemical properties of rice depending on \nthe amylose content of rice varieties. The qualitative analysis of the phytochemical content in different rice varieties showed \nthe presence of tannin, flavonoid, alkaloid, and terpenoid in Marsi and BR. While anthraquinone and saponin were negative \nfor all the rice varieties, protein and glycoside were found to be present in all the rice varieties. Also, the pigmented rice \nvarieties were found to have high nutritional components compared to the non-pigmented rice varieties. All the data observed \nin the study was found to be statistically significant (p < 0.05).  \n\nKeywords: Pigmented Rice, Antioxidant, phytochemicals, Jumli Marsi \n\n Corresponding author, email: pakuwalevance@gmail.com \n\nIntroduction \nCereal grains are consumed worldwide and are \n\nconsidered an important dietary source of proteins, \n\ncarbohydrates, vitamins, minerals, and fiber [1]. Rice is \n\nthe cereal grain that is consumed as a major staple food \n\naround the world. Rice (Oryza sativa L) belongs to the \n\nfamily Poaceae. The contribution of rice as a percentage \n\nof total dietary energy supplied in developing countries, \n\nwhich is 715 kcal/day, 27% of total dietary energy \n\nsupply, 20% of dietary protein, and 3% of dietary fat [2,3, \n\n4].  \n\nThere are different varieties of rice available around the \n\nworld. Among those, pigmented rice varieties such as \n\nred, black, purple, and brown rice are known to be \n\nconsumed for a long time in Southeast Asian countries. \n\nThey are also known to be originating in Southeast Asia \n[1]. In the case of Nepal, there are three different regions \n\n(Tarai, mountains, and hilly) classified according to their \n\ngeographic location. Different varieties of rice are \n\ncultivated from Tarai (lowland plains) to high Hilly \n\nregions.  \n\nJumla (2300 MSL) is the key site for growing cold-tolerant \n\nrice genotypes for the hilly regions of Nepal. The major \n\narea for the production of Jumli Marsi is Tila and Sinja \n\nvalleys in Jumla, which are the world heritage sites \n\npresent in Nepal [5]. Marsi (RR), sometimes spelled as \n\nMarsee is a variety of rice cultivated in the highest altitude \n\nin the world, Chhumchaur, Jumla [6]. It is commonly \n\nknown as nutritionally rich in compounds such as \n\nprotein, polyphenol, flavonoids, and antioxidants. The \n\nMarsi rice is classified by the farmers in different groups \n\nnamely, Kali Marsi, Raato Marsi (can be seen in \n\nphotograph 4), Seto Marsi, Daarime Marsi, Mehele Marsi, \n\netc. Marsi is also known to have medicinal value [7,8].", "start_char_idx": 48, "end_char_idx": 3597, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1b47f7bb-2300-4c9c-a8f3-b63c2d747f9b": {"__data__": {"id_": "1b47f7bb-2300-4c9c-a8f3-b63c2d747f9b", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3504ed8c-faac-461f-a10b-9474cf398230", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "ea8d46fd8a0465af710864b012a3315f976c40e11add7ae534a90088434ee1cf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "edf013c2-7ec8-483d-a5f0-a8f4abfc5c94", "node_type": "1", "metadata": {}, "hash": "9e99c38dde5826716fede82b58b3a13bd7840512483abdf1318cb8025b3764ee", "class_name": "RelatedNodeInfo"}}, "text": "Marsi is also known to have medicinal value [7,8]. \n\nBlack rice (BR) is a type of pigmented rice that has black \n\nbran covering the endosperm of rice grain. They are black \n\n(shown in Photograph 1) and turn purple when cooked. \n\nThe color intensities of pigmented rice are known to be \n\nrelated to the value of lightness, redness, and yellowness. \n\nIt takes a longer time to cook and has stickier consistency \n\ncompared to white rice [9]. The supplementation of diets \n\nwith black rice and the anthocyanin-rich extract of the \n\nblack rice is found to have various health benefits [10].  \n\nThe composition of rice variety is important for health-\n\nconscious consumers as well as useful to reduce fuel \n\nconsumption during the process of cooking. Amylose \n\ncontent can be a factor that can influence the cooking time \n\nof rice. These are different in different varieties of rice \n\n[11]. Amylose is a branched carbohydrate mainly based \n\non \u03b1 (1\u20134) bonds. Amylose content is an important factor \n\nin determining the cooking properties of a rice variety \n\n[12,13]. The amylose content in different rice varieties is \n\nclassified into waxy, low, intermediate, and high \n\naccording to the percentage of amylose present in the \n\nrice, which is classified as waxy (0-2% amylose), low (10- \n\n20% amylose), intermediate (20-25% amylose), and high \n\n(>25% amylose) [14]. \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0001-5333-5291\nmailto:pakuwalevance@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  43 \n\nThe cooking quality of rice depends on the components \n\nof the rice variety such as proteins and amylopectin [15]. \n\nThe antioxidant activities of black and red rice and their \n\ncrude extracts have given results that show the addition \n\nof the pigmented rice could increase antioxidant property \n\nin daily meals. The pigment in rice is the indicator of \n\nbioactive compounds present in the rice [9]. These \n\nvarieties are studied and found to have high anticancer, \n\nanti-inflammatory properties as well as antioxidant \n\ncontent. Some phytochemicals such as phenolic \n\ncompounds, bioflavonoids, terpenoids, and alkaloids are \n\nalso found in pigmented rice [8,16]. The grains with red \n\nand black (darker) colored pericarp grains have higher \n\nmolecular weight compared to the light brown pericarp \n\ncolor [16]. \n\nCarotenoids, vitamin E, terpenoids, and phenolics are \n\nsome important groups of phytochemicals found in \n\nwhole grains [17]. Phenolics are produced by the process \n\nof secondary metabolism in plants, which are also found \n\nto have a positive impact on human health [18]. The \n\nanthocyanins and pro-anthocyanidins/condensed \n\ntannins are some of the commonly found pigments in \n\nblack rice and red rice, respectively. The flavonoids like \n\nluteolin, apigenin, quercetin, isorhamnetin, kaempferol, \n\nand myricetin are found in rice. Black rice has high \n\nphenolic and antioxidant compounds, which contribute \n\nto multiple biological activities leading to an increase in \n\ntoday\u2019s market demand [19,20].  \n\nThe Taichung-176 (TR) rice variety (shown in Photograph \n\n3) is one of the commonly used rice varieties in Nepal and \n\nit is used to prepare traditional fermented food varieties \n\nsuch as Chyang, selroti, etc. Marsi and Black rice varieties \n\nare expensive compared to the other two rice varieties. \n\nKhumal-4 (KR) (shown in Photograph 2) rice is affordable \n\nand is consumed by the locals for a daily meal [21].", "start_char_idx": 3547, "end_char_idx": 7196, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "edf013c2-7ec8-483d-a5f0-a8f4abfc5c94": {"__data__": {"id_": "edf013c2-7ec8-483d-a5f0-a8f4abfc5c94", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1b47f7bb-2300-4c9c-a8f3-b63c2d747f9b", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "af0435cf9a5be8f879abf8af243fcc8284cd0afb71440775da7b38da91aa53cf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f6fd2f65-5a2c-46dc-828e-526f7cba51d1", "node_type": "1", "metadata": {}, "hash": "e4b5d38945c8b9a84ef3cf860a35b9aecc35fd4ae536ab666b0136c1156d857b", "class_name": "RelatedNodeInfo"}}, "text": "There \n\nhave been various research studies performed to study \n\ndifferent rice varieties, however, no researches have been \n\nperformed to compare Red rice (RR), also known as \n\nMarsi, which is mainly found in the altitudes of Nepal, \n\nwith other rice varieties. Therefore, in this study, the \n\nmain objective was to comparatively study the \n\nnutritional characteristics of Marsi rice with other rice \n\nvarieties available in Nepal.  \n\nMaterials and Methods \nSample Collection and identification \nFour different varieties of rice (Taichung-176 rice, Khumal-\n\n4 rice, Black rice, and Red rice/ Jumli Marsi rice) were \n\ncollected from the local market of Khumaltar and \n\nKathmandu Organics (as suggested by Food Research \n\nDivision, NARC, Khumaltar, Nepal).  \n\nDetermination of physical properties of rice  \nDetermination of length, breadth was done by randomly \n\nselecting 10 grains from each sample using a slide caliper \n\n[22]. \nLength = Main scale reading + Vernier scale reading \u00d7 \n\nVernier constant  \n\nBreadth = Main scale reading + Vernier scale reading \u00d7 \n\nVernier constant  \n\nWeight of kernels  \nKernel grains (1000 grains counted manually) from each \n\nvariety were randomly selected and weight was recorded \n\nin grams [23]. \n\nDetermination of cooking characteristics of \n\nrice varieties  \n\nSwelling property/ Water uptake  \nFor the determination of swelling property, finely \n\nground rice was mixed in distilled water (0.35 g in 12.5 \n\nml). The mixture was then heated in the water bath for 30 \n\nminutes at a temperature of 60\u00b0C. It was then centrifuged \n\nat 3500 rpm for 20 minutes. The supernatant was \n\ndecanted in a pre-weighed dish. The temperature was \n\nmaintained at 100 \u00b0C for drying (20 minutes) [24]. The \n\nresidue was weighed. The swelling power was calculated \n\nby: \n\nSwelling Property = Weight of the Residue /Original \n\nweight \n\nCooking time  \nRice (2 g) of each variety was cooked in distilled water (20 \n\nml) separately, and the temperature was maintained at \n\n90\u00b0C in a water bath. Two glass slides were used to press \n\ncooked rice samples in between (to find out the minimum \n\ncooking time) [23,24]. \n\nProximate Analysis of rice varieties  \nDetermination of moisture and ash was done based on \n\nthe standard method. The reducing sugar content was \n\ndetermined by the anthrone method. The carbohydrate \n\ncontent was determined using the anthrone method for \n\ncarbohydrate estimation. Spectrophotometric analysis \n\nwas done at 630 nm [24,25,26].  \n\nCalculation:   \nAmount of carbohydrate present in 100mg of the sample \n\n=   Glucose (in mg)/ Volume of test sample \u00d7 100  \n\n% carbohydrates = (concentration sample * dilution \n\nfactor)/ mass sample X 100  \n\nAmylose content  \nThe rice flour was mixed with ethanol (95%) and 1 N \n\nNaOH. A mixture was prepared by thoroughly mixing it \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  44 \n\nin a volumetric flask. The mixture was heated in a boiling \n\nwater bath for 10 minutes. It was then cooled to room \n\ntemperature, to gelatinize the starch present in the rice \n\nsample. Five milliliters of the solution was then \n\ntransferred to another volumetric flask. One ml of 1N \n\nacetic acid and iodine solution was added to the solution. \n\nThe distilled water was used to adjust the volume to 100 \n\nml. The vortex mixing method was used to mix it again. \n\nIt was then allowed to stand for the time duration of 20 \n\nminutes. The absorbance of the sample solution was \n\nmeasured at 620 nm using a UV Spectrophotometer. \n\nFinally, the determination of amylose content in samples \n\nwas done by using the amylase standard curve [23]. \n\nDetermination of Reducing Sugar by using 3, \n\n5- dinitrosalicylic acid / DNS method \nFor the rice sample, 100 mg of the sample was weighed \n\nand centrifuged with the hot 80% ethanol twice (5 ml each \n\ntime).", "start_char_idx": 7197, "end_char_idx": 11059, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f6fd2f65-5a2c-46dc-828e-526f7cba51d1": {"__data__": {"id_": "f6fd2f65-5a2c-46dc-828e-526f7cba51d1", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "edf013c2-7ec8-483d-a5f0-a8f4abfc5c94", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "47f905706771b705da143bcc4903b6547d02f8a78cebe7ec0aa9abad01ab3deb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0c658ed3-45b8-4210-b9d5-6f4109eb0b14", "node_type": "1", "metadata": {}, "hash": "24c571de55516c6cdfbb1b35ebd953962209ff3d882474d654ede8524ed9b2b0", "class_name": "RelatedNodeInfo"}}, "text": "The supernatant was collected and evaporated by \n\nkeeping it in a water bath at 80\u00b0C. 10 ml water was added \n\nto dissolve the sugars. 0.5 to 3 ml was pipette out of the \n\nextract in test tubes and volume was equalized to 3 ml \n\nwith water in all the tubes. The standard sugar solution \n\nwas prepared in the range of 0 to 3 ml in different test \n\ntubes. The final volume was made up to 3 ml with \n\ndistilled water, which made the concentrations ranging \n\nfrom 0 to 750 mg. 3 ml of DNS reagent was added. The \n\ncontents were heated in a boiling water bath for 5 \n\nminutes. It was then cooled and the intensity of dark \n\ncolor was read at 540nm. Reducing sugar in rice varieties \n\nwas determined by using the 3, 5- dinitrosalicylic acid / \n\nDNS method by spectrophotometric analysis at 540 nm \n\n[27]. \n\n\u25cf Antioxidant and phenolic content of rice \n\nvarieties  \nThe antioxidant content was determined by using the \n\nDPPH assay method. The phenolic content was \n\ndetermined by the Folin-Ciocalteu method using \n\nspectrophotometric analysis (gallic acid as the standard).  \n\nAntioxidant activity (DPPH Assay method)   \nThe antioxidant activity of rice samples was evaluated \n\nspectrophotometrically, as a measure of radical \n\nscavenging activity by using DPPH [24,25]. \n\nThe capability to scavenge the DPPH radical (% \n\ninhibition) was calculated using the following equation:  \n\nScavenging ability % =  \n\n[Absorbance of control (515 nm) - Absorbance of sample \n\n(515 nm)/Absorbance 515 nm of control] x 100  \n\nPhenolic content (Folin-Ciocalteu method) \nTotal phenolic content was determined by the standard \n\ngraph of gallic acid, which was prepared by dissolving 1 \n\nmg of dry gallic acid, and absorbance was measured at \n\n760 nm [26]. \n\nPhytochemical properties of rice varieties  \nThe qualitative analysis of different rice varieties was \n\ndone for the phytochemicals: terpenoid, flavonoid, \n\ntannin, anthraquinone, saponin, and glycoside [9].  \n\nExtraction  \nEach variety of rice was milled into flour and then the rice \n\nflour was kept in 20 ml of 80% methanol for 24 hrs at \n\nroom temperature and then centrifuged at 4000 g for 15 \n\nminutes.  \n\nTerpenoid: \nRice extract (5 ml) was mixed with 2 ml of chloroform. \n\nThe concentrated H2SO4 (3 ml) was added to the solution. \n\nReddish-brown coloration formed in between two liquid \n\nphases indicated the presence of terpenoids.  \n\nFlavonoid: \nCrude rice sample (2 g) was mixed with 10 ml of ethyl \n\nacetate heated in a water bath (5 minutes). The Whatman \n\npaper no.1 was used to filter the solution. Then, the \n\nfiltrate (4 ml) and the dilute ammonia solution (10%) \n\nwere mixed well and observed for color change \n\n(yellowish coloration), which indicated the presence of \n\nflavonoids.  \n\nTannin:  \nA few drops of 0.1% FeCl3 solution were mixed with the \n\n(10 ml) rice extract filtrate. The formation of bluish-black \n\nprecipitation indicated the presence of tannin.  \n\nPhlobatannin: \nThe concentrated HCl was added to the rice extract \n\nfiltrate (10 ml) in a test tube, and boiled for 1 minute. It \n\nwas then observed for the formation of a red precipitate, \n\nwhich indicated the presence of phlobatannins.  \n\nProtein: \nRice extract (2 ml) was taken in a test tube was taken. One \n\nml of 40% NaOH solution was added to it. The solution \n\nwas then mixed properly. One to two drops of CuSO4 \n\nsolution were added to the solution. The change of color \n\nto violet indicated the presence of proteins.   \n\n\n\nNepal J Biotechnol.", "start_char_idx": 11060, "end_char_idx": 14529, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0c658ed3-45b8-4210-b9d5-6f4109eb0b14": {"__data__": {"id_": "0c658ed3-45b8-4210-b9d5-6f4109eb0b14", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f6fd2f65-5a2c-46dc-828e-526f7cba51d1", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "a9cb4b3c5ea651eb7c561d69ea0ef3bf64587d1798869f8ccf1d62749761b434", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e185b5b6-a9b3-48ba-aabc-3072a9ae3e73", "node_type": "1", "metadata": {}, "hash": "7d0977497f9bfcb736f1ec3b1fc63ce7924596c530618bc67b2512cb9a13434b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  45 \n\nSaponin: \nThe crude sample (0.5 g) was mixed in distilled water, \n\nand boiled by using a water bath. After that, it was \n\nshaken well. The sample was then observed for the \n\nformation of froth, which indicated the presence of \n\nsaponin.  \n\nAnthraquinone: \nThe crude sample and benzene were mixed in a conical \n\nflask. A magnetic stirrer was used to mix it properly for 4 \n\nhrs. The filtrate (10 ml) was taken and mixed with 0.5 ml \n\nammonia solution (10%). The change in color of the \n\nsolution indicated the presence of anthraquinone. \n\nGlycoside: \nThe rice extract (5 ml) was mixed with a mixture of glacial \n\nacetic acid (2ml), 2% FeCl3 solution and 1 ml concentrated \n\nH2SO4 (added slowly when mixed). The mixture was \n\nobserved for the formation of a brown ring, which \n\nindicated the presence of glycoside.   \n\nData Analysis \nAll the tests (except length, breadth of rice, and amylose \n\ncontent) were performed on triplicates (n =3). The length \n\nand breadth of rice were measured in n=10 by randomly \n\nselecting grains from each sample. The determination of \n\nthe level of significance of various parameters of different \n\nrice varieties was performed by using Minitab Version 18. \n\nOne factor analysis of variance was performed for a \n\ncompletely randomized design to check the level of \n\nsignificance. \n\nResults \nFour different varieties of rice TR, KR, RR, and BR were \n\ncollected from four different places. Table 1 shows that \n\nthe antioxidant was highest for BR (61.58\u00b10.02%), lowest \n\nfor TR (8.13%) and the phenolic content was highest for \n\nBR (22.75 \u00b10.02GAE/100g), lowest for TR \n\n(9.35\u00b10.01GAE/100g).  \n\nThe physical characteristics and cooking characteristics \n\nof different rice varieties were studied. The rice kernel \n\nweight (g) (in Table 2) was 19.21\u00b10.23, 17.12\u00b10.05, \n\n18.04\u00b10.03, and 17.03\u00b10.02 for TR, KR, RR, and BR \n\nrespectively. \n\nThe results showed that the length and breadth of rice \n\nvarieties ranged between 4 mm to 6 mm and 2 mm to 4 \n\nmm. The swelling capacity ranged from 1.80 w/w to 3.83 \n\nw/w. The cooking time ranged minimum for KR (20 \n\nminutes) and maximum for BR (40 minutes). The \n\nmoisture content, ash content, and amylose content, as \n\nwell as amylopectin, which was determined for each \n\nvariety of rice (in Table 3). The moisture content of TR, \n\nKR, RR, and BR 9.05\u00b1 0.01%, 7.9\u00b1 0.00%, 10.2\u00b1 0.03%, and \n\n12.15\u00b1 0.01% respectively.  \n\nTable 1. Bioactive compounds in different rice varieties \n\nS.N. Type of rice  Antioxidant (%inhibition) Phenolic Content (GAE/100G) \n\n1. Taichung-176 (TR) 8.13\u00b10.01 9.35\u00b10.01 \n2. Khumal-4 (KR) 16.38\u00b10.00 11.74\u00b10.01 \n\n3. Marsi/ Red rice (RR) 53.06\u00b10.01 15.47\u00b10.03 \n\n4. Black rice (BR) 61.58\u00b10.02 22.75\u00b10.02 \n\nNote: Phenolic content was expressed in the unit of mg GAE/100g. All the values are mean \u00b1 standard deviation of triplicate analysis. The data \npresented were found to be significant (p < 0.05).", "start_char_idx": 14510, "end_char_idx": 17511, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e185b5b6-a9b3-48ba-aabc-3072a9ae3e73": {"__data__": {"id_": "e185b5b6-a9b3-48ba-aabc-3072a9ae3e73", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0c658ed3-45b8-4210-b9d5-6f4109eb0b14", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "81a4271691825c99ccd5d19a64c33829c07cad295d0d7e8cd487a1ecbe9d27dd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f90b914a-6dc8-40ab-bb23-46329826404b", "node_type": "1", "metadata": {}, "hash": "be8ebb0dbbcab10e408adffac36ccbf46f47b5ea4b843ee984450e60997c28b9", "class_name": "RelatedNodeInfo"}}, "text": "The data \npresented were found to be significant (p < 0.05). \n\nTable 2.Physical characteristics of different rice varieties \n\nS. \nN. \n\nRice \nType \n\nKernel \nweight (g) \n\nLength \n(mm) \n\nBreadth \n(mm) \n\nCooking \n\ntime (Min) \n\nAmylose \nContent (%) \n\nAmylose \nType \n\nAmylo-\n\npectin (%) \n\nSwelling \nproperty \n\n1. TR 19.21\u00b10.23 4\u00b10.01 3\u00b10.2 23\u00b12.0 27.62 High 72.40 3.83\u00b10.25 \n\n2. KR 17.12\u00b10.05 5\u00b10.20 3\u00b10.1 20\u00b10.5 24.12 Medium 75.88 3.25\u00b10.02 \n\n3. RR 18.04\u00b10.03 6\u00b10.25 4\u00b10.25 35\u00b11.0 20.67 Medium 79.33 2.32\u00b10.18 \n\n4. BR 17.03\u00b10.02 6\u00b10.25 3\u00b10.5 40\u00b12.0 5.28 Low 94.72 1.80\u00b10.15 \n\nNote: All the tests (except length and breadth of rice) were performed in triplicate and the data listed are the mean \u00b1 standard deviation of \ntriplicate readings. The data presented were found to be significant (p < 0.05). \n\nTable 3. Proximate analysis of different rice varieties \n\nS. N.  Tests performed    Types of Rice      \n\nTR   KR   RR   BR   \n\n1.   Ash content (%) 1.62\u00b10.01  0.74\u00b10.01  1.56\u00b10.01   1.70\u00b10.01   \n\n2.   Moisture (%)   9.05\u00b10.01   7.91\u00b1 0.00   10.2\u00b1 0.03  12.15\u00b1 0.01   \n\n3.   Carbohydrate (%)   82.5\u00b10.02   65.3\u00b1 0.01   74.5\u00b1 0.01   73.2\u00b10.01   \n\n4.   Reducing Sugar (%)   2.74\u00b10.01   1.42\u00b1 0.01   1.12\u00b1 0.02   1.87\u00b1 0.03   \n\nNOTE: X\u00b1Y= Mean \u00b1 S.D of triplicate values. All the experimental values were generated three times and the mean was then calculated. The \ndata presented were found to be statistically significant (p < 0.05).", "start_char_idx": 17451, "end_char_idx": 18879, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f90b914a-6dc8-40ab-bb23-46329826404b": {"__data__": {"id_": "f90b914a-6dc8-40ab-bb23-46329826404b", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e185b5b6-a9b3-48ba-aabc-3072a9ae3e73", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "af57dcb5166dcbf828b0338865e0ad7dd9aa3bb2a0fba566cd57b26c405d7c1a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6a7f7b7d-3fa0-44ee-ab61-0d3777163f95", "node_type": "1", "metadata": {}, "hash": "f1f1d1bd3373c0431d73d3b18a5bbfab291e350b2a696efb65c03d390daac31d", "class_name": "RelatedNodeInfo"}}, "text": "The \ndata presented were found to be statistically significant (p < 0.05).  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  46 \n\n The ash was highest in BR 1.70\u00b10.01%. Amylose content \n\nwas highest in TR (27.62\u00b10.01%) and lowest in BR \n\n(5.28\u00b10.01%). The reducing sugar was found highest in \n\nTR (2.74\u00b10.01%), and lowest in RR (1.12\u00b10.03%). The \n\nphytochemical properties of different varieties of rice \n\nsamples (Table 4). Tannin, alkaloids, flavonoids, and \n\nterpenoids were present only in red rice and black rice. \n\nAnthraquinone and saponin were absent in all the \n\nsamples. Glycoside and protein were present in all the \n\nrice samples. \n\nDiscussion \nThe antioxidant activity was determined by the DPPH \n\nassay method by determining the percentage inhibition \n\nfor every rice variety. The antioxidant activity of Marsi \n\nwas compared with TR, KR, and BR. BR had the highest \n\nantioxidant property and the phenolic content which was \n\n61.58\u00b1 0.02 % and 22.75\u00b1 0.02 GAE/100 g respectively. \n\nThe lowest antioxidant activity was found in TR, which \n\nwas 8.13\u00b1 0.01%. The DPPH radical scavenging activity \n\nwas found higher in BR variety (59.02 to 75.52%) with the \n\nhighest observed in aromatic black rice Poireiton chakhao \n\n(75.52%) [26]. Another study reported that the DPPH \n\nscavenging activity of red rice, parboiled rice, and Sona \n\nMasuri were 57.06%, 9.13%, and 16.38%, respectively \n\n[18,29].  \n\nPhotograph 1. Black rice variety \n\nTable 4.  Qualitative analysis of phytochemical properties in rice sample \n\nS. N.  Tests performed  \n \n\nTypes of Rice \nTaichung-176 (TR)  Khumal-4 (KR) Red rice/ Marsi (RR)  Black rice (BR)  \n\n1. Tannin   - - + + \n2. Flavonoid   - - + + \n3. Alkaloids   - - + + \n4. Terpenoid   - - + + \n5. Protein   + + + + \n6. Glycoside   + + + + \n7. Anthraquinone   - - - - \n8. Saponin   - - - - \n  Note: (+) denotes the presence of the phytochemical and (\u2013) denotes the absence of phytochemical \n\nFlowchart 1. Schematic representation of study of different rice varieties", "start_char_idx": 18805, "end_char_idx": 20866, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6a7f7b7d-3fa0-44ee-ab61-0d3777163f95": {"__data__": {"id_": "6a7f7b7d-3fa0-44ee-ab61-0d3777163f95", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f90b914a-6dc8-40ab-bb23-46329826404b", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "cfcf990c344effa3ca11d97eeb0a53b9baab92fc95d92a05e10bb35b5c127882", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e6c37922-30ce-4e8d-917f-07d7cafa2f84", "node_type": "1", "metadata": {}, "hash": "a0b89a2db6f0b0b0338f9d76b9db7d762088a1ea6c61c6d5a493d40a1f34969f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  47 \n\n \nPhotograph 2. Khumal-4 rice variety \n\n \nPhotograph 3. Taichin rice (Taichung-176) \n\n \n\nPhotograph 4. Red rice variety (Jumli Rato Marsi) \n\nThe phenolic content of Marsi was compared to that of \n\nother rice varieties. Only BR had a higher phenolic \n\ncontent than that of Marsi rice variety, which was 22.75\u00b1 \n\n0.02 GAE/100 g. The lowest phenolic content was found \n\nin TR, which was 9.36\u00b1 0.01 GAE/100 g. Similar findings \n\nof the phenolic content and antioxidant were found in \n\nanother study on pigmented rice varieties [27] [29]. All \n\nthe results from this study were found to be similar to the \n\nresult of previous experiments, and also were found to be \n\nstatistically significant (p < 0.05).  \n\nThe moisture content of Marsi rice was compared to that \n\nof TR, KR, and BR. The moisture content of Marsi was \n\n10.2% and for TR, KR, and BR, it was 9.05%, 7.9%, and \n\n12.15% respectively. Only BR was found to have more \n\nmoisture than Marsi. Only BR was found to have the \n\nhighest ash content compared to that of Marsi, which was \n\nfound to be 1.70%. In a similar study, the ash value was \n\nhigher in black rice varieties compared to the other \n\nvarieties of rice, which is the result of a similar trend as \n\nin our study [28]. In another study, black rice was \n\ncompared to white, brown, glutinous, and basmati rice, \n\nblack rice was found to have higher ash content than \n\nother varieties [18][30]. Carbohydrate content was found \n\nto be 74.5\u00b1 0.01% in Marsi rice variety and only TR had \n\ncarbohydrate content higher than that of Marsi which \n\nwas 82.5\u00b10.02%. Similar findings were reported in a \n\nprevious study for the Jumli Marsi rice [7]. \n\nAmylose content plays an important role in determining \n\nthe cooking and pasting properties of a rice variety \n\n[13,15]. The cooking quality of rice depends on the \n\ncomponents of the rice variety such as proteins and \n\namylopectin varieties [31][32]. Amylose content in TR, \n\nKR, Marsi rice, and BR were found to be 27.6\u00b1 0.01%, \n\n24.12\u00b1 0.01 %, 20.67\u00b1 0.01%, and 5.28\u00b1 0.01% respectively. \n\nIn this study, the relation was found to be positive as the \n\nrice varieties with a high amount of amylose had shorter \n\ncooking time duration (Table 2). Similar trends in results \n\nwere found in another study varieties [14,24,31].  \n\nIn this study, a qualitative analysis of phytochemical \n\nproperties was also performed in all the different \n\nvarieties of rice. Tannin, alkaloids, flavonoids, and \n\nterpenoids were present only in red rice and black rice. \n\nAnthraquinone and saponin were absent in all the \n\nsamples. Glycoside and protein were present in all the \n\nsamples (pigmented and non-pigmented rice). Similar \n\nresults were observed in a study performed on different \n\nrice varieties [9]. \n\nThe purpose of the study was to compare different \n\ncharacteristics of varieties of rice available in Nepal. \n\nMarsi rice and BR are expensive when compared to the \n\nother two varieties, and they are mainly consumed for \n\ntheir nutritional benefits. The reason for choosing these \n\nfour rice varieties is that they are of different price range \n\nand also have different colors as well as characteristics. \n\nThe pigmented and non-pigmented range of rice \n\nvarieties were taken for the study because of how they \n\nare promoted in the Food market these days. Pigmented \n\nrice varieties were found to be more nutritious as they \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.", "start_char_idx": 20871, "end_char_idx": 24448, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e6c37922-30ce-4e8d-917f-07d7cafa2f84": {"__data__": {"id_": "e6c37922-30ce-4e8d-917f-07d7cafa2f84", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6a7f7b7d-3fa0-44ee-ab61-0d3777163f95", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "8fb5189fda5f45c4130607bb48335d08ffcd5230705486535404ea8828072cd6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ab1c33be-0cb1-4c32-ab02-3fa18a0d8da1", "node_type": "1", "metadata": {}, "hash": "4441f270814ce71c3a1784770ffac046d4a17dd68c71d5d6e8f472003745e2e3", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  48 \n\nwere found to have more amount of antioxidant content \n\nas well as phenolic content. This could be because the \n\npigments of the rice varieties are directly linked to the \n\namount of nutrition. Marsi and black rice get their \n\npigment from anthocyanin, which is known to have \n\nantioxidant properties. \n\nConclusion  \nIn conclusion, Jumli Marsi rice variety is highly valued in \n\nNepal for its unique color and high nutritional content. \n\nFrom the result of this study, Marsi rice was found to be \n\nrich in antioxidants, phenolics when compared to the \n\nother two non-pigmented rice varieties but the black rice \n\nvariety was found to be richest in the bioactive \n\ncompounds. For a detailed comparison of these rice \n\nvarieties, the determination of anthocyanin, tannin \n\ncontent, and flavonoid could be done. Since methanol is \n\nused as an extracting solvent for most of the polar \n\ncomponents, there may be the presence of non-polar \n\ncomponents which may not have been measured by \n\nusing this method. Different methods could be used to \n\nquantify the non-polar content present in different rice \n\nvarieties.  \n\nAuthor\u2019s Contribution \nThe study was performed by Ms. Evance Pakuwal under \n\nthe supervision of Mr. Prakash Manandhar. \n\nCompeting Interest \nThere are no competing interests involved in this study. \n\nFunding \nThere was no funding provided by any internal or \n\nexternal sources. \n\nEthical Approval and Consent \nNot Applicable. \n\nAcknowledgment \nI would like to acknowledge my supervisor, Lecturer Mr. \n\nPrakash Manandhar for his constant encouragement, and \n\nvaluable supervision at every stage of this work. I would \n\nlike to thank our Head of the Department of \n\nMicrobiology, Mr. Sudhakar Pant, all the faculty \n\nmembers and staff of the Department of Microbiology \n\n(St. Xavier\u2019s College) for their constant technical and \n\nacademic support.  \n\n References \n1. The Rice Department MOAC. Thailand Rice Cultivation Areas. \n\n2018. Available from: www. ricethailand.go.th/rkb3/Eb_024.pdf \n2. FAOSTAT. Food and Agriculture Organization of the United \n\nNations. 2001. Statistical Databases. Available from: \nhttp://www.fao.org/faostat/en/#data/QC. \n\n3. FAOSTAT. Food and Agriculture Organization of the United \nNations. 2017. Available from: \nhttp://www.fao.org/faostat/en/#data/QC. \n\n4. Mohanty S, Wassmann R, Nelson A, Moya P, and Jagadish SV. Rice \nand climate change: Significance for food security and \nvulnerability. 2012. p 1-9. \n\n5. Khatiwada SP, Kushwaha UKS, and Upreti HK. Evaluation of rice \ngenotypes for the high hill region of Nepal. Proceedings of the 28th \nNational Summer Crop Workshop 17-18. 2015: 102-107 \n\n6. Surarit W, Jansom C, Lerdvuthisopon N, Kongkham S, Hansakul P. \nEvaluation of antioxidant activities and phenolic subtype of \nethanolic bran extracts of Thai pigmented rice varieties through \nchemical and cellular assays. International Journal of Food Science \nand Technology. 2015;50(4):990-998. DOI: 10.1111/ijfs.12703  \n\n7. Joshi Bk, Ojha P, Gauchan, D, and Chaudhary P. Jumli Maarsee Rice \nEvolved in Jumla, Nepal: Nature\u2019s Choices for High Mountains \nwith Nutrition Dense Landrace. Tools and Research Results of the \nUNEP GEF Local Crop Project (2020): 71-74. \n\n8. Acharya PP. 2019. Jumli Maarsee Dhaan. Sambridhi Media Bizypan \nAgency, Kathmandu (in Nepali language) \n\n9. Bhattacharyya S and Roy S. Qualitative and Quantitative \nassessment of bioactive phytochemicals in Gobindobhog and Black \nrice, cultivated in west (September).", "start_char_idx": 24452, "end_char_idx": 27946, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ab1c33be-0cb1-4c32-ab02-3fa18a0d8da1": {"__data__": {"id_": "ab1c33be-0cb1-4c32-ab02-3fa18a0d8da1", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e6c37922-30ce-4e8d-917f-07d7cafa2f84", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "15a967cc34f0f0e02129acd5aaf5043e21caef9007020a295882e350327b021b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0ec0f77a-44e6-4ba2-90f8-20820576411d", "node_type": "1", "metadata": {}, "hash": "646bb37b0401fdcf74dd8d7343b34e855bf64f78b06bc10ce0e26f3fab69b823", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of \nPharmaceutical Sciences and Research. 2018; (9): 3845-3851. \nDOI: 10.13040/IJPSR.0975-8232.9(9).3845-51 \n\n10. Zohoun EV, Tang EN, Soumanou MM, Manful J, Akissoe NH, \nBigoga J, et al. Physicochemical and nutritional properties of rice as \naffected by parboiling steaming time at atmospheric pressure and \nvariety. Food Sci Nutr. 2018 May; 6(3): 638\u2013652. \nDOI: 10.1002/fsn3.600 \n\n11. Adu-Kwarteng E, Ellis WO, Oduro I, and Manful JT. Rice grain \nquality: A comparison of local varieties with new varieties \nunderstudy in Ghana. Food Control 2003; 14: 507\u2013514. \nhttps://doi.org/10.1016/S0956-7135(03)00063-X \n\n12. Asghar S, Anjum FM, Amir MR and Khan MA. Cooking and eating \ncharacteristics of Rice (Oryza sativa L.)-A review. Pakistan Journal \nof Food Sciences. (2012); 22:128-132 \n\n13. Cai Y, Liu C, Wang W. and Cai K. Differences in physicochemical \nproperties of kernels of two rice cultivars during grain formation. \nJournal of Science of Food and Agriculture. (2011);91: 1977\u20131983 \nhttps://doi.org/10.1002/jsfa.4404  \n\n14. Chatterjee, L. and Das, P., Study on Amylose Content of Ten Rice \nVarieties Recommended for Assam, Int. J. Pure App. Biosci. 6(2): \n1230-1233 (2018). DOI: http://dx.doi.org/10.18782/2320-7051.6491 \n\n15. Goffman FD and Bergman CJ. Rice kernel phenolic content and its \nrelationship with antiradical efficiency. J Sci Food Agr, (2004). 84, \n1235-1240. https://doi.org/10.1002/jsfa.1780 \n\n16. Liu RH. Whole grain phytochemicals and health. J Cereal Sci \n2007;46(3):207\u2013219. https://doi.org/10.1016/j.jcs.2007.06.010 \n\n17. Fraga CG. Plant phenolics and human health: biochemistry, \nnutrition, and pharmacology, vol 1. John Wiley & Sons, Hoboken. \n(2009). ISBN: 978-0-470-28721-7 \n\n18. Chanu C, Yenagi S NB, Math KK. Nutritional and functional \nevaluation of black rice genotypes. Journal of Farm Sciences. 2016; \n29(1). \n\n19. Laokuldilok, Thunnop & Shoemaker, Charles & Jongkaewwattana, \nSakda & Tulyathan, Vanna. Antioxidants and Antioxidant Activity \nof Several Pigmented Rice Brans. Journal of agricultural and food \nchemistry. 2011; 59:193-199. DOI:10.1021/jf103649q.  \n\n20. Zubair MA, Rahman MS, Islam MS, Abedin MZ and Sikder MA. A \nComparative study of the proximate composition of selected rice \nvarieties in Tangail, Bangladesh Department of Food Technology \nand Nutritional Science Department of Biochemistry and Molecular \nBiology. 2015; 8(2): 97\u2013102. \nhttps://doi.org/10.3329/jesnr.v8i2.26874 \n\n21. Tamang, J.P. (2010). Himalayan fermented Foods. CRC Press Taylor \nand Francis Group. USA. 187-227. \n\n22. Varnamkhasti MG, Mobli H, Jafari A, Keyhani AR, Soltanabadi \nMH, Rafiee S and Kheiralipour K. Some physical properties of \nrough rice (Oryza sativa L.) grain. Journal of Cereal Science.", "start_char_idx": 27948, "end_char_idx": 30697, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0ec0f77a-44e6-4ba2-90f8-20820576411d": {"__data__": {"id_": "0ec0f77a-44e6-4ba2-90f8-20820576411d", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ab1c33be-0cb1-4c32-ab02-3fa18a0d8da1", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "7692e0193d300e0e5c029d82d11f525b2825361ef88124917f3dc424e54581b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a579f6bc-c917-4b13-aab5-db713f85391c", "node_type": "1", "metadata": {}, "hash": "4fb6ffb2a84aac228f803bf2aed25a85c9045025a917043f05f3eee2ed324d1e", "class_name": "RelatedNodeInfo"}}, "text": "grain. Journal of Cereal Science. 2008; 47: \n496\u2013501. DOI: 10.1016/j.jcs.2007.05.014  \n\n23. Bagchi TB, Sanghamitra P, Berliner J, Chattopadhyay K, Sarkar A, \nKumar A, et al. Assessment of physicochemical, functional and \n\nhttp://doi.org/10.1111/ijfs.12703\nhttps://www.researchgate.net/deref/http%3A%2F%2Fdx.doi.org%2F10.13040%2FIJPSR.0975-8232.9(9).3845-51\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980200/\nhttps://dx.doi.org/10.1002%2Ffsn3.600\nhttps://doi.org/10.1016/S0956-7135(03)00063-X\nhttps://doi.org/10.1002/jsfa.4404\nhttp://dx.doi.org/10.18782/2320-7051.6491\nhttps://doi.org/10.1002/jsfa.1780\nhttps://doi.org/10.1016/j.jcs.2007.06.010\nhttps://doi.org/10.1016/j.jcs.2007.05.014", "start_char_idx": 30664, "end_char_idx": 31352, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a579f6bc-c917-4b13-aab5-db713f85391c": {"__data__": {"id_": "a579f6bc-c917-4b13-aab5-db713f85391c", "embedding": null, "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-112", "node_type": "4", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "4179b3ef7157eaf033b42538c0a1c8723c561bc4e6c699fb450f02b9b64645e5", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0ec0f77a-44e6-4ba2-90f8-20820576411d", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "47ec12f057f931cecccbeadaba700605478cdb958b830486db2ff9279185c85d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2cc943c0-0381-48a0-ad75-b4fe073877be", "node_type": "1", "metadata": {}, "hash": "0bd5eb4c06b68773032d49540cb02ad731d0d1892b8bdebe05000589937a94c1", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 4 2 - 4 9               Pakuwal and Manandhar.  \n\n\u00a9NJB, BSN  49 \n\nnutritional properties of raw and traditional popped rice. Indian \nJournal of Trad. Knowledge. 2016;15(October):659-668. \n\n24. Singh N, Sodhi NS, Kaur M. and Saxena SK. Physico-chemical, \nmorphological, thermal, cooking and textural properties of chalky \nand translucent rice kernels. Food Chemistry. 2003; 82: 433\u2013439. \n\n25. AOAC. Official Methods of Analysis. Association of Official. \nAnalytical Chemists. Washington, D.C. USA. (2000). \n\n26. David T. Plummer. An Introduction to Practical Biochemistry, \n1990;179. Third Edition. \n\n27. Shrestha B. Practical Biochemistry and Biotechnology. SNEMP, \nSwoyambhu, Kathmandu, 2009; 128-131. \n\n28. Mishra K, Ojha H, Chaudhury NK. Estimation of antiradical \nproperties of antioxidants using DPPH- assay: A critical review and \nresults. Food Chemistry [Internet]. 2012;130(4):1036\u201343. Available \nfrom: http://dx.doi.org/10.1016/j.foodchem.2011.07.127  \n\n29. Arab F, Alemzadeh L, Maghsoudi V. Determination of antioxidant \ncomponent and activity of rice bran extract. Scientia Iranica. \n2011;18(6):1402-1406. https://doi.org/10.1016/j.scient.2011.09.014 \n\n30. Blainski A, Lopes GC, and Palazzo de Mello JC. Application and \nAnalysis of the Folin Ciocalteu Method for the Determination of the \nTotal Phenolic Content from Limonium Brasiliense L. Molecules \n2013;18: 6852-6865. doi:10.3390/molecules18066852 \n\n31. Thomas R, Wan-Nadia WA and Bhat R. Physicochemical \nproperties, proximate composition and cooking qualities of locally \ngrown and imported rice varieties marketed in Penang, Malaysia. \nInt. Food Res. J. 2013; 20(3): 1345-1351. \n\n32. Singh N, Kaur L, Singh SN and Sekhon KS. Physicochemical, \ncooking and textural properties of milled rice from different Indian \nrice cultivars. Food Chemistry. 2005; 89: 253\u2013259. DOI: \n10.1016/J.Foodchem.2010.05.115 \n\n \n\nhttp://dx.doi.org/10.1016/j.foodchem.2011.07.127\nhttps://www.sciencedirect.com/science/article/pii/S1026309811001945#!\nhttps://www.sciencedirect.com/science/article/pii/S1026309811001945#!\nhttps://www.sciencedirect.com/science/article/pii/S1026309811001945#!\nhttps://www.sciencedirect.com/science/journal/10263098\nhttps://doi.org/10.1016/j.scient.2011.09.014", "start_char_idx": 31355, "end_char_idx": 33632, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2cc943c0-0381-48a0-ad75-b4fe073877be": {"__data__": {"id_": "2cc943c0-0381-48a0-ad75-b4fe073877be", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a579f6bc-c917-4b13-aab5-db713f85391c", "node_type": "1", "metadata": {"identifier": "njb-112", "author": "Pakuwal, Evance; Manandhar, Prakash", "title": "Comparative Study of Nutritional Profile of Rice Varieties in Nepal", "date": "2021-07-31", "file": "njb-112.pdf"}, "hash": "99b817ecada5640bfffaae3d62a4293751b30dbe3992ce937ae7c3df662bbb92", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "45c871ab-b334-49df-8b3d-8fb410497c89", "node_type": "1", "metadata": {}, "hash": "5f8cd6f8086f53b39e7c04d3f1d8642153c3741e4231f290319094170f499972", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):24-41 Review article         DOI: https://doi.org/10.3126/njb.v9i1.38647  \n\n\u00a9NJB, BSN 24 \n\nAvian/Bird flu: A review: H5N1 outbreaks in Nepal \nDhiraj Shrestha1, Balkrishna Bhattachan2, Hiramani Parajuli3, and Sujata Shrestha4 * \n1Department of Microbiology, Shi-Gan International College of Science and Technology, Kathmandu, Nepal. \n2Biotechnology Society Nepal, BSN Bulletin, Bhaktapur, Nepal. \n3Department of Microbiology, Tri-Chandra Multiple College, Kathmandu, Nepal. \n4Department of Laboratory, Central Jail Hospital, Kathmandu, Nepal. \n\nReceived: 11 Dec 2019; Revised: 03 Feb 2021; Accepted: 26 Feb 2021; Published online: 31 Jul 2021 \n\nAbstract \nAvian/Bird flu is a viral disease of birds, caused by avian influenza virus (AIV). A highly pathogenic avian influenza (HPAI) \nH5N1 has breached the barrier of species to humans and other animals escalating the pandemic threat. If the H5N1 evolves \nto a human-to-human transmissible virus retaining its pathogenicity, it can trigger an influenza pandemic. H5N1 has a \nmortality rate of about 60%, varying with strains. Meaningful antigenic alteration in hemagglutinin (HA) and/or \nneuraminidase (NA) results in recurring pandemics. The HPAI H5N1 subtype alone has outreached more than 77 nations \naround the world since the first human case and death was reported in 1997. Wild and migratory birds are the AIV reservoirs. \nPoultry is primarily impacted by incidents and outbreaks of the disease. A wide range of serological and molecular methods \nhave substantially aided in the identification of bird flu in humans. Candidate vaccines have been developed, yet are not \nready for widespread use. Oseltamivir (brand name: Tamiflu) is the preferred drug for the management of human Influenza-\nlike illness (ILI). Surveillance, mass awareness, and pandemic preparedness abiding WHO recommendations are of \nparamount importance for the prevention of bird flu outbreaks. \n\nKeywords: Avian Influenza Virus (AIV), Avian Flu, Bird Flu, H5N1, Nepal \n\n Corresponding author, email: ssuju.046@gmail.com \n\nIntroduction \nThe word \u2018influenza\u2019 has Italian origin meaning \n\n\u2018influence\u2019; from the belief that epidemics were due to the \n\ninfluence of the stars [1]. It became a term for a particular \n\ndisease after the 1743 epidemic. But, the virus was \n\nisolated from a human only in 1933 [2, 3]. Avian influenza \n\nvirus (AIV), better known as bird flu, is a type-A \n\ninfluenza virus of the Orthomyxoviridae family. \n\nTheoretically, owing to the combination of antigens, \n\nhemagglutinin (HA) and neuraminidase (NA), type-A \n\ninfluenza comprises thousands of distinct antigenic \n\nsubtypes [4]. To date, 18 subtypes of HA and 11 subtypes \n\nof NA have been identified, while two extra subtypes of \n\nHA and NA have been identified in bats [5, 6]. World \n\nOrganisation for Animal Health (OIE) defines AIV as \u201can \n\ninfection of poultry by any influenza A virus, including \n\nby subtypes H5 and H7\u201d [7]. OIE requires notification for \n\nall low-pathogenic avian influenza (LPAI) virus \n\noutbreaks, i.e. H7 and H5 subtypes as they can mutate \n\ninto highly pathogenic avian influenza (HPAI) viruses, as \n\ndocumented in some poultry outbreaks. Non-H5 and \n\nnon-H7 LPAI are not deemed notifiable [8].", "start_char_idx": 48, "end_char_idx": 3316, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "45c871ab-b334-49df-8b3d-8fb410497c89": {"__data__": {"id_": "45c871ab-b334-49df-8b3d-8fb410497c89", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2cc943c0-0381-48a0-ad75-b4fe073877be", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "77a2375f171eb5da34024acb619f69012b93639e26faefdf7e4754445cffff7e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "beedabe5-8c4f-4268-bed0-1b0972c2cde9", "node_type": "1", "metadata": {}, "hash": "6dfe524c97d71d7021e35fb0c75603d36a478d5bac8e5656d9a4a13c2c995147", "class_name": "RelatedNodeInfo"}}, "text": "Non-H5 and \n\nnon-H7 LPAI are not deemed notifiable [8]. In certain \n\npoultry, such as ducks, some HPAI virus (e.g., H5N1) \n\nhave been shown to cause no illness. HPAI has been \n\nassociated with AIV subtypes H5 and H7, including the \n\nviruses H5N1, H7N7, and H7N3. Human infections have \n\nvaried from moderate (H7N3, H7N7) to severe and lethal \n\n(H5N1) infections [9]. \n\nThe HPAI H5N1 virus has raised concerns around the \n\nworld as it threatens poultry, especially chickens; also it \n\nhas shown the potential to pass from poultry to humans \n\nand caused severe infection and death [10]. The first \n\nreported human infection of H5N1 occurred in 1997 in \n\nHong Kong [11]. The virus has been endemic in several \n\ncountries after the re-emergence of H5N1 in Asia, Africa, \n\nthe Pacific region, Europe, and the Middle East in 2003, \n\nand continues to cause poultry outbreaks [12]. In Dhaka, \n\nthe presence of the H5N1 virus was confirmed in March \n\n2007 [13].  The first case of AIV was identified at a remote \n\nnon-commercial poultry farm in Kakarvitta, Eastern \n\nNepal, on January 16, 2009 in Nepal [14, 15].  More than \n\n256 H5N1 outbreaks has been witnessed in poultry since \n\n2009 [16]. \n\nIncidence and Prevalence \nClimate change can bring a quick ecosystem shift which \n\nalters the evolution and ecology of infectious diseases \n\nincluding AIV. This ecosystem shifts had played a \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-3521-2279\nmailto:ssuju.046@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  25 \n\ncumulative effect in the outbreak of the influenza \n\npandemic in the past century [17]. Furthermore, studies \n\nshow an increase in temperature of freshwater in \n\ntemperate and Arctic regions had a persisting impact \n\naltering the suitable habitat variation in prevalence rate \n\nand viral spreading [18]. Several studies have posited \n\nagricultural system, poultry trade, availability of water \n\ntemperature, salinity as factors resulting in an alteration \n\nof AIV ecology [19, 20]. Studies have shown that the \n\noccurrence of new AIVs is supported by either point \n\nmutation, partial genes recombination, or genetic re-\n\nassortment of the whole genome. Most new strains of \n\nAIV evolved due to point mutations while genetic re-\n\nassortment performs a key task in the genesis of H5N1 \n\nand H7N9 strains [21]. The pathogenicity of AIV is \n\nassociated with efficient virus replication. Host immune \n\nresponses and the genetic markers are determinants for \n\nefficient virus replication [22]. The occurrence of human \n\ninfluenza A (H5N1) often parallels with massive \n\noutbreaks of avian H5N1 influenza A, while avian \n\noutbreaks in 2004 and 2005 have seldom led to human \n\ninfection [23]. Different types of studies based on \n\ngeographical distribution and outbreak of AIVs revealed \n\nthat prevalence is related to the migratory route and \n\nstages of the itinerary of migrating birds [24, 25]. Studies \n\nhave concluded that Asia is the prevalent continent for \n\nthe avian influenza virus due to a unique ecosystem of \n\nvarious lakes, wetlands, creeks, and rivers that constitute \n\nmigratory bird wintering areas [26]. Due to consequence \n\nand the risk represented to human health, AIV is of great \n\nconcern and are widely studied in recent times.", "start_char_idx": 3261, "end_char_idx": 6720, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "beedabe5-8c4f-4268-bed0-1b0972c2cde9": {"__data__": {"id_": "beedabe5-8c4f-4268-bed0-1b0972c2cde9", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "45c871ab-b334-49df-8b3d-8fb410497c89", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "076590fc2c23849da3095d7284e8995bad8320282b26e1935fa0e3abf311d5f6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0fd4414f-3ea2-401b-8676-36e72de67ef3", "node_type": "1", "metadata": {}, "hash": "98feea054cf3fcc4f3b35b17a4ac40ed177deed07794fee26d041baa68cc1825", "class_name": "RelatedNodeInfo"}}, "text": "Wild \n\nbirds of genera Antidae (ducks, geese, and swans) and \n\nLaridae (gulls, terns, and kittiwakes) have been in focus \n\nfor decoding the epidemiological links between hosts and \n\ntransmissions of AIV [27]. \n\nEtiology \nThe pandemic influenza virus has its origins in avian \n\ninfluenza [28]. Its genomic material is composed of eight \n\nsegmented negative strand RNAs. The influenza A virus \n\nshows external spikes when examined with an electron \n\nmicroscope. Various techniques can distinguish two \n\nkinds of spikes. The HA molecules made up one type and \n\nthe NA molecules the other. There are roughly 5 times as \n\nmany HA spikes as NA spikes [29] (Figure 1). The 18 \n\ndifferent subtypes of HA and 11 different subtypes of NA \n\nexist allowing for 198 potential different viral strains. As \n\nof 2019, only 131 subtypes have been detected in nature \n\n[30]. \n\n \n\nFigure 1. Schematic diagram of the Influenza A virus with the \nvirus components. Note: NA=neuraminidase, \nHA=hemagglutinin, M1=matrix protein-1, M2=matrix protein-\n2, ss RNA=single stranded RNA, PB2=polymerase basic-2, \nPB1=polymerase basic-1, PA=polymerase acidic, \nNP=nucleoprotein, NS1=non-structural protein-1, NS2=non-\n\nstructural protein-2. \n\nGenomic variation \nGenetic analyses have indicated that since 1997 the H5N1 \n\nvirus has evolved into multiple genotypes [31]. Whether \n\nthe genetic modifications in the HA protein are the result \n\nof immune escape or are related to host adaptation is not \n\nclear [32]. Hence, the best H5N1 epitopes is difficult to \n\npredict to target with vaccines without understanding \n\nthe antigenicity of emerging strains. As H5N1 viruses \n\nhave expanded their geographical and host ranges, it has \n\nbecome increasingly important to determine acquired \n\nfeatures that permit successful human-to-human \n\ntransmission. The transmission of H5N1 viruses to \n\nhumans has been ineffective, arising either by direct \n\ninteraction with infected poultry or through ingestion of \n\nundercooked meat or blood of infected birds [33]. In \n\ninfluenza A viruses, antigenic drift is a continuous \n\nmechanism where point mutations occurs during \n\nreplication of the viral genome. This mechanism is \n\napparent in all the influenza A virus gene products; \n\nhowever, it is most pronounced in the HA and NA \n\nglycoproteins\u2019 antibody-binding sites. These mutations \n\ncannot be repaired because of the lack of a proofreading \n\nmechanism, and the resulting aggregation of variations \n\nin the amino acid sequence transforms these antigenic \n\nsites in such a way that they are less detectable by the host \n\nantibody response. Thus, these virus strains are naturally \n\nselected as host antibodies no longer no longer \n\nneutralized them, and they lead to increased viral fitness \n\n[34, 35]. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  26 \n\nAntigenic Variation  \nBoth the HA and the NA undergo antigenic variation \n\n[36]. Antigenic shift is a major change in antigenicity, \n\nwhile antigenic drift is a minor change sufficient to spark \n\na new outbreak. In the human population, pandemics \n\nhappened in 1957 (Asian influenza) and 1968 (Hong \n\nKong influenza); both are associated with the presence of \n\nantigenically transformed HA molecules. The HA \n\nantigenic variation is known to be more important \n\nquantitatively than that of the NA. The human pandemic \n\nvirus of 1918 (Spanish influenza) was the common \n\nancestor of human and classical swine H1N1 influenza \n\nvirus [37, 38]. \n\nEpidemiology \nAvian flu or bird flu was initially known as Fowl Plague, \n\nand later Fowl Plague virus was discovered as an \n\ninfluenza virus.", "start_char_idx": 6721, "end_char_idx": 10364, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0fd4414f-3ea2-401b-8676-36e72de67ef3": {"__data__": {"id_": "0fd4414f-3ea2-401b-8676-36e72de67ef3", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "beedabe5-8c4f-4268-bed0-1b0972c2cde9", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "74c92c8c6ef26f6808dcb9eedc3a46a28f5d657d5c81811e5ce6a12a54a638a9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "650d21df-b775-410e-aa17-d851f4548125", "node_type": "1", "metadata": {}, "hash": "8fd894d9a22b038c8cd4a487007a1abf6c29523a60586fcf5fbcfeb4fcbeb922", "class_name": "RelatedNodeInfo"}}, "text": "The outbreak of avian flu was first \n\ndocumented in 1878 in Italy. Later on, in 1924 and 1929 \n\ntwo massive outbreaks in poultry were recorded in the \n\nUnited States [39]. Since the first reported human case \n\nand death by the influenza virus in 1997 in Hong Kong, \n\nthe eruption of the disease has been documented from \n\nwild and domestic birds including humans. HPAI H5N1 \n\nalone has been reported from over 77 countries [40, 41]. \n\nTill the date, a total of 860 human cases have been \n\nreported since 2003 with more than 50% deaths by \n\nH5N1[42]. Since 2013, 1,568 human cases and 616 deaths \n\nwere reported worldwide attributed to the novel H7N9 \n\n[43]. Geographically the disease is globally prevalent \n\ndominating the Asia continent showing the higher \n\noutbreaks in China, Vietnam, India, Taiwan, Israel, \n\nJapan, and South Korea [26]. \n\nHost range, transmission, and spread \nAIV is found distributed in a wide variety of hosts. \n\nPredominantly free-flying water birds, like geese, ducks, \n\nshorebirds, and gulls, are major reservoirs of AIVs. \n\nBesides this, AIV can infect both wild and domestic birds \n\nlike chicken, turkeys, partridges, pheasants, quails, \n\npigeons, and ostriches [44]. However, the AIVs primarily \n\nassociated with transmission in chickens but not in ducks \n\nwere found to be adapted to ducks by acquiring genes \n\nfrom duck influenza viruses [45]. AIV is essentially a \n\ndisease of birds, but evidence has shown that the \n\ninfection can be transmitted in cats, dogs, eagles, ferrets, \n\nhamsters, horses, humans, macaques, marine mammals, \n\nmice, minks, pigs, and tigers; but the zoonotic infection \n\nhad been only reported in China [46]. The transmission \n\nand spread of disease were primarily related to poultry \n\ncontact, yet human-to-human transmission is also \n\npossible [47]. Moreover, the outbreak of avian flu is also \n\nassociated with the vicinity of water source and 97.5% of \n\nreported cases showed the proximity of water source \n\n[26]. \n\nVirus replication \nThe influenza A virus genome consists of segmented \n\nRNA encoding 10 proteins [48]. These 10 viral proteins \n\nare necessary for a successful infection cycle within \n\nimmuno-competent hosts. On the surface of the host \n\ncells, the HA protein binds to sialic acid, enabling the \n\nvirus to enter [49]. The host cell endocytoses the virus \n\nparticles. The endosome maturation lowers the pH \n\ntriggering a HA conformational change. This results in \n\nthe membrane fusion of the endosome and the virion. \n\nThe viral matrix protein-2 (M2) acts as an ion channel that \n\nfurther lowers the pH of the virus particle. This aids in \n\nthe dissociation of the matrix protein-1 (M1). The virus \n\nribonucleoproteins (vRNPs), including polymerase basic-\n\n1 (PB1), polymerase acidic (PA), and polymerase basic-2 \n\n(PB2), are released into the cytosol [50]. For viral \n\nreplication and transcription, the vRNPs are then moved \n\ninto the host cell nucleus. The nuclear export protein \n\n(NEP) and M1 traffic out freshly produced vRNPs into \n\nthe cytoplasm and then to the plasma membrane after \n\nreplication, transcription, and protein synthesis. Several \n\nviral proteins, including M1 and M2, contribute to \n\nbudding. Finally, in both the viral and host cell \n\nmembrane, NA removes sialic acid from glycoproteins. \n\nThis stops HA and host cells from interacting, releasing \n\nnew infective virus particles [1]. Nonstructural protein 1 \n\n(NS1) counteracts the innate host-cell defense within the \n\ninfected cell, including interferon, for efficient virus \n\nreplication [51]. New proteins generated by co-\n\ntranscription or co-translation with the host are being \n\ncontinuously identified [52].", "start_char_idx": 10365, "end_char_idx": 14032, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "650d21df-b775-410e-aa17-d851f4548125": {"__data__": {"id_": "650d21df-b775-410e-aa17-d851f4548125", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0fd4414f-3ea2-401b-8676-36e72de67ef3", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "9ee96c2da5a705ed1cb2d5d7141f2cc48dc174a314f28c12a1a767cfa41b9154", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ed9377c3-4a4d-45b6-860c-a92d5ab9a53a", "node_type": "1", "metadata": {}, "hash": "639f82dd3d9e367e1b266234b3aa5ca2b7ec709455f228ba37ce7cfb8abc02e8", "class_name": "RelatedNodeInfo"}}, "text": "Pathogenesis  \nVirulence factors comprise the highly cleavable HA that \n\nseveral cellular proteases can activate, a specific \n\nsubstitution in the polymerase basic protein-2 enhances \n\nviral replication [53]. Non-structural protein-1 \n\nsubstitution confers improved resistance to interferons \n\nand tumor necrosis factor-alpha inhibition [54]. The \n\nH5N1 viruses potently induce proinflammatory \n\ncytokines in macrophages, the most notable being tumor \n\nnecrosis factor-alpha, which may contribute to the \n\nunusual severity in humans [55]. With antigenicity \n\nchange [56], constellation of internal gene, expanded \n\nrange of avian species [57], enhanced pathogenicity [58], \n\nand increased environmental stability, these viruses keep \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  27 \n\nevolving. Despite extensive exposure to infected poultry, \n\ncomparatively low frequencies of H5N1 infection in \n\nhumans suggest that the species barrier to acquisition of \n\nthis avian virus is important [33].  \n\nDisease in birds \nAvian flu has been reported in many wild and domestic \n\nbirds. Chickens, ducks, swans, gooses, quails, crows, \n\nstorks, etc. have been known to have been infected with \n\nAIV. It is understood that H5N1 infections show distinct \n\nclinical presentations in chickens and ducks. The \n\ninfection is characterized by clinical signs and high \n\nmortality in chickens, whereas the infection is generally \n\nasymptomatic in ducks. This leads to an underestimation \n\nof the prevalence of the disease [59]. Infected ducks can \n\nthus help to sustain and spread the virus \u201csilently\u201d to \n\nother vulnerable hosts [60]. The H5N1 infection in ducks \n\nhas been considered a threat to the poultry flock and \n\npublic health due to its silent nature [57]. Thus, \n\nmonitoring and surveillance based on ducks is \n\nsubstantial for AIV control [61]. Since ducks may become \n\ninfected and co-infected with multiple AIVs, re-\n\nassortment of the viral genes is likely [62]. Spatial \n\nevidence on HPAI outbreaks in Southeast Asia has \n\nshown that scavenging ducks lead to HPAI outbreaks in \n\ndomestic poultry [63]. Evidence shows that ducks play a \n\nmajor role in the transmission of HPAI since they do not \n\nexhibit the disease symptoms. \n\nCharacterization of virus  \nIn 1996, the HPAI H5N1 virus was found in Gundong \n\nprovince, China which was thought to have originated \n\nfrom the H5 virus in migratory birds through both drift \n\nand shift mutation mechanisms [64]. Since then the \n\ninfluenza virus in human cases has evolved and is \n\nclassified in a different list based on hemagglutinin \n\nphylogeny known as a clade. This list of the clade is \n\nupdated for continuous change [65]. There are 13 major \n\nclades in the list distinguished by the hemagglutinin gene \n\nof the H5N1 representative subtype of HPAI. These \n\nclades are determined by sequencing and measuring the \n\naverage pairwise nucleotide distance (APD) [66]. These \n\nclades were further split by measuring the within-clade \n\naverage pairwise distance to determine the sub-clade \n\nresulting in more than 32 clades or sub-clades [67]. \n\nClade 0 comprises viruses that were first identified to \n\ncause human infections in Gundong province, China \n\n1996 (A/goose/Guandong/1/96 linage). In the early \n\nphase of epidemic (2004-2005), clade 1 viruses \n\npredominated in Vietnam, Thailand, and Cambodia, and \n\nclade 2.1 viruses are endemic in Indonesia. The clade \n\n2.1.3.2b was found to be restricted to a certain \n\ngeographical area i.e. Java, Indonesia [68].", "start_char_idx": 14035, "end_char_idx": 17584, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ed9377c3-4a4d-45b6-860c-a92d5ab9a53a": {"__data__": {"id_": "ed9377c3-4a4d-45b6-860c-a92d5ab9a53a", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "650d21df-b775-410e-aa17-d851f4548125", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "acbd51576771ad412de56d211ddfa2861ce6a1b57b068a46637e96f583368434", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4abe46b6-943a-42db-9c12-61a312c11a21", "node_type": "1", "metadata": {}, "hash": "80fc16bd5a452f3a64cf64ad43dcbc830727c8f161b6c4db00c1bde69bc93e24", "class_name": "RelatedNodeInfo"}}, "text": "Java, Indonesia [68]. A major \n\noutbreak of H5N1 disease in migratory birds has been \n\nassociated with clade 2.2 and the virus is distinct from Z \n\ngenotype virus and was first detected in Russia, \n\nKazakhstan and later spread, causing avian disease in \n\nAfrica, Central Asia, Europe, the Middle East, South Asia, \n\nand human disease in western Africa, Asia, and the \n\nMiddle East resulting pandemic in 77 countries [69].  In \n\nsouthern China, clade 2.3 has been dominant and has also \n\nbeen reported in adjacent countries.  Later on, clade \n\n2.3.2.1c was detected in Canada and Austria. Clade 7 was \n\nrestricted to China and Vietnam in the year 2006-2009 \n\n[70]. \n\nThe pattern of viral replication \nThe characterization of virologic course of AIV H5N1 is \n\nyet to be done. Many studies reported prolonged viral \n\nreplication. AIV can be detected in nasopharyngeal or \n\nlower respiratory sites ranging from 1 to 16 days. \n\nNasopharyngeal replication has been lower in humans as \n\ncompared to lower respiratory tract replication [71]. AIV \n\nhas also been documented to replicate in the \n\ngastrointestinal tract [72, 73]. The invasive infection has \n\nbeen documented in humans [72]. The HA cleavage site\u2019s \n\npolybasic amino acid sequence is linked with visceral \n\ndissemination in birds [33]. \n\nHost immune response (Innate type) \nDespite widespread exposure to infected poultry, the \n\ncomparatively low disease frequencies in humans \n\nillustrate the species barrier of AIV [33]. H5N1\u2019s ability to \n\ninfect birds or humans tends to be partially determined \n\nby the HA binding specificity. Generally, HAs of human \n\nstrains of influenza virus preferentially bind sialic acids \n\nbound to the terminal galactose of the oligosaccharides \n\non the cell surface by \u03b1 2,6 linkage (SA \u03b1 2,6), which is \n\nabundant in human respiratory epithelia [74]. The HA of \n\navian strains, on the other hand, binds preferentially to \u03b1 \n\n2,3-linked sialic acids (SA \u03b1 2,3). These linkages are \n\ncommon in the avian intestinal tract [75]. For host cell \n\ninfection and the dissemination and virulence of \n\ninfluenza viruses, \n\nFor host cell infection and the dissemination and \n\nvirulence of influenza viruses, HA interaction with \n\nsialylated glycans on the cell surface is important [76]. \n\nMutations altering the specificity of the receptor binding \n\nof avian viruses may be essential for avian to human \n\ncrossover, and for enabling direct human-to-human \n\ntransmission [77]. Pathogenesis of disease is contributed \n\nby the innate immune response [33]. Elevated blood \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  28 \n\nlevels of interleukin-6, interleukin-8, interleukin-1 b, \n\nsoluble interleukin-2 receptor, tumor necrosis factor \n\n(TNF), TNF-a, interferon-g, monokines induced by \n\nchemokines interferon-inducible protein 10, interferon-g, \n\nand monocyte chemoattractant protein 1 were observed \n\nin the fatal cases [71, 78]. Such responses can be attributed \n\nto the sepsis syndrome, acute respiratory distress \n\nsyndrome (ARDS), and multiorgan failure. \n\nCorticosteroids are used to minimize such responses. \n\nIndividuals developing specific humoral immune \n\nresponses can survive [33]. \n\nTransmission \nTransmission of AIV occurs through direct contact via the \n\ninhalation of droplets and droplet nuclei. Transmission \n\nalso occurs perhaps through indirect (fomite) contact and \n\nself-inoculation onto the upper respiratory tract or \n\nconjunctival mucosa [79]. The data is consistent with \n\nbird-to-human, potentially environmental-to-human, \n\nand limited human-to-human transmission to date. \n\nAnimal to human transmission is mainly attributed to \n\nexposure to live ill poultry and butchering of birds [80].", "start_char_idx": 17563, "end_char_idx": 21302, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4abe46b6-943a-42db-9c12-61a312c11a21": {"__data__": {"id_": "4abe46b6-943a-42db-9c12-61a312c11a21", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ed9377c3-4a4d-45b6-860c-a92d5ab9a53a", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "e6cd4eee82f32bfd3074dae56abc707e7be4e29e4433fa6b5804ac61ea8c62dd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f1bf225a-853d-4fdb-b946-0520c4101af5", "node_type": "1", "metadata": {}, "hash": "0257272980209218cc73c251db1aefef42f288f3e923b69d3ab5298256b1f564", "class_name": "RelatedNodeInfo"}}, "text": "In addition to plucking and preparing infected birds, \n\nhandling of fighting cocks, handling poultry notably \n\nasymptomatic infected ducks, and eating of undercooked \n\npoultry or duck\u2019s blood has also been proposed for \n\ntransmission of AIV. Also, feeding tigers and leopards in \n\nzoos with raw, infected chickens has been proposed for \n\ntransmission of AIV [81]. Human-to-human transmission \n\nhas also been manifested in recent years. But no case of \n\nhuman-to-human transmission through aerosols of small \n\nparticles or through social contact has been reported. \n\nStill, severe illness has been reported in clinicians \n\nexposed to an infected patient. The survival of H5N1 in \n\nthe environment outlines the possible mode of \n\ntransmission. Direct intranasal or conjunctival \n\ninoculation, and oral intake of contaminated water \n\nduring swimming is the possible mode of transmission. \n\nAlso, contamination of hand and subsequent self-\n\ninoculation is the possible mode of transmission. The use \n\nof untreated poultry feces as fertilizer has increased the \n\nrisk of environmental transmission [82]. \n\nClinical features \n\nIncubation: \nThe incubation period of H5N1 infection is 2\u20134 days after \n\nexposure to infected, sick, or dead poultry [83]. But \n\nincubation time can be prolonged up to 8 days [33, 84]. \n\nThe degree of virus shedding during this time is still \n\nunknown [85-87]. \n\nInitial symptoms \nThe most common and initial symptoms that appear after \n\nH5N1 infection is respiratory distress. Milder cases show \n\nuncomplicated flu-like illness [88]. Pneumonia is often of \n\nviral origin with no bacterial superinfection, but may \n\ndiffer from cases to cases [78]. Crackles on examination of \n\nthe chest are also seen [89]. Other frequently occurring \n\nsymptoms include a high fever with >38\u00b0C, sore throat, \n\ncough, shortness of breath, rhinorrhoea, etc. [33]. Some \n\ncases suffer from diarrhea, vomiting, and abdominal pain \n\napart from typical clinical manifestation [90]. \n\nConjunctivitis or upper respiratory infections are not \n\ncommon [87]. Further complication includes multi-organ \n\nfailure like renal disorder, cardiac malfunction, and \n\npulmonary hemorrhage. Reye\u2019s syndrome, \n\npneumothorax, ventilator-associated pneumonia, and \n\nARDS are also seen in some cases [33]. Though central \n\nnervous system involvement is rare there are cases where \n\nconvulsion and progressive coma are reported leading to \n\nthe death if not treated on time [90, 91]. Two patients in \n\nVietnam were detected with encephalitis only [92]. \n\nClinical course \nA patient infected with H5N1 has a rapid clinical course, \n\ndeveloping progressive lower respiratory tract disease \n\nand viral pneumonia. Mechanical ventilation may be \n\nneeded depending upon the severity of the case [33]. \n\nARDS following the multi-organ failure develops in 68% \n\nof patients within six days of disease onset [91]. The \n\naverage duration from onset of disease to hospital \n\nadmission is four days and from onset to death in critical \n\ncases is nine days [93]. Few cases of seropositive patients \n\nbut without typical manifestation are also reported [94]. \n\nMortality/Morbidity \nThe mortality rate of H5N1 is significantly high and is at \n\nan alarming rate accounting for about 60% [42]. But the \n\nrate may increase up to 73% in the youth of the age group \n\n10-19 years followed by 18% in the age group 50 years \n\n[95]. The mortality rate was even 90% in severe cases [96].  \n\nMost of the death cases are frequent with late admission \n\n[97, 98].  \n\nDiagnosis  \n\nSpecimen for upper respiratory tract \n\ninfections   \n\nNasal swab/nasal wash \nPolyester or Dacron swab is recommended with an \n\naluminum or plastic shaft. The dry swab is inserted in the \n\ninternal nares below the inferior turbinate. It is then \n\nallowed to absorb secretion for some time and is rotated \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.", "start_char_idx": 21305, "end_char_idx": 25216, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f1bf225a-853d-4fdb-b946-0520c4101af5": {"__data__": {"id_": "f1bf225a-853d-4fdb-b946-0520c4101af5", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4abe46b6-943a-42db-9c12-61a312c11a21", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "44d087f044a01da663542270e9ae74e006a64fbd5b2c6591183d2c0f197be968", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8e58da4e-d2f6-4dcf-9c2f-4ab88112f92a", "node_type": "1", "metadata": {}, "hash": "691813b41832dcbb7e3a0ce5cfde8b4d55c6f02112e043d562c321ef56d38b96", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  29 \n\naround the area before the withdrawal. The tip is then \n\nbroken and dipped in viral transport media (VTM) vial. \n\nFor nasal wash, physiological saline is used [99]. The \n\npatient is advised to close the pharynx by saying the letter \n\n\u201cK\u201d and the saline is introduced in nostrils one at a time \n\nwith the head tilted backward and later the saline is \n\ncollected in a cup or dish by tilting the head forward. The \n\nobtained wash is then mixed with the VTM [100]. \n\nThroat swabs \nA throat swab is considered to be the high yielding \n\nspecimen for upper respiratory tract infections and \n\nshould be obtained within three days after symptoms \n\nappeared for the best result [100]. By using the tongue \n\ndepressor or blade, polyester or Dacron swab is gently \n\nrubbed several times in the posterior pharynx. The tip is \n\nthen broken and dipped in a VTM vial [99]. \n\nNasopharyngeal secretions  \nA catheter with a mucus trap is inserted in nostrils. By \n\nuse of a vacuum, the catheter is withdrawn with a \n\nrotating motion. After that, the catheter is flushed with a \n\ntransport medium in a 1:2 ratio [99].  \n\nSpecimen for lower respiratory tract infections   \nAn endotracheal aspirate or bronchoalveolar lavage is the \n\nbest specimen for lower respiratory tract illness. For \n\nincreasing the potential of viral isolation, multiple \n\nspecimens can be taken from multiple respiratory sites \n\nfor at least two consecutive days [72]. \n\nBlood samples  \n Both acute and convalescent serum samples are \n\nrecommended if feasible. Within the first 3-5 days after \n\nthe onset of symptoms, acute serum samples should be \n\ntaken. Convalescent serum samples collected after 3-4 \n\nweeks would be of great use if collected in conjunction \n\nwith acute samples [99, 101]. \n\nOther specimens \nIt may be either plasma or rectal swab in diarrhea for the \n\ndetection of viral. Spinal fluid/ tap is also preferred in the \n\ncase of meningitis [100]. Autopsy specimens along with \n\npara-mortem biopsies are also recommended [28]. \n\nTransportation and storage of specimens \nVTM contains balanced salt solutions, a protein \n\nstabilizer, bovine albumin, and a spectrum of antibiotics \n\nto minimize bacterial and fungal growth. Non-phosphate \n\nbased VTM is selected if the specimen is only suggested \n\nfor the PCR test. The collected specimen is placed in VTM \n\nand refrigerated at 4\u00b0C or with refrigerated packs for \n\ntransportation to the designated laboratories [100]. \n\nThe clinical specimens should be well labeled and sent to \n\nthe laboratory as soon as possible. If specimens cannot be \n\nprocessed, then it should be either stored at 4\u00b0C or frozen \n\nat \u2264-70\u00b0C. Samples should not be repeatedly freezed0 and \n\nthawed [102]. In the case of viral antigen detection by \n\nimmunofluorescence staining, specimen processing \n\nshould be done as soon as possible not more than 1-2 \n\nhours after collection [83]. A guide for field operations is \n\nrecommended regarding the collection, storage, \n\nprocessing, and even transportation of samples for H5N1 \n\ndetection [100]. \n\nLaboratory diagnosis \nSince most of the respiratory infections show similar \n\nmanifestations, H5N1 can only be diagnosed in account \n\nwith endemic areas and contact with dead or infected \n\npoultry or with confirmed cases. \n\nDirect detection of virus or viral antigen \nVirus isolation or reverse transcriptase PCR (RT-PCR) \nVirus isolation is considered to be the gold standard \n\nmethod and can be carried out in the laboratory either by \n\ninoculation of embryonated eggs or by using cell lines \n\nlike Mardin-Darby Canine Kidney (MDCK). The \n\nobtained viral isolate can be later used for the study of \n\npathogenicity, antiviral resistance, and DNA sequencing \n\nand analysis. But culture needs a biosafety level 3 (BSL-\n\n3) which is not available at ease. So, RT-PCR is used as \n\nthe first diagnostic tool in contrast to culture [85, 86]. \n\nReal-time PCR or quantitative PCR (qPCR) \nqPCR methods are preferred to conventional RT-PCR.", "start_char_idx": 25220, "end_char_idx": 29199, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8e58da4e-d2f6-4dcf-9c2f-4ab88112f92a": {"__data__": {"id_": "8e58da4e-d2f6-4dcf-9c2f-4ab88112f92a", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f1bf225a-853d-4fdb-b946-0520c4101af5", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "e27a0a056a5c51ed15e2fba79524f78faa65c6279b599b7004e238be6518aa3d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f0b2d314-e09b-4eca-979c-292b55023d42", "node_type": "1", "metadata": {}, "hash": "c547a5e5b53ece91a37d29454be3a540c68e5365a577f24738389f4780177e6a", "class_name": "RelatedNodeInfo"}}, "text": "There is a panel of qPCR assays covering even the specific \n\ndetection of different NA genes and HA subtypes like H5, \n\nH3, and H1. H5 and N1 specific primers are used as they \n\nexclude false-negative results due to mutation in genes \n\n[72, 103]. Loop-mediated isothermal amplification \n\n(LAMP) tests are no longer in use [104, 105]. \n\nSerology \nThe Antigen detection \nFor the detection of viral antigens by using specific \n\nmonoclonal antibodies against H5 and N1 direct \n\nimmunofluorescence and enzyme immunoassay (EIA) \n\nare commonly used. The EIA method is considered \n\nsimple, convenient, sensitive, and even applicable as \n\nPoint of Care testing (POCT). The sensitivity of EIA is \n\n1,000-folds low compared to viral isolation. Thus, \n\nsubtype-specific diagnostic methods like RT-PCR should \n\nbe carried out simultaneously [106].   \n\nAntibody detection \nSince seroconversion is delayed and needs paired sera, \n\nantibody detection provides a retrospective scenario of \n\ninfection. It can be done by detecting a four-fold rise \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  30 \n\nwhile comparing acute and convalescent sera. \n\nHemagglutination Inhibition (HI) assay is conventional \n\nyet the preferred method. But HI has low sensitivity for \n\nthe detection of subtype-specific antibodies [107-109]. \n\nThe use of horse erythrocytes while detecting antibodies \n\nagainst H5N1 shows effective results [110]. One of the \n\nstudies done in 1997 during the Hong Kong outbreak \n\nreported similar results and reported that the micro-\n\nneutralization test is more effective, reliable, and \n\nsensitive as compared to HI [111]. Western blot analysis \n\nwith recombinant H5 is also done for the conformation \n\n[112-114]. \n\nHematological Profile  \n\nIt includes leukopenia, relative lymphopenia, and even \n\nthrombocytopenia. Disseminated intravascular \n\ncoagulation occurs but is rare [33, 84]. \n\nBiochemical parameters \n\nLevels of liver enzymes are elevated, along with lactate \n\ndehydrogenase and creatinine kinase, serum glutamate \n\noxaloacetate transaminase (SGOT) and serum glutamate \n\npyruvate transaminase (SGPT) [85, 86].  \n\nOthers \n\nBlood culture, sputum culture, and CSF analysis are \n\ncarried out in case of serious complications. Chest X-ray \n\nreveals effusions, multifocal consolidation, \n\nlymphadenopathy, and even shows diffuse ground-glass \n\nappearance as in the case of ARDS [71, 78]. \n\nDifferential diagnosis \n\nDiagnosis of AIV should be differentiated with atypical \n\npneumonia, community-acquired pneumonia (CAP), \n\ncorona-virus disease 2019 (COVID-19), endemic \n\nrespiratory infections, hantavirus pulmonary syndrome, \n\nmiddle east respiratory syndrome (MERS), pediatric \n\npneumococcal infections, pneumococcal infections \n\n(Streptococcus pneumoniae), respiratory syncytial virus \n\ninfection, seasonal influenza, and severe acute \n\nrespiratory syndrome (SARS) [83, 94, 115]. \n\nDefinition of exposures to poultry and \n\nhumans \nHuman exposure definition differs from the \n\nenvironments and the situation (Table 1). \n\n \n\nTable 1. Definition of exposures to poultry and humans [116].", "start_char_idx": 29202, "end_char_idx": 32333, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f0b2d314-e09b-4eca-979c-292b55023d42": {"__data__": {"id_": "f0b2d314-e09b-4eca-979c-292b55023d42", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8e58da4e-d2f6-4dcf-9c2f-4ab88112f92a", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "b57c5e41a85379d9978773bdb31841b94839a3f18e53948a61acfa6de74f233b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "714a11bb-6dd9-4608-a5bc-28ffae153efe", "node_type": "1", "metadata": {}, "hash": "414b9d0c136e3bbddf96f2a8fac21b6283a20395c3a6c766bdbd94dd08999a5b", "class_name": "RelatedNodeInfo"}}, "text": "Table 1. Definition of exposures to poultry and humans [116]. \n\nExposure to live poultry in occupations \n\n Poultry-related exposure at workplace (e.g., persons \n\nengaged with poultry raising, trafficking, selling, and \n\nquarantine) within 2 weeks of the start of symptoms \n\nExposure to poultry at live bird markets \n\n Visiting a live poultry or bird wholesale or retail market \n\nwithin 2 weeks before the start of symptoms \n\nExposure to sick or dead poultry \n\n Close or direct physical contact with infected or dead \n\npoultry or poultry products (e.g., meat) or feces within 2 \n\nweeks before the start of symptoms \n\nExposure to backyard poultry \n\n Close or direct contact with poultry raised in the backyard \n\nwithin 2 weeks before the start of symptoms \n\nAny exposure to poultry \n\n Close or direct or indirect contact to healthy, or sick, or dead \n\npoultry (including birds-e.g., chickens, ducks, geese, pet \n\nbirds, pigeons) in live bird markets, or backyards, or farms, \n\nor neighborhoods, or consumption of improperly processed \n\npoultry products \n\nExposure through patient contact \n\n A patient with a history of close contact within 2 weeks \n\nbefore the start of symptoms with a person with a confirmed \n\nor suspected influenza H5N1 virus infection (at any time \n\nfrom the day before the onset of symptom to death, or \n\nduring the period during which the patient was \n\nhospitalized) \n\nCase definitions [117, 118] \nFor prompt diagnosis and treatment, WHO has defined \n\nH5N1 infection as: \n\nPerson under investigation  \nA person who is decided to be investigated for possible \n\nH5N1 infection by assigned public health authorities  \n\nSuspected H5N1 case \nA person with fever (>38\u00baC) exhibiting unexplained acute \n\nlower respiratory distress along with cough, shortness of \n\nbreath, or dyspnoea.  \n\nAND \n\nIn the 7 days prior to symptom onset, one or more of the \n\nfollowing exposures: \n\na) Contact with a person within 1-meter distance during \ncaring, speaking, or touching whether s/he is a \nsuspected, probable, or confirmed H5N1 case. \n\nb) Exposure either to poultry in an area suspected or \nconfirmed with H5N1 infections not more than a \nmonth or wild animals during handling, slaughtering, \nde-feathering, etc. and even contact with the \ncontaminated feces. \n\nc) Intake of poultry products raw or undercooked in an \narea where animal or human H5N1 infections is \nreported or confirmed in the past month. \n\nd) Close contact with an animal infected with confirmed \nH5N1 other than poultry or wild birds (e.g. cat or pig)  \n\ne) Handling animal or human samples in a laboratory or \nother facility suspected of having the H5N1 virus. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  31 \n\nProbable H5N1 case (notify WHO) \nProbable definition 1: \n\nA person who satisfies the conditions for a suspected \n\ncase   \n\nAND  \n\nAdditional one of the following criteria: \n\na) Infiltrates or proof of acute chest radiograph \npneumonia, with proof of respiratory failure \n\n(hypoxemia, severe tachypnea)  \n\nOR  \n\nb) Confirmed laboratory influenza A infection but \ninadequate evidences in laboratory. \n\nProbable definition 2: \n\nA person dying from an unidentified acute respiratory \n\nillness who is known to be epidemiologically linked to a \n\nprobable or confirmed case of H5N1 by time, place, and \n\nexposure. \n\nConfirmed H5N1 case (notify WHO) \nA person who satisfies the condition of a suspected \n\nor probable case  \n\nAND  \n\nEither of the following findings in a national, regional, or \n\ninternational laboratory of influenza whose H5N1 test \n\nresults are recognized as confirmatory by WHO: \n\na) H5N1 virus isolation;  \n\nb) H5 PCR positive by tests using two separate \n\ntargets of PCR, e.g.", "start_char_idx": 32272, "end_char_idx": 35996, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "714a11bb-6dd9-4608-a5bc-28ffae153efe": {"__data__": {"id_": "714a11bb-6dd9-4608-a5bc-28ffae153efe", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f0b2d314-e09b-4eca-979c-292b55023d42", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "0d2ff76ff4906c544b390b4ebed1f07f1da7ed40e8a34f6fb2f7edd6a040bf3c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "05c9025f-24d0-4457-8527-7e0d72c0a59d", "node_type": "1", "metadata": {}, "hash": "50ba083f3f2001bc2297ba10250014423a987e26438e72051669527eb5530f20", "class_name": "RelatedNodeInfo"}}, "text": "influenza A and H5 HA \n\nspecific primers; \n\nc) On test of an acute serum sample (collected 7 days \n\nor less after the start of symptom) and a \n\nconvalescent serum sample, the H5N1 \n\nneutralization titer must be at least fourfold rise. \n\nThe convalescent neutralizing antibody titer must \n\nalso be at least 1:80; \n\nd) In a single serum sample collected at day 14 or \n\nlater after the start of symptom a \n\nmicroneutralization H5N1 antibody titer must be \n\nat least 1:80 and a positive in a different \n\nserological assay, for e.g. titer of at least 1:160 in a \n\nhorse red blood cell HI or western blot specific to \n\nH5. \n\nManagement/Treatment \nHospitalization \nH5N1 can be a serious disease in humans that needs \n\nhospitalization, isolation, and intensive care [119]. \n\nAntiviral agents \nThe primary therapy is the use of antiviral medication. \n\nThe WHO and CDC guidelines (2015) recommend use of \n\na neuraminidase inhibitor [92]. \n\nAmantadine \nAmantadine interferes with M2 protein and affects the \n\nrelease of infectious viral nucleic acid [120]. It is found to \n\nbe effective for both prophylaxis and short-term \n\ntreatment [121]. Since most of the H5N1 virus shows \n\nresistant patterns to amantadine or rimantadine, \n\ncombination therapy with oseltamivir is recommended \n\n[92]. \n\nRimantadine (brand name: Flumadine) \nIt inhibits uncoating affecting viral replication. But H5N1 \n\ndevelops resistance to it [92]. \n\nOseltamivir (brand name: Tamiflu) \nIt affects the receptor of host cells towards viral HA [92]. \n\nBut the spread of oseltamivir-resistant H5N1 from 2007 \n\nto 2009 surprised the influenza community [122]. Failures \n\nof treatment have been reported in single-drug \n\noseltamivir regimens due to resistance. There are current \n\nresearches on the relative success of high-dose and/or \n\nextended oseltamivir treatment courses [83]. If the use of \n\nhigh-dose tends to be more effective, it will affect the \n\nsupply of antiviral drugs in the case of a large epidemic, \n\nin addition to medical considerations for patients who are \n\nmoderately or seriously ill [92]. \n\nZanamivir (brand name: Relenza) \nIn an animal model, it shows better results but has not \n\nbeen tested in humans. But in severe cases, inhaled \n\nzanamivir may not be able to reach the distal airways [83, \n\n92].  \n\nUricosuric Agents \nProbenecid can be used as adjunctive therapy [92]. But no \n\nstudy has been carried out to confirm the suitable dose. \n\nStudies are still going on other drugs like arbidol and \n\nperamivir [123].  \n\nImmunomodulators \nLow dose corticosteroids are used to treat acute lung \n\ninjury (ALI)/ARDS caused due to H5N1 [83, 124]. But the \n\nlong term and high dose use may result in serious \n\ncomplications increasing the probability of opportunistic \n\ninfections [92]. Other immunomodulators used are non-\n\nsteroid anti-inflammatory drugs (NSAIDs), antipyretics, \n\ngrowth hormones, etc. But the use of such intervention \n\nmodalities has not been proven any significant benefits \n\n[125, 126]. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  32 \n\nMiscellaneous supportive therapy in a critical case \nThese include oxygen therapy, ventilatory support [127], \n\nand non-ventilatory treatments for ALI/ARDS [128].", "start_char_idx": 35997, "end_char_idx": 39244, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "05c9025f-24d0-4457-8527-7e0d72c0a59d": {"__data__": {"id_": "05c9025f-24d0-4457-8527-7e0d72c0a59d", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "714a11bb-6dd9-4608-a5bc-28ffae153efe", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "58b0e0ce8fe961e86ede764c43cc4a056b1222b87ba933e75d900b92c2ffa9a0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "92c7e1ec-617a-47d4-bc3c-522b60dabef6", "node_type": "1", "metadata": {}, "hash": "ed22d1abb0881e5e3a3aca7316a9d5202a5293e7eba10e84f264f2552d0bb1ca", "class_name": "RelatedNodeInfo"}}, "text": "Prevention and control \nImmunization \nBased on different studies, three vaccines of H5N1 were \n\nlicensed as Sanofi Pateur's vaccine, (monovalent killed \n\nvaccine approved by the US Food and Drug \n\nAdministration (FDA), United States), Glaxo Smith \n\nKline's vaccine-Prepandrix (approved by the European \n\nUnion), and CSL Limited's vaccine-Panvax (approved by \n\nAustralia) [129-131]. Even though none of the vaccines \n\nare available for the civilians yet [132]. Since the egg \n\ninoculation method is not applicable, tissue or cell culture \n\nand recombinant viruses are used for the production of \n\nvaccine and the adjuvants like aluminum hydroxide can \n\nbe used to increase immunogenicity [133]. The \n\ninactivated vaccines produced by using the H5N1 was \n\nfound to be immunogenic with high doses of HA [134]. \n\nBut due to rapid mutation, immunization is not quite \n\neffective and practicable [135]. For travelers, prophylaxis \n\nby antiviral is not recommended, instead avoiding \n\ncontact with dead birds and poultry, use of properly \n\ncooked food, and if needed use of N95 respirator mask, \n\ngoggles while traveling in outbreak areas is \n\nrecommended [136]. \n\nDisease management for preventing outbreaks \nThe most effective way to prevent an outbreak is to avoid \n\nthe source of exposure. People should avoid contact with \n\nthe wild as well as suspected domestic birds and even \n\nshould avoid contact with contaminated feces or surface. \n\nActive surveillance and strengthening biosecurity done \n\nin poultry workers, animals, and the environment in \n\nsuspected sites contribute to a great extent. [136]. One \n\nhealth concept or collaboration of different sectors play a \n\nvital role in early detection and prevention. Proper \n\nsupervision of animal transportation, inspection on the \n\nborder while transporting, and provision of isolation \n\nstations on different sites play a vital role [137]. Skilled \n\nmanpower in the field of veterinary to help with \n\nidentifying the diversity and emerging disease will help \n\nin early warning [136]. Stamping-Out, proper carcass \n\ndisposal, and surveillance and monitoring of animal \n\nhealth are needed [138]. Minimizing the occupational risk \n\nof exposure and vaccination of exposed poultry workers \n\nand animals should be prioritized [136]. \n\nPandemic preparedness \nPandemic preparedness can be implemented effectively \n\nonly if it is planned by a collaboration of global, federal, \n\nand state or local levels. It requires the involvement of \n\npublic health personnel, different health care \n\nprofessionals, researchers, and even private sectors to \n\nprepare effective measures for pandemic situations. \n\nThus, the instant response becomes possible that will \n\nhelp to tackle the pandemic scenario in a relevant way \n\n[139]. Pandemic preparedness includes mass \n\nsurveillance, continuous monitoring, early diagnosis, \n\nformulation of triage policies [140], development and \n\ndistribution of different medical interventions like \n\ntherapeutics, personal protective equipment (PPE), the \n\nrapid response of health care system, and proper \n\ninteraction [139].  \n\nPrecautionary measures for general publics \nAs a general precaution, avoiding contact with suspected \n\nwild birds, contaminated poultry products, \n\ncontaminated water, and proper hand hygiene is \n\nrecommended for the public. Besides, awareness \n\nprograms on the public level regarding the transmission \n\nand different preventive measures should be made \n\navailable by the government [136, 138].  \n\nHospital infection control  \nSpecific measures taken in a hospital setting not only \n\nprotect the health care workers but also guide them to \n\ncontrol the infection that in turn prevents the spread of \n\ndisease in the community. Health workers must ensure \n\nthe environment cleaning, proper disinfection and must \n\nbe trained about occupational hazards and various \n\nmodes of virus transmission [141].  N95 masks are more \n\neffective than multiple surgical masks. Pre-exposure \n\nprophylaxis should be considered if there is evidence of \n\ntransmission of the virus or likely to be at risk of exposure \n\n[33, 142]. Chemoprophylaxis by oseltamivir is \n\nrecommended for a person at high risk. The appropriate \n\ndose is 75mg of oseltamivir, a single dose for 7-10 days \n\n[143, 144].", "start_char_idx": 39247, "end_char_idx": 43519, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "92c7e1ec-617a-47d4-bc3c-522b60dabef6": {"__data__": {"id_": "92c7e1ec-617a-47d4-bc3c-522b60dabef6", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "05c9025f-24d0-4457-8527-7e0d72c0a59d", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "108af3edb717e47961041514a408b093654dba9fec311334d5977ec7430585a8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8bbfcd2c-408d-4ee3-a997-78a8b9713127", "node_type": "1", "metadata": {}, "hash": "c7f08bf5f077717b61d32c16b2ef1b0d6768fed593b5bc284cc64c1950e768dc", "class_name": "RelatedNodeInfo"}}, "text": "Household and close contacts \nThere are many routes of transmission for the H5N1 virus \n\nas such there are different protective measures to avoid \n\nthe infection. Proper handwashing plays a significant \n\nrole in avoiding the self-inoculation of the virus, thus \n\npreventing the disease [145]. Besides, households with \n\nillness should follow extra specific measures of post-\n\nexposure chemoprophylaxis that include oseltamivir \n\n[146, 147]. \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  33 \n\nTable 3. Major events related to avian influenza in Nepal \n\n[16, 42, 150-154]. \n\nTime Major events \n\n2004 Surveillance of influenza started from Jhapa, \n\neastern Nepal. \n\nOct 2005 First report of serologic evidence of AIV \n\ninfection (H9N2) in poultry in Nepal. \n\n2009 Domestic ducks seropositive for antibodies \n\nagainst H5 and H9, but not H7. \n\n2009 With the deployment of Real-Time PCR (qPCR) \n\nat the National Public Health Laboratory \n\n(NPHL), a molecular diagnostic assay-based \n\ninfluenza surveillance was initiated. \n\nJan 2009 The first case of H5N1 detected in the Jhapa \n\ndistrict, eastern Nepal \n\nJun 2009 During the 2009 pandemic influenza virus \n\noutbreak, detection and molecular \n\ncharacterization of pandemic influenza virus A \n\nH1N1 in a human specimen obtained at \n\nTribhuvan International Airport. \n\nOct 2009 In Kathmandu valley, the community spread of \n\npandemic H1N1 2009 was identified. \n\nApr \n\n2010 \n\nHighly equipped National Influenza Centre was \n\nestablished at NPHL. \n\nApr \n\n2011 \n\nFor the isolation and characterization of \n\ninfluenza and parainfluenza viruses, the Madin \n\nDarby Canine Kidney (MDCK) cell line was \n\nsuccessively cultured and propagated at NPHL. \n\n  \n\nJun 2011 From a clinical specimen obtained and stored at \n\nNPHL, the influenza virus was isolated and \n\ncharacterized successively. \n\nNov \n\n2011 \n\nThe NPHL isolated and characterized a total of \n\n28 influenza viruses and sent them to the WHO \n\nCollaborating Centre (WHOCC), National \n\nInstitute of Infectious Diseases, Japan. \n\nMar \n\n2019 \n\nThe first human death by H5N1 in Nepal. \n\nAvian influenza in the context of Nepal \nEpidemiology and outbreaks history \nNepal had not observed HPAI H5N1 until 2009, although \n\nadjoining India and China reported several episodes of \n\noutbreaks. In Jhapa, a district bordering India and close \n\nto Bangladesh, the first HPAI outbreak was identified in \n\nJanuary 2009. Although Nepal has undergone sporadic \n\nHPAI outbreaks since January 2009, until January 2012, \n\nno clinical outbreaks were identified in the capital city of \n\nKathmandu. As per the National Agriculture census \n\n2011, Nepal has a population of domestic poultry of \n\naround 53.6 million birds (Table 2). The 45% of this \n\npoultry are commercial birds and 55% are backyard \n\nbirds. In Chitwan and Kathmandu Valley, the majority of \n\nthe layers and broilers are raised. There were an \n\nestimated 58 hatcheries and 800 poultry producers in \n\nNepal [148]. Most of the parent stocks are imported to \n\nNepal from foreign countries. Occasional HPAI \n\noutbreaks has occurred in some of these stocks. In most \n\nof these countries, parent flocks are, however, subjected \n\nto strict biosecurity measures and national HPAI \n\nsurveillance. It is extremely unlikely that parent stocks \n\nare contaminated with the HPAI virus. The import from \n\nTibet is negligible. However, smuggling from India poses \n\na greater risk of HPAI infected parent stock across the \n\nborder. Also, there are minimal biosecurity practices \n\nacross the industry, and migratory wild birds also use the \n\nsame bodies of water used by domestically raised ducks.", "start_char_idx": 43522, "end_char_idx": 47185, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8bbfcd2c-408d-4ee3-a997-78a8b9713127": {"__data__": {"id_": "8bbfcd2c-408d-4ee3-a997-78a8b9713127", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "92c7e1ec-617a-47d4-bc3c-522b60dabef6", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "53a86b7fd139210d342882af87519531b35f8140cdb7e98c9e9d99c409e8d2f0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "510efb03-431e-4187-bbbb-ff6ca73efefd", "node_type": "1", "metadata": {}, "hash": "6b0f5ba70316ac29681081ec9f58e9578ceb37a21b3ca1afc3a7fb0f9a95aa1f", "class_name": "RelatedNodeInfo"}}, "text": "With arrays of wildlife reserves and national parks, \n\nmigratory birds and wild birds often visit the water \n\nbodies in Nepal [149].  \n\nTFigure 2: Map of Nepal showing risk districts and wild water \n\nbird zones. Level of risk as represented by a different color: \n\nBlack color represents the 27 high-risk districts, blue color \n\nrepresents the 18 medium-risk districts, white color represents \n\nthe 32 low-risk districts, 6 green bubbles represent the wild \n\nwater bird zones [Figure adapted from Reference 149].  \n\nBased on the national HPAI surveillance plan, \n\nKathmandu and Chitwan are identified as high-risk \n\ndisease areas in Nepal (Figure 2). The high-risk \n\ndesignation is based on the higher commercial poultry \n\ndensity, the higher poultry influx from other districts,  \n\nTable 2. Poultry population and products in Nepal [148]. \n\nPoultry Population \n\nFowl 45,171,185 \n\nDucks 376,916 \n\nLaying hens 7,907,468 \n\nLaying ducks 174,978 \n\nMeat Weight in metric tonnes \n\nChickens 40,346 \n\nDucks 217 \n\nEggs Numbers in 1000 \n\nHens 788,310 \n\nDucks 13,060", "start_char_idx": 47188, "end_char_idx": 48243, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "510efb03-431e-4187-bbbb-ff6ca73efefd": {"__data__": {"id_": "510efb03-431e-4187-bbbb-ff6ca73efefd", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8bbfcd2c-408d-4ee3-a997-78a8b9713127", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "6477bab1cfcbadee03a6c75bd6cae620950e597755e41a7fd2c18120c87f7a64", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1444b384-ffbe-49db-920b-d119fb240e5b", "node_type": "1", "metadata": {}, "hash": "c93dfbd43f91660cefe23fdcdabbf7b27221420d1e1e8b68366ba9a233b65729", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  34 \n\nthe larger free-ranging ducks, the larger natural and man-\n\nmade ponds and lakes visited by migratory birds \n\nannually, the existence of live bird markets, and the lower \n\nbio-security of commercial poultry farms [16, 153]. Since \n\nthe first outbreak of 2009, 256 outbreaks have been \n\nreported to OIE by Nepal. Outbreaks peaked during 2012 \n\nfollowed by silent 4 years. Outbreaks have been \n\nresurgent in recent years. Most of the infected population \n\nincludes commercial broilers and commercial layers \n\n(Table 4) (Figure 3) [16]. Recently Epidemiology and \n\nDisease Control Division, Ministry of Health and \n\nPopulation confirmed the first human death by H5N1 on \n\n2nd May 2019. The 21 years old male was admitted on 24th \n\nMarch 2019 and died on 29th March 2019. It has been the \n\nfirst and the only case of identified human death by \n\nH5N1 infection in Nepal [42, 154]. \n\nIsolation and study of influenza in \n\nhuman/birds/poultry \nNational Influenza Centre at National Public Health \n\nLaboratory (NPHL) is currently recognized by WHO and \n\nso is a member of the WHO Global Influenza Surveillance \n\nNetwork. Initially, influenza viruses were detected in \n\nsuspected cases by Rapid Diagnostic Test (RDT), but now \n\nqPCR is employed. The center is primarily focused on \n\ncollecting appropriate clinical specimens from patients, \n\nstoring, transporting, and processing. Initial \n\nidentification of virus type and subtype is done and \n\nisolates are forwarded to the WHO Collaborating Centre \n\nfor Reference and Research on Influenza and alert the \n\nWHO Global Influenza Programme [152].  \n\nFigure 3. Map showing avian influenza outbreak clusters since \n\n2009 in Nepal and its periphery [Figure adapted from Reference \n155]. \n\nTo date, 256 outbreaks have been reported by Nepal to \n\nOIE. Besides these notifications, several surveillance \n\nstudies have been done in a bird population in Nepal. The \n\nfirst report of serologic evidence of AIV infection in \n\npoultry in Nepal was reported in October 2005 which was \n\nlater determined to be the H9N2 subtype. This was clear \n\nevidence of the introduction of the virus in Nepal before \n\n2005. The antibodies to influenza A were detected in the \n\nTable 4: Avian influenza outbreak history in Nepal [16]. \n\nYears \nTotal \n\noutbreaks \n\nNew \n\noutbreaks \nDistrict Species Affected population Cases \n\nKilled and \n\ndisposed \n\n2019 15 \n\n14-Feb Makwanpur Birds Commercial layers 9,769 41,125 \n\n28-Feb Kathmandu Birds Layers 2,600 2,600 \n\n06-Nov Bhaktapur Birds Commercial broilers 1,535 3,961 \n\n17-Mar Kathmandu Birds House crow 200 0 \n\n2018 3 \n03-May Chitwan Birds Commercial layers 1,500 12,091 \n\n20-May Kathmandu Birds Ducks 270 240 \n\n2017 4 \n\n17-Feb Kaski Birds Chicken and ducks 98 297 \n\n01-Mar Sunsari Birds Commercial layers 3,650 2,550 \n\n02-Mar Sunsari Birds Swans and storks 15 0 \n\n2016 0 - - - - 0 0 \n\n2015 0 - - - - 0 0 \n\n2014 1 13-Feb Sunsari Birds Commercial layers 570 1,430 \n\n2013 0 - - - - 0 0 \n\n2012 210 27-Aug Lalitpur Birds Commercial layers 2,500 0 \n\n2011 13 10-Nov Bhaktapur Birds Chicken and ducks 88 308 \n\n2010 8 \n26-Jan Kaski Birds Chicken and ducks 153 11,128 \n\n25-Oct Chitwan Birds Commercial poultry 66 11,437 \n\n2009 2 08-Jan Jhapa Birds Poultry 14 24,689 \n\nTotal 256     23,028 111,856 \n\n\n\nNepal J Biotechnol.", "start_char_idx": 48248, "end_char_idx": 51615, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1444b384-ffbe-49db-920b-d119fb240e5b": {"__data__": {"id_": "1444b384-ffbe-49db-920b-d119fb240e5b", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "510efb03-431e-4187-bbbb-ff6ca73efefd", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "32bdbab4c87d051a2d7047b9e40ee664e52a0bc8d93409bc397d92352df9b04a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7ec31275-2d2e-4ebc-9518-5e29ffce4909", "node_type": "1", "metadata": {}, "hash": "0f0636f419b267e52667ff721b81d6f02933ac5cd0fd04c86d055b5a262d786f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  35 \n\nchicken while ducks and pigeons were still serologically \n\nnegative for the virus. This also outlines the absence of \n\nHPAI and notifiable H5 and H7 subtypes until that time. \n\nThis also proved the brewing adaptation and \n\nintroduction of another influenza A viruses in Nepal \n\n[150]. H5 subtype was not detected in 2007 by the RT-\n\nPCR test in suspected dead birds [156]. Similarly, \n\ndomestic ducks were found seropositive for antibodies \n\nagainst H5 and H9 but not against H7 in 2009. Though \n\nnone of the seropositive ducks were symptomatic for \n\nHPAI or LPAI virus infection [151]. H9N2 was reported \n\nin a fecal sample of migratory birds in southern Nepal. \n\nHowever, H7N9 and other HPAI viruses were not \n\ndetected [157]. Different studies reported pandemic \n\nH1N1 in Nepal [158, 159]. Pandemic H1N1 detected in \n\nNepal was the lineage of the novel influenza H1N1 virus \n\n(A/California/07/2009) [159]. \n\nHPAI virus has been a major threat to the Nepalese \n\npoultry industry since its outbreak in India in 2003. A \n\nsubstantial number of customers temporarily stopped \n\nbuying chicken meat and switched to other sources of \n\nmeat. The loss to entire value chain actors was estimated \n\nat NRs 1,154 million per year ($1=NRs. 73.06, the average \n\nexchange rate from 2001 to 2010) [160] from 2001 to 2010 \n\n[161]. Since the 2009 outbreak, the impacts have spilled \n\nacross the country. The economic loss is even \n\ndevastating. Loss of more than NRs 4.5 million has been \n\nattributed to the Pokhara bird flu outbreak alone, the \n\nthird outbreak in Nepal. The commercial and backyard \n\nfarmers including butchers on a small scale are primarily \n\naffected by the outbreak. Nepal government had tried to \n\ncompensate for the loss during the outbreak, but the \n\ncompensation offered is lower as compared to the gate \n\nprice of the poultry and products [161, 162]. \n\nOutbreak Preparedness in Nepal \nA pandemic imposes an increased pressure on \n\ninfrastructures to contain the disease, be it a developed \n\nnation or a developing nation. Developing nations face \n\nmuch more stress in resource mobilization which in the \n\ncase is already scarce. But in global context, pandemic \n\nhelp to better understand the disease and effects of \n\npreventive measures. Pandemic preparedness includes \n\nthe holistic approach from virologists, epidemiologists, \n\nanimal and human health professionals, military and \n\nparamilitary forces, press, media, and administrations. \n\nContinuous coordination among all parties is warranted \n\nfor the execution of swift countermeasures in case of a \n\nnew pandemic outbreak. But as is the case in most \n\ndeveloping countries, this co-ordination is still lacking in \n\nNepal [163]. Besides this, AIV is dynamically evolving \n\nand adapting to newer hosts so there is still the need to \n\nestablish tests that are quick and easy to use to \n\ncharacterize new influenza strains [164]. Cross country \n\nmovements of birds and products should strictly follow \n\nOIE recommendations. Farms should be designed to \n\nminimize the interaction with wild bird populations and \n\nthere should be maximum biosecurity practices [165-\n\n167]. \n\nPublic awareness level about the disease in \n\nNepal \nDifferent studies reported that most of the Nepalese \n\npopulation were aware of AIV and that poultry workers \n\nwere at risk of infection. Televisions, radios, newspapers, \n\nand social media had been conveying the message to the \n\ngeneral population about AIV. However, on preventive \n\nmeasures, only hand washing was widely accepted by \n\nmost folks even though the majority of the population \n\nwas familiar with most of the preventive behaviors. Thus, \n\nthere is a strong degree of acceptance of many specific \n\ngovernment regulation policies in public.", "start_char_idx": 51596, "end_char_idx": 55434, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7ec31275-2d2e-4ebc-9518-5e29ffce4909": {"__data__": {"id_": "7ec31275-2d2e-4ebc-9518-5e29ffce4909", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1444b384-ffbe-49db-920b-d119fb240e5b", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "685476b4b3ec46667b6e8a64205a8a5530d204e7f6a4c75a31cedd8673bd54c3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "32591215-e656-4afd-8708-00e66260d494", "node_type": "1", "metadata": {}, "hash": "a781922312b80d62179763a9c41da4c05d45ea5eb87fec7094d194a6eb086073", "class_name": "RelatedNodeInfo"}}, "text": "But the \n\nimplemented control measures were not considered \n\nsufficient even though preventive measures are \n\nfrequently conveyed by the government (Table 4) [168, \n\n169]. \n\nPolicy in controlling outbreaks in Nepal \nBetween 2007 and 2011, the World Bank-funded the AIV \n\nsurveillance and awareness campaign as the Avian \n\nInfluenza Control Project (AICP) in Nepal. Since 2011, \n\nNepal has continued the AICP with its internal resources. \n\nThe management program focuses largely on outbreak-\n\nassociated culling of poultry in tandem with washing and \n\ndisinfection. control policy is mainly focused on the \n\nTable 4: Excerpt of the preventive measures released by the \n\nMinistry of Health and Population, Nepal in the public \n\ninterest [154]. \n\nFrequent hand washing using soap. \n\nPersonal hygiene and cleanliness. \n\nEnvironmental cleanliness. \n\nAvoid close contact with sick poultry and wild birds. \n\nImmediately visit nearby health institutions on suspicion of \n\nill birds or persons in the vicinity. \n\nKeep children far away from dead and sick birds. \n\nSafe handling and personal safety should be considered \n\nwhile preparing poultry meat. \n\nThe handler should clean hands and tools after preparing \n\nmeat. \n\nConsume only well-prepared poultry meat (cooked to at \n\nleast 70\u00baC). \n\nDon\u2019t do drugs without consultation with physicians. \n\nAvoid close contact with dead birds or their droppings. Bury \n\nthe dead birds carefully if found. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  36 \n\noutbreak-related culling of poultry in conjunction with \n\ncleaning and disinfection. However, despite of current \n\nAICP, the continuing incidence of outbreaks has raised \n\nconcerns about the effectiveness of the money expended. \n\nPoultry and product export from Nepal is negligible. But \n\ndomestic demand is nearly self-sufficient, though parent \n\nstocks and vaccines are imported. Thus, it is crucial to \n\npreserve the poultry industries as a pandemic can wipe \n\nout the industry owing to the small geography of the \n\ncountry. Thus, AICP alone cannot be fully relied upon, \n\ninstead, the vaccination control program can be \n\nimplemented along with bio-security, as proven effective \n\nin Pakistan [170]. Nevertheless, there is always a \n\npossibility of the circulation of the asymptomatic virus \n\nand the future spread of infection [171]. \n\nControl and management strategies in Nepal \nThe control and management of AIV are primarily \n\nfocused on outbreak zone/s in Nepal. Once the outbreak \n\nis identified, the area is sealed and poultry production is \n\nbanned for 45 days. Surveillance is intensified in the \n\nproximity of epidemic regions and other regions at \n\nhigher risks. The suspected poultry are quarantined and \n\nculled. Since the first outbreak, 111,856 poultry have been \n\nkilled to control the further spread. Thus, occasional \n\noutbreaks in different areas have seriously hampered the \n\npoultry industry (Table 2). The control and management \n\nstrategies are thus targeted to outbreak areas only after \n\nthe outbreak has been identified, rather than focusing on \n\nsurveillance and adopting preventive measures in the \n\noutbreak prone and high-risk areas. The control \n\nmeasures were adopted by Nepal to curb AIV outbreaks, \n\nas reported to OIE (Table 5).  \n\nTable 5. Measures taken by Nepal to curb AIV outbreaks \n(as reported to OIE). \nControl of movement in the country \n\nSurveillance outside containment and/or protection zone \n\nSurveillance within containment and/or protection zone \n\nScreening \n\nTraceability \n\nQuarantine \n\nOfficial destruction of animal products \n\nOfficial disposal of carcasses, by-products, and wastes \n\nStamping out \n\nDisinfection \n\nVaccination prohibited \n\nNo treatment of affected animals \n\nConclusion \nMore than 20 years ago, H5N1 in humans was first \n\nidentified. The reservoirs and key sources of human \n\nH5N1 infections are infected birds. Human-to-human \n\ntransmission is very low and initial manifestations of \n\nillness are non-specific so detailed histories along with \n\npossible travel in endemic areas should be considered for \n\ncase detection. Outbreaks have been recurrent in recent \n\nyears in Nepal. Commercial rapid antigen tests are \n\ninsensitive and only suitable for screening.", "start_char_idx": 55435, "end_char_idx": 59702, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "32591215-e656-4afd-8708-00e66260d494": {"__data__": {"id_": "32591215-e656-4afd-8708-00e66260d494", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7ec31275-2d2e-4ebc-9518-5e29ffce4909", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "566fe4f567534668bc635f24366de4647e09ca35d52ca32e97d3f260aefef22c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fc009ffc-d027-4d49-a4be-29ffd22009f9", "node_type": "1", "metadata": {}, "hash": "966f3d81259ff4d42c53aa8e5b6737513e4474d9098c23a0c22021b7f7ca23d0", "class_name": "RelatedNodeInfo"}}, "text": "Commercial rapid antigen tests are \n\ninsensitive and only suitable for screening. Confirmatory \n\ndiagnosis can only be done by molecular techniques. \n\nOseltamivir has been warranted for the treatment of \n\nsevere cases. The understanding of epidemiology, \n\nnatural history, and human H5N1 disease control is still \n\ninadequate; thus, warranting the need for co-ordination \n\nin clinical and epidemiological research among \n\ninstitutions globally. \n\nAuthor\u2019s Contribution \nBB and HP compiled the literature review. DS and SS \n\nprepared the initial draft. DS finalized the draft. All \n\nauthors read and approved the final draft. \n\nCompeting Interest \nThe authors declare no conflict of interest or competing \n\ninterest. \n\nAcknowledgements \nNone \n\nReferences \n1. Dictionary M-W: Influenza. In: Merriam-Webster Dictionary. \n\nMerriam-Webster Corporation; 2019. \n2. Smith W, Andrewes CH, Laidlaw PP: A virus obtained from \n\ninfluenza patients. The Lancet 1933, 222(5732):66-68. \nhttps://doi.org/10.1016/S0140-6736(00)78541-2 \n\n3. Verhoeyen M, Fang R, Jou WM, Devos R, Huylebroeck D, Saman \nE, Fiers W: Antigenic drift between the haemagglutinin of the \nHong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. \nNature 1980, 286(5775):771-776. https://doi.org/10.1038/286771a0 \n\n4. Abolnik C: A current review of avian influenza in pigeons and \ndoves (Columbidae). Veterinary Microbiology 2014, 170(3):181-196. \nhttps://doi.org/10.1016/j.vetmic.2014.02.042 \n\n5. Tong S, Li Y, Rivailler P, Conrardy C, Castillo DAA, Chen L-M, \nRecuenco S, Ellison JA, Davis CT, York IA et al: A distinct lineage \nof influenza A virus from bats. Proceedings of the National Academy \nof Sciences 2012, 109(11):4269. \nhttps://doi.org/10.1073/pnas.1116200109 \n\n6. Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen \nX, Recuenco S, Gomez J et al: New world bats harbor diverse \ninfluenza A viruses. PLoS Pathog 2013, 9(10):e1003657-e1003657. \nhttps://doi.org/10.1371/journal.ppat.1003657 \n\n7. World organisation for Animal Health (OIE): Manual of \nDiagnostic Tests and Vaccines for Terrestrial Animals \n(mammals, birds and bees). In.; 2015. \n\n8. What is Avian Influenza. [https://www.oie.int/en/animal-\nhealth-in-the-world/avian-influenza-portal/what-is-avian-\ninfluenza/] \n\n9. Centers for Disease Control and Prevention (CDC): Spread of \nAvian Influenza Viruses among Birds, USA. In.; 2008. \n\n10. Roberton SI, Bell DJ, Smith GJD, Nicholls JM, Chan KH, Nguyen \nDT, Tran PQ, Streicher U, Poon LLM, Chen H et al: Avian \ninfluenza H5N1 in viverrids: implications for wildlife health \nand conservation. Proceedings of the Royal Society B: Biological \nSciences 2006, 273(1595):1729-1732. \nhttps://doi.org/10.1098/rspb.2006.3549 \n\n11. WHO Global Influenza Program Surveillance Network, World \nHealth Organization (WHO): Evolution of H5N1 avian influenza \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.", "start_char_idx": 59621, "end_char_idx": 62532, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fc009ffc-d027-4d49-a4be-29ffd22009f9": {"__data__": {"id_": "fc009ffc-d027-4d49-a4be-29ffd22009f9", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "32591215-e656-4afd-8708-00e66260d494", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "4cdca9e51f47b8224147dd012e8c0eb9341fc1ece12b755a4d8c09031dd42ae4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2550c7f8-5910-45cc-8b82-b59ed02f444c", "node_type": "1", "metadata": {}, "hash": "7d207bb29dba95a52caa08ae35c25140f0d28d1f683a42e78c33b0be295f4104", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  37 \n\nviruses in Asia. Emerg Infect Dis 2005, 11(10):1515. \nhttps://doi.org/10.3201/eid1110.050644 \n\n12. Melidou A, Gioula G, Exindari M, Chatzidimitriou D, Diza-\nMataftsi E: Influenza A(H5N1): an overview of the current \nsituation. 2009, 14(20):19216. \nhttps://doi.org/10.2807/ese.14.20.19216-en \n\n13. Giasuddin M, Alam J, Samad M, Al-Mamun M, Taimur J: Highly \nPathogenic Avian Influenza: Bangladesh Situation. Bangladesh \nLivestock Research Institute 2006. \n\n14. World Health Organization (WHO): Geographical spread of \nH5N1 avian influenza in birds-update 28. . In.; 2005. \n\n15. UN Office for the Coordination of Humanitarian Affairs (IRIN): \nNepal: bird flu returns. In.; 2011. \n\n16. World Animal Health Information Database (WAHIS Interface)-\nVersion 1 \n[https://www.oie.int/wahis_2/public/wahid.php/Countryinfo\nrmation/Countryreports] \n\n17. Morin CW, Stoner-Duncan B, Winker K, Scotch M, Hess JJ, \nMeschke JS, Ebi KL, Rabinowitz PM: Avian influenza virus \necology and evolution through a climatic lens. Environment \ninternational 2018, 119:241-249. \nhttps://doi.org/10.1016/j.envint.2018.06.018 \n\n18. Roche B, Lebarbenchon C, Gauthier-Clerc M, Chang CM, Thomas \nF, Renaud F, Van Der Werf S, Guegan JF: Water-borne \ntransmission drives avian influenza dynamics in wild birds: the \ncase of the 2005\u20132006 epidemics in the Camargue area. Infection, \nGenetics and Evolution 2009, 9(5):800-805. \nhttps://doi.org/10.1016/j.meegid.2009.04.009 \n\n19. Gilbert M, Xiao X, Robinson TP: Intensifying poultry production \nsystems and the emergence of avian influenza in China: a \u2018One \nHealth/Ecohealth\u2019epitome. Archives of public health 2017, 75(1):48. \nhttps://doi.org/10.1186/s13690-017-0218-4 \n\n20. Liu Q, Cao L, Zhu X: Major emerging and re-emerging zoonoses \nin China: a matter of global health and socioeconomic \ndevelopment for 1.3 billion. International Journal of Infectious \nDiseases 2014, 25:65-72. https://doi.org/10.1016/j.ijid.2014.04.003 \n\n21. Bui C, Bethmont A, Chughtai AA, Gardner L, Sarkar S, Hassan S, \nSeale H, MacIntyre CR: A systematic review of the comparative \nepidemiology of avian and human influenza A H5N1 and \nH7N9\u2013lessons and unanswered questions. Transboundary and \nemerging diseases 2016, 63(6):602-620. \nhttps://doi.org/10.1111/tbed.12327 \n\n22. de Wit E, Munster VJ, van Riel D, Beyer WEP, Rimmelzwaan GF, \nKuiken T, Osterhaus ADME, Fouchier RAM: Molecular \ndeterminants of adaptation of highly pathogenic avian influenza \nH7N7 viruses to efficient replication in the human host. Journal \nof Virology 2010, 84(3):1597-1606. \nhttps://doi.org/10.1128/JVI.01783-09 \n\n23.", "start_char_idx": 62536, "end_char_idx": 65138, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2550c7f8-5910-45cc-8b82-b59ed02f444c": {"__data__": {"id_": "2550c7f8-5910-45cc-8b82-b59ed02f444c", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fc009ffc-d027-4d49-a4be-29ffd22009f9", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "3bc075189e7968fbf2156ae0ec0c3fd71b91eb430510597d87c88fd035fbbd4b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "efd00087-9f75-4c5e-9694-a774c8658c5f", "node_type": "1", "metadata": {}, "hash": "4b2e71ac4ec9ff219754637675149c555752bbea557e7ef9407b95655b6bc769", "class_name": "RelatedNodeInfo"}}, "text": "Chen H, Smith GJD, Zhang SY, Qin K, Wang J, Li KS, Webster RG, \nPeiris JSM, Guan Y: H5N1 virus outbreak in migratory waterfowl. \nNature 2005, 436(7048):191-192. \nhttps://doi.org/10.1038/nature03974 \n\n24. Gilbert M, Jambal L, Karesh WB, Fine A, Shiilegdamba E, Dulam \nP, Sodnomdarjaa R, Ganzorig K, Batchuluun D, Tseveenmyadag \nN: Highly pathogenic avian influenza virus among wild birds in \nMongolia. PloS one 2012, 7(9):e44097. \nhttps://doi.org/10.1371/journal.pone.0044097 \n\n25. Shin JH, Woo C, Wang SJ, Jeong J, An IJ, Hwang J-K, Jo S-D, Do Yu \nS, Choi K, Chung HM: Prevalence of avian influenza virus in \nwild birds before and after the HPAI H5N8 outbreak in 2014 in \nSouth Korea. Journal of Microbiology 2015, 53(7):475-480. \nhttps://doi.org/10.1007/s12275-015-5224-z \n\n26. Chatziprodromidou IP, Arvanitidou M, Guitian J, Apostolou T, \nVantarakis G, Vantarakis A: Global avian influenza outbreaks \n2010\u20132016: a systematic review of their distribution, avian \nspecies and virus subtype. Systematic reviews 2018, 7(1):17. \nhttps://doi.org/10.1186/s13643-018-0691-z \n\n27. Arnal A, Vittecoq M, Pearce-Duvet J, Gauthier-Clerc M, Boulinier \nT, Jourdain E: Laridae: A neglected reservoir that could play a \nmajor role in avian influenza virus epidemiological dynamics. \nCritical reviews in microbiology 2015, 41(4):508-519. \nhttps://doi.org/10.3109/1040841X.2013.870967 \n\n28. Peiris JSM, de Jong MD, Guan Y: Avian influenza virus (H5N1): a \nthreat to human health. Clin Microbiol Rev 2007, 20(2):243-267. \nhttps://doi.org/10.1128/CMR.00037-06 \n\n29. Wrigley N: Electron microscopy of influenza virus. British medical \nbulletin 1979, 35(1):35-38. \nhttps://doi.org/10.1093/oxfordjournals.bmb.a071539 \n\n30. Influenza (Flu): Types of Influenza Viruses \n[https://www.cdc.gov/flu/about/viruses/types.htm] \n\n31. Guan Y, Peiris JSM, Lipatov AS, Ellis TM, Dyrting KC, Krauss S, \nZhang LJ, Webster RG, Shortridge KF: Emergence of multiple \ngenotypes of H5N1 avian influenza viruses in Hong Kong SAR. \nProceedings of the National Academy of Sciences 2002, 99(13):8950. \nhttps://doi.org/10.1073/pnas.132268999 \n\n32. Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, Sakai-Tagawa \nY, Muramoto Y, Ito M, Kiso M, Horimoto T et al: Haemagglutinin \nmutations responsible for the binding of H5N1 influenza A \nviruses to human-type receptors. Nature 2006, 444(7117):378-382. \nhttps://doi.org/10.1038/nature05264 \n\n33. The Writing Committee of the World Health Organization (WHO) \nConsultation on Human Influenza A/H5: Avian Influenza A \n(H5N1) Infection in Humans.", "start_char_idx": 65139, "end_char_idx": 67678, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "efd00087-9f75-4c5e-9694-a774c8658c5f": {"__data__": {"id_": "efd00087-9f75-4c5e-9694-a774c8658c5f", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2550c7f8-5910-45cc-8b82-b59ed02f444c", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "b0ff2a261a39139a1eccddef2ad67d689848c5f550b48878169696132fd4941c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5e8648c4-ca5d-4c9c-8f71-6788609b3221", "node_type": "1", "metadata": {}, "hash": "dedfe505887c5479ced4cc9d98cced8b3a42b04dd19857c5684071a41698af92", "class_name": "RelatedNodeInfo"}}, "text": "N Engl J Med 2005, 353(13):1374-\n1385. https://doi.org/10.1056/NEJMra052211 \n\n34. Boni MF: Vaccination and antigenic drift in influenza. Vaccines \n(Basel) 2008, 26(Suppl 3):C8-C14. \nhttps://doi.org/10.1016/j.vaccine.2008.04.011 \n\n35. Treanor J: Influenza Vaccine \u2014 Outmaneuvering Antigenic Shift \nand Drift. N Engl J Med 2004, 350(3):218-220. \nhttps://doi.org/10.1056/NEJMp038238 \n\n36. Webster RG, Laver WG, Air GM, Schild GC: Molecular \nmechanisms of variation in influenza viruses. Nature 1982, \n296(5853):115-121. https://doi.org/10.1038/296115a0 \n\n37. Hajjara SA, McIntoshb K: The first influenza pandemic of the 21st \ncentury. Ann Saudi Med 2010, 30(1):1-10. \nhttps://doi.org/10.5144/0256-4947.59365 \n\n38. Taubenberger JK: The Origin and Virulence of the 1918 \n\u201cSpanish\u201d Influenza Virus. Proc Am Philos Soc 2006, 150(1):86-112.  \n\n39. Waliya P, Gupta DK, Sheikh AA, Rashid R, Dar RB, Mishra A: \nAvian Influenza-A pandemic threat. The Pharma Innovation \nJournal 2017, 6(11):526-531. \n\n40. Avian Influenza \"at a glance\" [https://www.oie.int/en/animal-\nhealth-in-the-world/web-portal-on-avian-influenza/] \n\n41. Duan L, Bahl J, Smith GJD, Wang J, Vijaykrishna D, Zhang LJ, \nZhang JX, Li KS, Fan XH, Cheung CL: The development and \ngenetic diversity of H5N1 influenza virus in China, 1996\u20132006. \nVirology 2008, 380(2):243-254. \nhttps://doi.org/10.1016/j.virol.2008.07.038 \n\n42. World Health Organization (WHO): Information on Avian \nInfluenza A (H5N1) Identified in Human in Nepal. In.; 2019. \n\n43. Agriculture and Consumer Protection Department, Food and \nAgriculture Organisation of the United Nations: H7N9 situation \nupdate. In.; 2019. \n\n44. Kang HM, Lee EK, Song BM, Jeong J, Choi JG, Jeong J, Moon OK, \nYoon H, Cho Y, Kang YM: Novel reassortant influenza A (H5N8) \nviruses among inoculated domestic and wild ducks, South \nKorea, 2014. Emerg Infect Dis 2015, 21(2):298. \nhttps://doi.org/10.3201/eid2102.141268 \n\n45. Ma MJ, Yang Y, Fang LQ: Highly pathogenic avian H7N9 \ninfluenza viruses: recent challenges. Trends in microbiology 2019, \n27(2):93-95. https://doi.org/10.1016/j.tim.2018.11.008 \n\n46. World Health Organization (WHO): Influenza at the Human-\nAnimal Interface, 8 December 2018\u201325 January 2018. In.  \n\n47. Lazarus R, Lim PL: Avian influenza: recent epidemiology, travel-\nrelated risk, and management. Current infectious disease reports \n2015, 17(1):456. https://doi.org/10.1007/s11908-014-0456-3 \n\n48. Ozawa M, Kawaoka Y: Taming influenza viruses.", "start_char_idx": 67679, "end_char_idx": 70143, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5e8648c4-ca5d-4c9c-8f71-6788609b3221": {"__data__": {"id_": "5e8648c4-ca5d-4c9c-8f71-6788609b3221", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "efd00087-9f75-4c5e-9694-a774c8658c5f", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "1950840c314413c66a015004811bc82f27f8dfbed7b83ea48d78d4a048ffdff4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8c14defe-6774-4bd9-8e95-25305598bdc0", "node_type": "1", "metadata": {}, "hash": "825e13f8cf9bd7fececee8399f67b9f8a997a84964fd368e1434f78816b9435a", "class_name": "RelatedNodeInfo"}}, "text": "Ozawa M, Kawaoka Y: Taming influenza viruses. Virus Research \n2011, 162(1):8-11. https://doi.org/10.1016/j.virusres.2011.09.035 \n\n49. Matrosovich M, Stech J, Klenk HD: Influenza receptors, \npolymerase and host range. Revue Scientifique et Technique \n(International Office of Epizootics) 2009, 28(1):203-217. \nhttps://doi.org/10.20506/rst.28.1.1870 \n\n50. Basler CF, Aguilar PV: Progress in identifying virulence \ndeterminants of the 1918 H1N1 and the Southeast Asian H5N1 \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  38 \n\ninfluenza A viruses. Antiviral Res 2008, 79(3):166-178. \nhttps://doi.org/10.1016/j.antiviral.2008.04.006 \n\n51. Hale BG, Randall RE, Ort\u00edn J, Jackson D: The multifunctional NS1 \nprotein of influenza A viruses. 2008, 89(10):2359-2376. \nhttps://doi.org/10.1099/vir.0.2008/004606-0 \n\n52. Zhang H, Hale BG, Xu K, Sun B: Viral and Host Factors Required \nfor Avian H5N1 Influenza A Virus Replication in Mammalian \nCells. 2013, 5(6):1431-1446. https://doi.org/10.3390/v5061431 \n\n53. Shinya K, Hamm S, Hatta M, Ito H, Ito T, Kawaoka Y: PB2 amino \nacid at position 627 affects replicative efficiency, but not cell \ntropism, of Hong Kong H5N1 influenza A viruses in mice. \nVirology 2004, 320(2):258-266. \nhttps://doi.org/10.1016/j.virol.2003.11.030 \n\n54. Heui SS, Hoffmann E, Webster RG: Lethal H5N1 influenza \nviruses escape host anti-viral cytokine responses. Nature Medicine \n2002, 8(9):950-954. https://doi.org/10.1038/nm757 \n\n55. Cheung CY, Poon LLM, Lau AS, Luk W, Lau YL, Shortridge KF, \nGordon S, Guan Y, Peiris JSM: Induction of proinflammatory \ncytokines in human macrophages by influenza A (H5N1) \nviruses: a mechanism for the unusual severity of human disease. \nThe Lancet 2002, 360(9348):1831-1837. \nhttps://doi.org/10.1016/S0140-6736(02)11772-7 \n\n56. Horimoto T, Fukuda N, Iwatsuki-Horimoto K, Guan Y, Lim W, \nPeiris M, Sugii S, Odagiri T, Tashiro M, Kawaoka Y: Antigenic \nDifferences between H5N1 Human Influenza Viruses Isolated \nin 1997 and 2003. Journal of Veterinary Medical Science 2004, \n66(3):303-305. https://doi.org/10.1292/jvms.66.303 \n\n57. Sturm-Ramirez KM, Ellis T, Bousfield B, Bissett L, Dyrting K, Rehg \nJE, Poon L, Guan Y, Peiris M, Webster RG: Reemerging H5N1 \ninfluenza viruses in Hong Kong in 2002 are highly pathogenic to \nducks. Journal of Virology 2004, 78:4892-4901. \nhttps://doi.org/10.1128/JVI.78.9.4892-4901.2004 \n\n58.", "start_char_idx": 70098, "end_char_idx": 72515, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8c14defe-6774-4bd9-8e95-25305598bdc0": {"__data__": {"id_": "8c14defe-6774-4bd9-8e95-25305598bdc0", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5e8648c4-ca5d-4c9c-8f71-6788609b3221", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "d79072ee770acbcc69f85279e13e0b7f06c16d07ae1c60e94f7de22228404c5c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0ae0e93b-eefb-4099-ae57-748f55e78d7d", "node_type": "1", "metadata": {}, "hash": "152fead50df20a1b83f73ba4071cabd43c01036972bf142d5db60567cdc43088", "class_name": "RelatedNodeInfo"}}, "text": "Govorkova EA, Rehg JE, Krauss S, Yen H-L, Guan Y, Peiris M, \nNguyen TD, Hanh TH, Puthavathana P, Long HT et al: Lethality \nto Ferrets of H5N1 Influenza Viruses Isolated from Humans and \nPoultry in 2004. Journal of Virology 2005, 79(4):2191. \nhttps://doi.org/10.1128/JVI.79.4.2191-2198.2005 \n\n59. Chantong W, Kaneene JB: Poultry raising systems and highly \npathogenic avian influenza outbreaks in Thailand: the situ-\nation, associations, and impacts. The Southeast Asian Journal of \nTropical Medicine and Public Health 2011, 42:596-608.  \n\n60. Henning J, Henning KA, Morton JM, Long NT, Ha NT, Vu LT, Vu \nPP, Hoa DM, Meers J: Highly pathogenic avian influenza (H5N1) \nin ducks and in-contact chickens in backyard and smallholder \ncommercial duck farms in Vietnam. Preventive Veterinary Medicine \n2011, 101:229-240. \nhttps://doi.org/10.1016/j.prevetmed.2010.05.016 \n\n61. Brown JD, Luttrell MP, Uhart MM, del Valle Ferreyra H, Romano \nMM, Rago MV, Stallknecht DE: Antibodies to type A influenza \nvirus in wild waterbirds from Argentina. Journal of Wildlife \nDiseases 2010, 46:1040-1045. https://doi.org/10.7589/0090-3558-\n46.3.1040 \n\n62. Chua TH: Studies on the diagnosis, epidemiology and control of \nhighly pathogenic H5N1 avian influenza. Murdoch University, \nPerth, Australia.; 2009.  \n\n63. Pfeiffer DU, Minh PQ, Martin V, Epprecht M, Otte MJ: An analysis \nof the spatial and temporal patterns of highly pathogenic avian \ninfluenza occur-rence in Vietnam using national surveillance \ndata. The Veterinary Journal 2007, 174:302-309. \nhttps://doi.org/10.1016/j.tvjl.2007.05.010 \n\n64. Lycett SJ, Duchatel F, Digard P: A brief history of bird flu. Philos \nTrans R Soc Lond B Biol Sci 2019, 374(1775):20180257. \nhttps://doi.org/10.1098/rstb.2018.0257 \n\n65. Harfoot R, Webby RJ: H5 influenza, a global update. Journal of \nMicrobiology 2017, 55(3):196-203. https://doi.org/10.1007/s12275-\n017-7062-7 \n\n66. Smith GJD, Donis RO, World Health Organization/World \nOrganisation for Animal Health/Food Agriculture Organization \nH5 Evolution Working Group: Nomenclature updates resulting \nfrom the evolution of avian influenza A(H5) virus clades 2.1.3.2a, \n2.2.1, and 2.3.4 during 2013\u20132014. 2015, 9(5):271-276. \nhttps://doi.org/10.1111/irv.12324 \n\n67. Kim S-H, Hur M, Suh J-H, Woo C, Wang S-J, Park E-R, Hwang J, \nAn I-J, Jo S-D, Shin J-H et al: Molecular characterization of highly \npathogenic avian influenza H5N8 viruses isolated from Baikal \nteals found dead during a 2014 outbreak in Korea. J Vet Sci 2016, \n17(3):299-306. https://doi.org/10.4142/jvs.2016.17.3.299 \n\n68. Miller RS, Sweeney SJ, Akkina JE, Saito EK: Potential \nIntercontinental Movement of Influenza A(H7N9) Virus into \nNorth America by Wild Birds: Application of a Rapid \nAssessment Framework.", "start_char_idx": 72516, "end_char_idx": 75268, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0ae0e93b-eefb-4099-ae57-748f55e78d7d": {"__data__": {"id_": "0ae0e93b-eefb-4099-ae57-748f55e78d7d", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8c14defe-6774-4bd9-8e95-25305598bdc0", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "b187676e6533e4fd5e3e459e69f12c42003b4a1277cb6803293fafda29216ad8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1a8c0cfc-1c20-459a-b4a0-73b9274546a4", "node_type": "1", "metadata": {}, "hash": "6b8174d6f0fc81e6111e209c8040adcd9d6f3c9bc39b772a305bf6cc4856c342", "class_name": "RelatedNodeInfo"}}, "text": "Transboundary and emerging diseases 2015, \n62(6):650-668. https://doi.org/10.1111/tbed.12213 \n\n69. Couacy-Hymann E, Kouakou VA, Aplogan GL, Awoume F, \nKouakou CK, Kakpo L, Sharp BR, McClenaghan L, McKenzie P, \nWebster RG et al: Surveillance for influenza viruses in poultry \nand swine, west Africa, 2006-2008. Emerging infectious diseases \n2012, 18(9):1446-1452. https://doi.org/10.3201/eid1809.111296 \n\n70. WHO/OIE/FAO H5N1 Evolution Working Group: Continued \nevolution of highly pathogenic avian influenza A (H5N1): \nupdated nomenclature. Influenza Other Respir Viruses 2012, 6(1):1-\n5. https://doi.org/10.1111/j.1750-2659.2011.00298.x \n\n71. Peiris JSM, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, \nNg TK, Chan KH, Lai ST, Lim WL et al: Re-emergence of fatal \nhuman influenza A subtype H5N1 disease. The Lancet 2004, \n363(9409):617-619. https://doi.org/10.1016/S0140-6736(04)15595-\n5 \n\n72. de Jong MD, Cam BV, Qui PT, Hien VM, Thanh TT, Hue NB, Beld \nM, Phuong LT, Khanh TH, Chau NVV et al: Fatal Avian Influenza \nA (H5N1) in a Child Presenting with Diarrhea Followed by \nComa. N Engl J Med 2005, 352(7):686-691. \nhttps://doi.org/10.1056/NEJMoa044307 \n\n73. Uiprasertkul M, Puthavathana P, Sangsiriwut K, et al: Influenza \nA H5N1 replication sites in humans. Emerg Infect Dis 2005, \n11:1036-1041. https://doi.org/10.3201/eid1107.041313 \n\n74. Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y: \nInfluenza virus receptors in the human airway. Nature 2006, \n440(7083):435-436. https://doi.org/10.1038/440435a \n\n75. Pillai SPS, Lee CW: Species and age related differences in the \ntype and distribution of influenza virus receptors in different \ntissues of chickens, ducks and turkeys. Virology Journal 2010, \n7(1):5. https://doi.org/10.1186/1743-422X-7-5 \n\n76. Skehel JJ, Wiley DC: Receptor Binding and Membrane Fusion in \nVirus Entry: The Influenza Hemagglutinin. Annual Review of \nBiochemistry 2000, 69(1):531-569. \nhttps://doi.org/10.1146/annurev.biochem.69.1.531 \n\n77. Neumann G, Kawaoka Y: Host range restriction and \npathogenicity in the context of influenza pandemic. Emerg Infect \nDis 2006, 12(6):881-886. https://doi.org/10.3201/eid1206.051336 \n\n78. To KF, Chan PK, Chan KF, Lee WK, Lam WY, Wong KF, Tang NL, \nTsang DN, Sung RY, Buckley TA et al: Pathology of fatal human \ninfection associated with avian influenza A H5N1 virus. Journal \nof medical virology 2001, 63(3):242-246. \nhttps://doi.org/10.1002/1096-9071(200103)63:3<242::AID-\nJMV1007>3.0.CO;2-N \n\n79.", "start_char_idx": 75269, "end_char_idx": 77744, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1a8c0cfc-1c20-459a-b4a0-73b9274546a4": {"__data__": {"id_": "1a8c0cfc-1c20-459a-b4a0-73b9274546a4", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0ae0e93b-eefb-4099-ae57-748f55e78d7d", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "3d69bd024837b6c543ec93050c21739429a15f52b7eaa0f6a5e83bb81cd8ca32", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "970c8d91-20b2-4427-9268-982e325e5907", "node_type": "1", "metadata": {}, "hash": "e6410bb75ec9d984c2d530bf288c0bbfffeb8b5e0a99e171f9f5c1ccb1a03e2c", "class_name": "RelatedNodeInfo"}}, "text": "Weinstein RA, Bridges CB, Kuehnert MJ, Hall CB: Transmission \nof influenza: implications for control in health care settings. \nClinical infectious diseases 2003, 37(8):1094-1101. \nhttps://doi.org/10.1086/378292 \n\n80. Mounts AW, Kwong H, Izurieta HS, Ho Y-y, Au T-k, Lee M, \nBridges CB, Williams SW, Mak KH, Katz JM et al: Case-Control \nStudy of Risk Factors for Avian Influenza A (H5N1) Disease, \nHong Kong, 1997. The Journal of Infectious Diseases 1999, 180(2):505-\n508. https://doi.org/10.1086/314903 \n\n81. Thanawongnuwech R, Amonsin A, Tantilertcharoen R, \nDamrongwatanapokin S, Theamboonlers A, Payungporn S, \nNanthapornphiphat K, Ratanamungklanon S, Tunak E, Songserm \nT et al: Probable tiger-to-tiger transmission of avian influenza \nH5N1. Emerg Infect Dis 2005, 11(5):699-701. \nhttps://doi.org/10.3201/eid1105.050007 \n\n82. Hayden F, Croisier A: Transmission of Avian Influenza Viruses \nto and between Humans. The Journal of Infectious Diseases 2005, \n192(8):1311-1314. https://doi.org/10.1086/444399 \n\n83. Writing Committee of the Second World Health Organization \nConsultation on Clinical Aspects of Human Infection with Avian \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  39 \n\nInfluenza A (H5N1) Virus, Adbdel-Ghafar AN, \nChotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, et al: \nUpdate on Avian Influenza A (H5N1) Virus Infection in \nHumans. N Engl J Med 2008, 358(3):261-273. \nhttps://doi.org/10.1056/NEJMra0707279 \n\n84. Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, \nChunsuthiwat S, Sawanpanyalert P, Kijphati R, Lochindarat S, \nSrisan P, Suwan P, Osotthanakorn Y et al: Human disease from \ninfluenza A (H5N1), Thailand, 2004. Emerg Infect Dis 2005, \n11(2):201-209. https://doi.org/10.3201/eid1102.041061 \n\n85. Kandun IN, Wibisono H, Sedyaningsih ER, Yusharmen, \nHadisoedarsuno W, Purba W, Santoso H, Septiawati C, \nTresnaningsih E, Heriyanto B et al: Three Indonesian Clusters of \nH5N1 Virus Infection in 2005. N Engl J Med 2006, 355(21):2186-\n2194. https://doi.org/10.1056/NEJMoa060930 \n\n86. Oner AF, Bay A, Arslan S, Akdeniz H, Sahin HA, Cesur Y, Epcacan \nS, Yilmaz N, Deger I, Kizilyildiz B et al: Avian Influenza A (H5N1) \nInfection in Eastern Turkey in 2006. N Engl J Med 2006, \n355(21):2179-2185. https://doi.org/10.1056/NEJMoa060601 \n\n87.", "start_char_idx": 77745, "end_char_idx": 80056, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "970c8d91-20b2-4427-9268-982e325e5907": {"__data__": {"id_": "970c8d91-20b2-4427-9268-982e325e5907", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1a8c0cfc-1c20-459a-b4a0-73b9274546a4", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "9717b0724bb14b9e063ac827b4e972808a9756eb451be714f4a712406fbeb7af", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "408f770a-f021-4e8b-87e1-7d038e1c71f5", "node_type": "1", "metadata": {}, "hash": "2d7e14ec461e815343eae344027055ac54f889b000175c5c618e3809f0982ae0", "class_name": "RelatedNodeInfo"}}, "text": "Hien TT, Liem NT, Dung NT, San LT, Mai PP, Chau NvV, Suu PT, \nDong VC, Mai LTQ, Thi NT et al: Avian influenza A (H5N1) in 10 \npatients in Vietnam. N Engl J Med 2004, 350(12):1179-1188. \nhttps://doi.org/10.1056/NEJMoa040419 \n\n88. Yuen KY, Chan PKS, Peiris M, Tsang DNC, Que TL, Shortridge KF, \nCheung PT, To WK, Ho ETF, Sung R et al: Clinical features and \nrapid viral diagnosis of human disease associated with avian \ninfluenza A H5N1 virus. The Lancet 1998, 351(9101):467-471. \nhttps://doi.org/10.1016/S0140-6736(98)01182-9 \n\n89. Yusuke SK, Kiyoshi F, Kazuo S: Mechanism of a Mutation in Non-\nStructural Protein 1 Inducing High Pathogenicity of Avian \nInfluenza Virus H5N1. Protein & Peptide Letters 2016, 23(4):372-\n378. https://doi.org/10.2174/0929866523666160204124406 \n\n90. Apisarnthanarak A, Kitphati R, Thongphubeth K, Patoomanunt P, \nAnthanont P, Auwanit W, Thawatsupha P, Chittaganpitch M, \nSaeng-Aroon S, Waicharoen S et al: Atypical avian influenza \n(H5N1). Emerg Infect Dis 2004, 10(7):1321-1324. \n\n91. Sandrock C, Kelly T: Clinical review: update of avian influenza \nA infections in humans. Critical care 2007, 11(2):209-209. \nhttps://doi.org/10.1186/cc5675 \n\n92. World Health Organization (WHO): Clinical management of \nhuman infection with avian influenza A (H5N1) virus. In.; 2015. \n\n93. World Health Organization (WHO): Update: WHO-confirmed \nhuman cases of avian influenza A (H5N1) infection, 25 \nNovember 2003-24 November 2006. Weekly Epidemiological Record \n2007, 82(06):41-47. \n\n94. Avian Influenza (Bird Flu) \n[http://emedicine.medscape.com/article/250002] \n\n95. World Health Organization (WHO): Epidemiology of WHO-\nconfirmed human cases of avian influenza A (H5N1) infection. \nWeekly Epidemiological Record 2006, 81(26):249-257. \n\n96. Gruber PC, Gomersall CD, Joynt GM: Avian influenza (H5N1): \nimplications for intensive care. Intensive Care Med 2006, 32(6):823-\n829. https://doi.org/10.1007/s00134-006-0148-z \n\n97. Chan PKS: Outbreak of Avian Influenza A(H5N1) Virus \nInfection in Hong Kong in 1997. Clinical Infectious Diseases 2002, \n34(Supplement_2):S58-S64. https://doi.org/10.1086/338820 \n\n98. Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RA, Amonsin \nA, Payungporn S, Noppornpanth S, Wattanodorn S, \nTheamboonlers A, Tantilertcharoen R: Avian influenza H5N1 in \ntigers and leopards. Emerg Infect Dis 2004, 10(12):2189. \nhttps://doi.org/10.3201/eid1012.040759 \n\n99. Regional Office for South-East Asia, World Health Organization \n(WHO): Guidelines on laboratory diagnosis of avian influenza. \nIn. New Delhi: WHO Regional Office for South-East Asia; 2007. \n\n100.", "start_char_idx": 80057, "end_char_idx": 82653, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "408f770a-f021-4e8b-87e1-7d038e1c71f5": {"__data__": {"id_": "408f770a-f021-4e8b-87e1-7d038e1c71f5", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "970c8d91-20b2-4427-9268-982e325e5907", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "8c38e2181b5d7d53bea4452c13fbc689b0dd3cfa520baf0610283829b8112584", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f4264fa5-d3ed-43f0-b308-d497a2a36ac3", "node_type": "1", "metadata": {}, "hash": "74512becb02a1d6c9f5f5370a781dd2df7c691c339d759cafb89c0bf82852aac", "class_name": "RelatedNodeInfo"}}, "text": "New Delhi: WHO Regional Office for South-East Asia; 2007. \n\n100. World Health Organization (WHO): Collecting, preserving and \nshipping specimens for the diagnosis of avian influenza \nA(H5N1) virus infection : guide for field operations. In. Geneva: \nWorld Health Organization; 2006. \n\n101. Madeley CR, World Health Organization (WHO): Guide to the \ncollection and transport of virological specimens : including \nchlamydial and rickettsial specimens. In. Geneva: World Health \nOrganization; 1977. \n\n102. Specimen Collection and Testing for patients with novel \ninfluenza A viruses with potential to cause severe disease in \nhumans [https://www.cdc.gov/flu/avianflu/severe-\npotential.htm] \n\n103. Ng EKO, Cheng PKC, Ng AYY, Hoang TL, Lim WWL: Influenza \nA H5N1 detection. Emerg Infect Dis 2005, 11(8):1303-1305. \nhttps://doi.org/10.3201/eid1108.041317 \n\n104. Poon LLM, Leung CSW, Chan KH, Lee JHC, Yuen KY, Guan Y, \nPeiris JSM: Detection of human influenza A viruses by loop-\nmediated isothermal amplification. J Clin Microbiol 2005, \n43(1):427-430. https://doi.org/10.1128/JCM.43.1.427-430.2005 \n\n105. Imai M, Ninomiya A, Minekawa H, Notomi T, Ishizaki T, Tashiro \nM, Odagiri T: Development of H5-RT-LAMP (loop-mediated \nisothermal amplification) system for rapid diagnosis of H5 avian \ninfluenza virus infection. Vaccines (Basel) 2006, 24(44-46):6679-\n6682. https://doi.org/10.1016/j.vaccine.2006.05.046 \n\n106. Chan KH, Lam S, Puthavathana P, Nguyen T, Long H, Pang C, \nChan KM, Cheung C, Seto WH, Peiris JS: Comparative analytical \nsensitivities of six rapid influenza A antigen detection test kits \nfor detection of influenza A subtypes H1N1, H3N2 and H5N1. \nJournal of clinical virology 2007, 38:169-171. \nhttps://doi.org/10.1016/j.jcv.2006.11.010 \n\n107. Kida H, Ito T, Yasuda J, Shimizu Y, Itakura C, Shortridge KF, \nKawaoka Y, Webster RG: Potential for transmission of avian \ninfluenza viruses to pigs. Journal of General Virology 1994, \n75(9):2183-2188. https://doi.org/10.1099/0022-1317-75-9-2183 \n\n108. Lu BL, Webster RG, Hinshaw VS: Failure to detect \nhemagglutination-inhibiting antibodies with intact avian \ninfluenza virions. Infect Immun 1982, 38(2):530-535. \nhttps://doi.org/10.1128/IAI.38.2.530-535.1982 \n\n109. Beare A, Webster R: Replication of avian influenza viruses in \nhumans. Archives of virology 1991, 119(1-2):37-42. \nhttps://doi.org/10.1007/BF01314321 \n\n110. Stephenson I, Wood JM, Nicholson KG, Charlett A, Zambon MC: \nDetection of anti-H5 responses in human sera by HI using horse \nerythrocytes following MF59-adjuvanted influenza \nA/Duck/Singapore/97 vaccine. Virus research 2004, 103:91-95. \nhttps://doi.org/10.1016/j.virusres.2004.02.019 \n\n111.", "start_char_idx": 82589, "end_char_idx": 85261, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f4264fa5-d3ed-43f0-b308-d497a2a36ac3": {"__data__": {"id_": "f4264fa5-d3ed-43f0-b308-d497a2a36ac3", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "408f770a-f021-4e8b-87e1-7d038e1c71f5", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "d7b1fe3a9c6f835f81152285892b3ce39dda4abbc4356070a1c4f88cf53bc5c6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4009556-38f3-42d2-bc6f-235f21a96989", "node_type": "1", "metadata": {}, "hash": "481e73c84c4b23326df5d9bc144c348611dc96ac75a8c1088dd11fbccb17ab47", "class_name": "RelatedNodeInfo"}}, "text": "Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim \nW, Fukuda K, Cox NJ, Katz JM: Detection of antibody to avian \ninfluenza A (H5N1) virus in human serum by using a \ncombination of serologic assays. J Clin Microbiol 1999, 37(4):937-\n943. https://doi.org/10.1128/JCM.37.4.937-943.1999 \n\n112. Bridges CB, Katz JM, Seto WH, Chan PKS, Tsang D, Ho W, Mak \nKH, Lim W, Tam JS, Clarke M et al: Risk of Influenza A (H5N1) \nInfection among Health Care Workers Exposed to Patients with \nInfluenza A (H5N1), Hong Kong. The Journal of Infectious Diseases \n2000, 181(1):344-348. https://doi.org/10.1086/315213 \n\n113. Bridges CB, Lim W, Hu-Primmer J, Sims L, Fukuda K, Mak KH, \nRowe T, Thompson WW, Conn L, Lu X et al: Risk of Influenza A \n(H5N1) Infection among Poultry Workers, Hong Kong, 1997\u2013\n1998. The Journal of Infectious Diseases 2002, 185(8):1005-1010. \nhttps://doi.org/10.1086/340044 \n\n114. Katz JM, Lim W, Bridges CB, Rowe T, Hu-Primmer J, Lu X, \nAbernathy RA, Clarke M, Conn L, Kwong H: Antibody response \nin individuals infected with avian influenza A (H5N1) viruses \nand detection of anti-H5 antibody among household and social \ncontacts. The Journal of Infectious Diseases 1999, 180(6):1763-1770. \nhttps://doi.org/10.1086/315137 \n\n115. Avian influenza A (H5N1) virus infection \n[https://online.epocrates.com/diseases/45535/Avian-influenza-\nA-H5N1-virus-infection/Differential-Diagnosis] \n\n116. Lai S, Qin Y, Cowling BJ, Ren X, Wardrop NA, Gilbert M, Tsang \nTK, Wu P, Feng L, Jiang H et al: Global epidemiology of avian \ninfluenza A H5N1 virus infection in humans, 1997\u20132015: a \nsystematic review of individual case data. The Lancet Infectious \nDiseases 2016, 16(7):e108-e118. https://doi.org/10.1016/S1473-\n3099(16)00153-5 \n\n117. Asian H5N1 Case Defination \n[https://www.cdc.gov/flu/avianflu/h5n1/case-definitions.htm] \n\n118. WHO case definitions for human infections with influenza \nA(H5N1) virus \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  40 \n\n[https://www.who.int/influenza/resources/documents/case_d\nefinition2006_08_29/en/] \n\n119. White NJ, Webster RG, Govorkova EA, Uyeki TM: What is the \noptimal therapy for patients with H5N1 influenza? PLoS Med \n2009, 6(6):e1000091-e1000091. \nhttps://doi.org/10.1371/journal.pmed.1000091 \n\n120. Jefferson T, Jones MA, Doshi P, Del Mar CB, Hama R, Thompson \nMJ, Spencer EA, Onakpoya IJ, Mahtani KR, Nunan D et al: \nNeuraminidase inhibitors for preventing and treating influenza \nin adults and children. Cochrane Database of Systematic Reviews \n2014, 2014(4). https://doi.org/10.1002/14651858.CD008965.pub4 \n\n121.", "start_char_idx": 85262, "end_char_idx": 87874, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4009556-38f3-42d2-bc6f-235f21a96989": {"__data__": {"id_": "d4009556-38f3-42d2-bc6f-235f21a96989", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f4264fa5-d3ed-43f0-b308-d497a2a36ac3", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "f9967a647aa804727dc995b21b8de34e5e3455bb3e7f97c9df08ed534bbc7e76", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "03b10f91-b6ea-4ea1-a663-fe29f086b371", "node_type": "1", "metadata": {}, "hash": "aea45483fc9674d253bd3acd903f5716c4d046e4b55d87a5f5898152f153387b", "class_name": "RelatedNodeInfo"}}, "text": "Allen UD, Aoki FY, Stiver HG: The use of antiviral drugs for \ninfluenza: recommended guidelines for practitioners. Canadian \nJournal of Infectious Diseases & Medical Microbiology 2006, 17(5):273-\n284. https://doi.org/10.1155/2006/165940 \n\n122. Poland GA, Jacobson RM, Ovsyannikova IG: Influenza virus \nresistance to antiviral agents: a plea for rational use. Clinical \ninfectious diseases 2009, 48(9):1254-1256. \nhttps://doi.org/10.1086/598989 \n\n123. Hill G, Cihlar T, Oo C, Ho ES, Prior K, Wiltshire H, Barrett J, Liu \nB, Ward P: The Anti-Influenza Drug Oseltamivir Exhibits Low \nPotential to Induce Pharmacokinetic Drug Interactions via Renal \nSecretion\u2014Correlation of in Vivo and in Vitro Studies. Drug \nMetabolism and Disposition 2002, 30(1):13. \nhttps://doi.org/10.1124/dmd.30.1.13 \n\n124. Hui DS, Lee N, Chan PK, Beigel JH: The role of adjuvant \nimmunomodulatory agents for treatment of severe influenza. \nAntiviral Res 2018, 150:202-216. \nhttps://doi.org/10.1016/j.antiviral.2018.01.002 \n\n125. World Health Organization (WHO): WHO rapid advice \nguidelines on pharmacological management of humans infected \nwith avian influenza A (H5N1) virus. In. Geneva: World Health \nOrganization; 2006.  \n\n126. Sch\u00fcnemann HJ, Hill SR, Kakad M, Bellamy R, Uyeki TM, Hayden \nFG, Yazdanpanah Y, Beigel J, Chotpitayasunondh T, Del Mar C et \nal: WHO Rapid Advice Guidelines for pharmacological \nmanagement of sporadic human infection with avian influenza \nA (H5N1) virus. The Lancet Infectious Diseases 2007, 7(1):21-31. \nhttps://doi.org/10.1016/S1473-3099(06)70684-3 \n\n127. World Health Organization (WHO): WHO interim guidelines on \nclinical management of humans infected by influenza A (H5N1). \n2004. \n\n128. The ARDS Network: Comparison of Two Fluid-Management \nStrategies in Acute Lung Injury. N Engl J Med 2006, 354(24):2564-\n2575. https://doi.org/10.1056/NEJMoa062200 \n\n129. Australia approves CSL's H5N1 vaccine. \n[http://www.cidrap.umn.edu/news-\nperspective/2008/06/australia-approves-csls-h5n1-vaccine] \n\n130. FDA Approves First Adjuvanted Vaccine for H5N1 Bird Flu. \n[https://www.medscape.com/viewarticle/814914] \n\n131. EU license first pre-pandemic bird flu vaccine London \n[https://www.reuters.com/article/us-birdflu-glaxo/eu-licenses-\nfirst-pre-pandemic-bird-flu-vaccine-idUSL1644576720080519] \n\n132. Elegant S: Is a human pandemic next? In: Time. vol. 163; 2004: 14-\n20. \n\n133. Baz M, Luke CJ, Cheng X, Jin H, Subbarao K: H5N1 vaccines in \nhumans. Virus research 2013, 178(1):78-98. \nhttps://doi.org/10.1016/j.virusres.2013.05.006 \n\n134. Altman L: Avian flu drug works in first tests. New York Times \n2005. \n\n135. Bird Flu. [https://www.britannica.com/science/bird-flu] \n136.", "start_char_idx": 87875, "end_char_idx": 90551, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "03b10f91-b6ea-4ea1-a663-fe29f086b371": {"__data__": {"id_": "03b10f91-b6ea-4ea1-a663-fe29f086b371", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4009556-38f3-42d2-bc6f-235f21a96989", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "4fe0ffc3fb0230fc636a1f6881ebad39393958eb3e046f1d54c9c4e89f3fd33c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9bba7779-4c6c-4be1-80ce-ea16b9c6083c", "node_type": "1", "metadata": {}, "hash": "b5003718d5739be662a53c353e672ece7585af797ab6010736b4c48afed5685b", "class_name": "RelatedNodeInfo"}}, "text": "[https://www.britannica.com/science/bird-flu] \n136. Prevention and treatment of Avian Influenza A Viruses in \n\nPeople [https://www.cdc.gov/flu/avianflu/prevention.htm] \n137. Lam TT-Y, Zhou B, Wang J, Chai Y, Shen Y, Chen X, Ma C, Hong \n\nW, Chen Y, Zhang Y et al: Dissemination, divergence and \nestablishment of H7N9 influenza viruses in China. Nature 2015, \n522(7554):102-105. https://doi.org/10.1038/nature14348 \n\n138. Lemon SM, Mahmoud A, Mack A, Knobler SL: The threat of \npandemic influenza: are we ready? workshop summary: \nNational Academies Press; 2005. \n\n139. WHO Global Influenza Programme, World Health Organization \n(WHO): Pandemic influenza preparedness and response: a WHO \n\nguidance document. In. Geneva: World Health Organization; \n2009. \n\n140. Gomersall CD, Loo S, Joynt GM, Taylor BL: Pandemic \npreparedness. 2007, 13(6):742-747. \nhttps://doi.org/10.1097/MCC.0b013e3282f1bafd \n\n141. Hans C: Infection control practices for avian influenza in \nhealthcare facilities. J Indian Med Assoc 2006, 104(7):388-392. \n\n142. Derrick JL, Gomersall CD: Protecting healthcare staff from severe \nacute respiratory syndrome: filtration capacity of multiple \nsurgical masks. Journal of Hospital Infection 2005, 59(4):365-368. \nhttps://doi.org/10.1016/j.jhin.2004.10.013 \n\n143. Hayden FG, Belshe R, Villanueva C, Lanno R, Hughes C, Small I, \nDutkowski R, Ward P, Carr J: Management of Influenza in \nHouseholds: A Prospective, Randomized Comparison of \nOseltamivir Treatment With or Without Postexposure \nProphylaxis. The Journal of Infectious Diseases 2004, 189(3):440-449. \nhttps://doi.org/10.1086/381128 \n\n144. Welliver R, Monto AS, Carewicz O, Schatteman E, Hassman M, \nHedrick J, Jackson HC, Huson L, Ward P, Oxford JS et al: \nEffectiveness of Oseltamivir in Preventing Influenza in \nHousehold ContactsA Randomized Controlled Trial. JAMA \n2001, 285(6):748-754. https://doi.org/10.1001/jama.285.6.748 \n\n145. World Health Organization (WHO): WHO guidelines on hand \nhygiene in health care (advanced draft): a summary: clean hands \nare safer hands. In.: Geneva: World Health Organization; 2005. \n\n146. World Health Organization (WHO): Human cases of avian \ninfluenza A(H5N1) in North-West Frontier Province, Pakistan, \nOctober-November 2007. In., vol. 83; 2008: 359-364. \n\n147. Interim Guidance on Follow-up of Close Contacts of Persons \nInfected with Novel Influenza A Viruses Associated with Severe \nHuman Disease and on the Use of Antiviral Medications for \nChemoprophylaxis [https://www.cdc.gov/flu/avianflu/novel-\nav-chemoprophylaxis-guidance.htm] \n\n148. Ministry of Agricultural Development, Nepal: Statistical \nInformation on Nepalese Agriculture. In.; 2011/2012. \n\n149. Department of Livestock Services, Ministry of Agriculture and \nCooperatives: National Surveillance Plan for Highly Pathogenic \nAvian Influenza (HPAI). In.; 2008. \n\n150.", "start_char_idx": 90500, "end_char_idx": 93346, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9bba7779-4c6c-4be1-80ce-ea16b9c6083c": {"__data__": {"id_": "9bba7779-4c6c-4be1-80ce-ea16b9c6083c", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "03b10f91-b6ea-4ea1-a663-fe29f086b371", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "45eb120c68c0f23fde6e13577b0fd16c2d3f1396bdba8d0e82a530e25a54af9d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aa45b8be-9b64-4383-bcd2-a683a2e832a6", "node_type": "1", "metadata": {}, "hash": "5bb8a818874da005f3b20f9b38996d2f1c8460398a5c1ae62636e2a1c6922ad3", "class_name": "RelatedNodeInfo"}}, "text": "In.; 2008. \n\n150. Pant GR, Selleck PW: Surveillance for Avian Influenza in Nepal \n2004-2005 (Vigilancia epidemiol\u00f3gica para influenza aviar en \nNepal durante los a\u00f1os 2004 y 2005). Avian Diseases 2007, \n51(1):352-354. https://doi.org/10.1637/7649-051806R.1 \n\n151. Pandey S, Sherchand SP, Manandhar P, Pokhrel N, Globig A, \nSherchan S: Serological Surveillance of Avian Influenza Virus in \nNepal. Journal of Human Virology and Retrovirology 2016, 4(1):00120. \nhttps://doi.org/10.15406/jhvrv.2016.04.00120 \n\n152. National Influenza Center \n[http://nphl.gov.np/department/influenza] \n\n153. Chaudhary S, Pahwa VK: Avain influenza. Journal of Universal \nCollege of Medical Sciences 2013, 1(3):1-2. \nhttps://doi.org/10.3126/jucms.v1i3.8750 \n\n154. Epidemiology and Disease Control Division, Department of \nHealth Services, Nepal: Press release on Influenza A (H5N1) by \nMinistry of Health and Population. In.; 2019. \n\n155. Disease outbreak maps. World Animal Health Information \nDatabase (WAHIS Interface) \u2013 Version 1. \n[https://www.oie.int/wahis_2/public/wahid.php/Diseaseinfor\nmation/Diseaseoutbreakmaps?disease_type_hidden=0&disease_\nid_hidden=15&selected_disease_name_hidden=Highly+path.+av\nian+influenza+%28-+-\n%29+&disease_type=0&disease_id_terrestrial=15&disease_id_aq\nuatic=-\n999&selected_start_day=1&selected_start_month=1&selected_sta\nrt_year=2013&selected_end_day=1&selected_end_month=12&sel\nected_end_year=2013&newlang=en] \n\n156. Dhakal D, Dulal P, Shrestha RM, Manandhar S, Lamichane J: \nStudy of Prevalence of Bird flu by using RT-PCR at Central \nVeterinary Laboratory, Nepal, 2007. BSN E-Bulletin 2009, 1. \n\n157. Karmacharya D, Manandhar S, Sharma A, Bhatta T, Adhikari P, \nSherchan AM, Shrestha B, Bista M, Rajbhandari R, Oberoi M et al: \nSurveillance of Influenza A Virus and Its Subtypes in Migratory \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 2 4 - 4 1      Shrestha et al.  \n\n\u00a9NJB, BSN  41 \n\nWild Birds of Nepal. PloS one 2015, 10(7):e0133035. \nhttps://doi.org/10.1371/journal.pone.0133035 \n\n158. Mahato RK, Bhandari GP, Shrestha JM, Basnet P: Pandemic \nInfluenza A (H1N1) 2009 outbreak investigation in Nepal. \nJournal of Nepal Health Research Council 2011, 8(17):75-77. \nhttps://doi.org/10.33314/jnhrc.v0i0.229 \n\n159. Adhikari BR, Shakya G, Upadhyay BP, Prakash Kc K, Shrestha SD, \nDhungana GR: Outbreak of pandemic influenza A/H1N1 2009 in \nNepal. Virology Journal 2011, 8(1):133. \nhttps://doi.org/10.1186/1743-422X-8-133 \n\n160.", "start_char_idx": 93329, "end_char_idx": 95778, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aa45b8be-9b64-4383-bcd2-a683a2e832a6": {"__data__": {"id_": "aa45b8be-9b64-4383-bcd2-a683a2e832a6", "embedding": null, "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-113", "node_type": "4", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "253e3ac66a19efb79cff295420cd5b3e1fc30b53386fd3be37e68f4b57f50366", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9bba7779-4c6c-4be1-80ce-ea16b9c6083c", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "24c88c12b836dcac6f8d190a7324028a35200c3044f3c05726fbfb576b00af53", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "43958917-9f3f-4d23-a461-580c228912a2", "node_type": "1", "metadata": {}, "hash": "f9fb3a1c0024b32b41841a2ad9cb458f40e667e0c63093d48b9b47f6baff6055", "class_name": "RelatedNodeInfo"}}, "text": "Foreign Exchange Rates Fixed by Nepal Rastra Bank (NRB) \n[https://archive.nrb.org.np/fxmexchangerate.php] \n\n161. Ghimire YN, Karki NPS, Shrestha K: Economic Loss due to \nOutbreak of Highly Pathogenic Avian Influenza in the Nepalese \nCommercial Poultry. Nepal Agric Res J 2012, 12:84-90. \n\n162. Karki S: Effects of highly pathogenic avian influenza H5N1 \noutbreak In nepal from financial and social perspectives: a case \nstudy. Nepalese Vet J 2017, 34:26-35. \nhttps://doi.org/10.3126/nvj.v34i0.22861 \n\n163. Dharma K, Chakraborty S, Tiwari R, Kumar A, Rahal A, Latheef S, \nWani M, Kapoor S: Avian/Bird Flu Virus: Poultry Pathogen \nHaving Zoonotic and Pandemic Threats: A Review. J Med Sci \n2013, 13(5):301-315. https://doi.org/10.3923/jms.2013.301.315 \n\n164. Mak PW, S. Jayawardena S, Poon LL: The evolving threat of \ninfluenza viruses of animal origin and the challenges in \ndeveloping appropriate diagnostics. Clin Chem 2012, 58:1527-\n1533. https://doi.org/10.1373/clinchem.2012.182626 \n\n165. World Health Organization (WHO): Avian influenza A (H5N1)-\nupdate 31: Situation (poultry) in Asia: Need for a long-term \nresponse, comparision with previous outbreaks. Epidemic and \nPandemic Alert and Response (EPR). In.; 2004. \n\n166. Dharma K, Chauhan RS, Kataria JM, Mahendran M, Tomar S: \nAvian Influenza: The current perspectives. J Immunol \nImmunopathol 2005, 7:1-33. \n\n167. Dharma KM, Mahendran M, Tomar S: Pathogens transmitted by \nmigratory birds: Threat perceptions to poultry health and \nproduction. Int J Poult Sci 2008, 7:516-525. \nhttps://doi.org/10.3923/ijps.2008.516.525 \n\n168. Neupane D, Khanal V, Ghimire K, Aro AR, Leppin A: Knowledge, \nattitudes and practices related to avian influenza among poultry \nworkers in Nepal: a cross sectional study. BMC Infectious Diseases \n2012, 12(1):76. https://doi.org/10.1186/1471-2334-12-76 \n\n169. Sah JK, Chiluwal S, Yadav SK, Jha D: A study on knowledge and \npreventive practices related to Avian Influenza among Higher \nSecondary School Students of Rajbiraj Municipality, Nepal. Al \nAmeen J Med Sci 2017, 10(4):276-280. \nhttps://doi.org/10.15406/mojph.2016.04.00091 \n\n170. Naeem K: The avian influenza H7N3 outbreak in South Central \nAsia. Avian Diseases 2003, 47:31-35.  \n\n171. Ellis TM, Leung CY, Chow MK, Bissett LA, Wong W, Guan Y, \nMalik Peiris J: Vaccination of chickens against H5N1 avian \ninfluenza in the face of an outbreak interrupts virus \ntransmission. Avian Pathology 2004, 33:405-412. \nhttps://doi.org/10.1080/03079450410001724012", "start_char_idx": 95779, "end_char_idx": 98274, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "43958917-9f3f-4d23-a461-580c228912a2": {"__data__": {"id_": "43958917-9f3f-4d23-a461-580c228912a2", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aa45b8be-9b64-4383-bcd2-a683a2e832a6", "node_type": "1", "metadata": {"identifier": "njb-113", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna; Parajuli, Hiramani; Shrestha, Sujata", "title": "Avian/Bird flu: A review: H5N1 outbreaks in Nepal", "date": "2021-07-31", "file": "njb-113.pdf"}, "hash": "48ddbb437ab9988e054a21ce740e93cd24e4bd6a3f7ec459b689522171eacf78", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "67ce506e-3a09-4d08-8c41-a88d2c96aadc", "node_type": "1", "metadata": {}, "hash": "a3c02c3b3aad445011b526525f6e78171e825d62622b64c2b5a658f34bf54373", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):18-23 Research article DOI: https://doi.org/10.3126/njb.v9i1.38646 \n\n\u00a9NJB, BSN 18 \n\nAssessment of phytochemicals, antimicrobial, antioxidant and \ncytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo) \nTara Shrestha         , Janardan Lamichhane \nDepartment of Biotechnology, Kathmandu University, Dhulikhel, Kavre, Nepal. Received: \n\n23 Feb 2021; Revised: 14 Jul 2021; Accepted: 19 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract\nTraditionally used medicinal plants are the major resources of biologically active metabolites which are widely used for the cure \n\nof numerous diseases especially in developing countries where health facilities are rare. Many plants are in use for centuries but \n\nthere is not enough scientific evidence and exploration. This research is focused on phytochemicals, antibacterial, antioxidant and \n\ncytotoxicity activity analysis of one of the most commonly used ethnomedicine Tinospora cordifolia collected from the \n\nKavrepalanchok district of Nepal. Phytochemicals analysis of methanol extract of T. cordifolia showed the presence of alkaloids, \n\ncoumarin saponins, glycosides, reducing sugar, and triterpenes. Antibacterial activity performed by disc diffusion method \n\nexhibited the highest activity against Streptococcus with a zone of inhibition are 10.3mm, 8.5mm, 6.5mm, and 6mm at 200mg/ml, \n\n100mg/ml, 50mg/ml, and 25mg/ml of concentration respectively. DPPH radical scavenging activity increased with increasing \n\nconcentration of extract. When compared with ascorbic acid at equivalent concentration, the extract shows a lower scavenging \n\nprofile (56.07% for the extract and 98.01% for ascorbic acid at 320 ppm). Cytotoxicity was evaluated in terms of LC50 (lethality \n\nconcentration). The result showed that the extract of T. cordifolia was found to be toxic with an LC50 value of 232.64\u03bcg/ml.  The \n\nbioactive component present in the plants could be the result of its pharmacological effects that support the traditional use of \n\nplants. \n\nKeywords: Tinospora cordifolia, Antioxidant activity, Antibacterial activity, Phytochemicals, DPPH, Brine Shrimp bioassay. \n\n Corresponding author, email: tarashrestha2@gmail.com \n\nIntroduction:  \nHerbal medicines are the biological resource of \n\nmedicines that have been used in different cultures \n\naround the world as safe therapeutic drugs. In addition, \n\nthey are also the enormous resource of dietary \n\nsupplements, popular drugs, pharmaceutical \n\nintermediaries, nutraceutical drugs, and chemical \n\nentities for synthesized drugs [1]. The activity of \n\nmedicinal plants is focused on the rich experiences of \n\ncountless healers over centuries, whether inherited from \n\nancestors, passed down from healer to healer, or \n\nestablished over time through personal experiences [2]. \n\nSecondary metabolites are non-nutrient plant chemical \n\ncompounds or bioactive components that protect the \n\nplant from microbial infections or insect infestations. \n\nThey are also known as phytochemicals or \n\nphytoconstituents [3]. \n\nT. cordifolia, also known as \"Guduchi,\" is well-known in \n\ntraditional ayurvedic literature for its extensive use in \n\nthe treatment of various diseases like jaundice, urinary \n\ndiseases, rheumatism, anemia, fever, vomiting, diabetes, \n\nskin disease, etc.  It is found mostly throughout It is a big \n\ndeciduous climbing shrub with a variety of coiling \n\nbranches that spreads widely. The plant's stem is \n\nsucculent, with long, fleshy, and climbing tendencies [4, \n\n5]. \n\nThe discovery of active components of the plant and \n\ntheir biological role in disease control has generated \n\nintense interest in the plant [6].", "start_char_idx": 50, "end_char_idx": 3741, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "67ce506e-3a09-4d08-8c41-a88d2c96aadc": {"__data__": {"id_": "67ce506e-3a09-4d08-8c41-a88d2c96aadc", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "43958917-9f3f-4d23-a461-580c228912a2", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "9feb1e391d739652407fd5fa022e092e6f57f898f5910097852c848265e2f131", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d1286df7-db95-4a95-974b-515c5a0bf471", "node_type": "1", "metadata": {}, "hash": "d72b3d3a1873c6dba995fbe67bdccf02c64fb178c6cd44e2f217d518254144f5", "class_name": "RelatedNodeInfo"}}, "text": "A wide number of \n\nchemical compounds like aporphine alkaloids, \n\nditerpenes, berberine, palmatine, tembertarine, \n\nmagniflorine, choline, and tinosporin, etc. have been \n\nisolated from this plant. [7]. T. cordifolia methanol \n\nextracts of stem [8] are effective against microbial \n\ninfections. It is reported that aqueous, ethanol, and \n\nacetone extract of leaves and stem of T. cordifolia \n\nshowed inhibitory activity against urinary pathogens \n\nsuch as Klebsiella pneumoniae and Pseudomonas \n\naeruginosa [9]. Methanolic, ethanolic and water extracts \n\n[10] of T. cordifolia has found to be significant \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-4191-2052\nmailto:tarashrestha2@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 18-23   Shrestha & Lamichhane .   \n\n\u00a9NJB, BSN 19 \n\nantioxidant compared to other solvents. It is suggested \n\nthat T. cordifolia methanol extract when taken orally has \n\nincreased the erythrocytes membrane lipid peroxide and \n\ncatalase activity [11] \n\nT. cordifolia is a well-known medicinal plant in \n\ntraditional medicine, and several recent scientific \n\ninvestigations have highlighted its potential value in \n\nmodern medicine. There have been several studies are \n\ncarried on, but T. cordifolia research in Nepal is limited. \n\nThe purpose of this research is to document T. cordifolia \n\nmedicinal characteristics as well as its potential for \n\nfurther scientific inquiry in the production of effective \n\ntherapeutic molecules. Isolation of phytochemicals, \n\nantibacterial activity, antioxidant activity, and \n\ncytotoxicity analysis of phytoconstituents derived from \n\nmethanol extract stem of T. cordifolia all are addressed \n\nin this research. The decision of solvent is made due to \n\nthe idea of solvent to separate wide assortments of \n\nhydrophilic and lipophilic substance constituents. \n\nMaterials and Methods \nSample collection \n The sample of   T. cordifolia stems was collected from \n\nBhakundebesi, Kavrepalanchok district of Nepal \n\nsituated at an altitude of 1120 msl with Latitude \n\n27.560677\u00b0 and Longitude 85.6409178\u00b0 shown in Figure \n\n1. The identification of the plant was done by Tirtha\n\nMaya Shrestha, taxonomist, Department of Pharmacy, \n\nKathmandu University.  \n\nFigure 1: The sample collection site, Kavrepalanchok \n\nDistrict, Nepal. \n\nSample preparation and extraction: \nThe collected plant was shade dried at 25\u00baC and finely \n\npowdered using a grinder. The sample extract was \n\nprepared using a cold methanol maceration process [12] \n\nin which 50 g of finely ground powder was infused with \n\n200 ml of methanol. The mixture was then stirred for \n\napproximately half an hour and stored for 48 hours. The \n\nsolution was filtered using the Whatman No. 1 filter \n\npaper after 48 hours. A rotary evaporator was used to \n\nevaporate the filtrate sample.", "start_char_idx": 3742, "end_char_idx": 6711, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d1286df7-db95-4a95-974b-515c5a0bf471": {"__data__": {"id_": "d1286df7-db95-4a95-974b-515c5a0bf471", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "67ce506e-3a09-4d08-8c41-a88d2c96aadc", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "ec82c2b3a196772f051ab002411ca26947e76dcfce28deef7014b779d24f7257", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f1baba6b-79b6-4a93-9b21-e44acb464b01", "node_type": "1", "metadata": {}, "hash": "c5a475f2ecd8d1f253de802817144b3ecdef59f69e30b9c343881c6c3f34bc59", "class_name": "RelatedNodeInfo"}}, "text": "A rotary evaporator was used to \n\nevaporate the filtrate sample. The extraction yield was \n\ncalculated using the formula: \n\n\ud835\udc38\ud835\udc65\ud835\udc61\ud835\udc5f\ud835\udc4e\ud835\udc50\ud835\udc61\ud835\udc56\ud835\udc5c\ud835\udc5b \ud835\udc66\ud835\udc56\ud835\udc52\ud835\udc59\ud835\udc51(%) =\n\ud835\udc64\ud835\udc52\ud835\udc56\ud835\udc54\u210e\ud835\udc61 \ud835\udc5c\ud835\udc53 \ud835\udc52\ud835\udc65\ud835\udc61\ud835\udc5f\ud835\udc4e\ud835\udc50\ud835\udc61 \ud835\udc5c\ud835\udc4f\ud835\udc61\ud835\udc4e\ud835\udc56\ud835\udc5b\ud835\udc52\ud835\udc51\n\n\ud835\udc61\ud835\udc5c\ud835\udc61\ud835\udc4e\ud835\udc59 \ud835\udc64\ud835\udc52\ud835\udc56\ud835\udc54\u210e\ud835\udc61 \ud835\udc5c\ud835\udc53 \ud835\udc60\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52 \ud835\udc62\ud835\udc60\ud835\udc52\ud835\udc51\n \u00d7 100\n\nPhytochemical screening \n Phytochemical screenings were performed for basic \n\nalkaloids, coumarins, saponins, glycosides, reducing \n\nsugars, sterols, triterpenes, and flavonoids [13]. \n\nAntimicrobial Screening \nAntimicrobial analysis was done by the disc diffusion \n\nmethod [14]. Test solution of 200mg/ml, 100mg/ml, \n\n50mg/ml, and 25mg/ml concentration of plant was \n\nprepared for antimicrobial test against the bacterial \n\npathogens namely Staphylococcus aureus, Klebsiella \n\npneumoniae, Bacillus subtilis, and Streptococcus in Muller \n\nHinton Agar. The human pathogenic strain was \n\nobtained from Dhulikhel Hospital, Kavrepalanchok, \n\nNepal. The plant extract was prepared in the No.1 \n\nWhatman filter. CIP30: Ciprofloxacin-30 \u00b5g/disc, T30: \n\nTetracyclin-30 \u00b5g/disc, and A10: Ampicillin-10 \u00b5g/disc \n\nwere used to equate the zone of inhibition with regular \n\nantibiotic discs. \n\nDPPH free radical scavenging assay \n The DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical \n\nscavenging method was used to test antioxidant activity \n\n[15, 16]. Ascorbic acid was used as a standard scavenger. \n\nDimethyl sulfoxide (DMSO) was used as a solvent to \n\nprepare a solution of ascorbic acid and plant extracts at \n\nconcentrations of 0.5, 2, 4, 8, 16, and 32 \u00b5g/ml. Then, \n\nusing a UV spectrophotometer set to 517nm, the IC50 (the \n\nsample needed to scavenge 50% of the DPPH free \n\nradical) was measured for each concentration of the \n\nsample. The equation for IC50 calculation:  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 18-23      Shrestha & Lamichhane .   \n\n\u00a9NJB, BSN 20 \n\n\ud835\udc43\ud835\udc52\ud835\udc5f\ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc4e\ud835\udc54\ud835\udc52\ud835\udc46\ud835\udc50\ud835\udc4e\ud835\udc63\ud835\udc52\ud835\udc5b\ud835\udc54\ud835\udc56\ud835\udc5b\ud835\udc54(\ud835\udc3c\ud835\udc3650) =\n\ud835\udc340 \u2212 \ud835\udc341\n\n\ud835\udc340\n \u00d7 100 \n\nwhere, A0 = DPPH solution absorbance, A1= DPPH \n\nabsorbance in relation to various extract concentrations. \n\nThe IC50 was determined by plotting a graph of \n\nconcentration versus percent inhibition and using a \n\nlinear trend line equation. Higher free radical \n\nscavenging activity was demonstrated by a lower \n\nabsorbance of the reaction mixture. \n\nBrine shrimp lethality assay \n The toxic property of T. cordifolia methanolic extract was \n\ndetermined using the brine shrimp lethality [17] \n\ntechnique. To obtain various concentrations, the extracts \n\nwere dissolved in DMSO and diluted with artificial sea \n\nsaltwater.", "start_char_idx": 6647, "end_char_idx": 9099, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f1baba6b-79b6-4a93-9b21-e44acb464b01": {"__data__": {"id_": "f1baba6b-79b6-4a93-9b21-e44acb464b01", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d1286df7-db95-4a95-974b-515c5a0bf471", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "3dc1af06e43aea2f73d4997893794e3fad7fc3ab18e477c5ed35cfba3f7cd603", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "61bf99ab-5845-4cf3-9963-fd4b3f4b7d4d", "node_type": "1", "metadata": {}, "hash": "505aa58091bbd8d8aba2757be001cdf987371650d4651c9ad0255edf1b3915da", "class_name": "RelatedNodeInfo"}}, "text": "The total volume was adjusted to 10 ml with \n\naerated seawater (1000, 100, and 10ppm).  Every tube \n\nheld ten nauplii, which were incubated for 24 hours. \n\nAfter that, the tubes were examined under a magnifying \n\nglass, and the number of survivor\u2019s nauplii in each tube \n\nwas counted. Experiments were carried out in a series of \n\nthree tubes per dose, with a monitor (DMSO in seawater) \n\nand various concentrations of the test substances. The \n\nlogarithm of the extract concentration was used (to make \n\ndealing with a wide variety of values simpler). Death \n\npercentage was calculated by: \n\n\ud835\udc37\ud835\udc52\ud835\udc4e\ud835\udc61\u210e \ud835\udc5d\ud835\udc52\ud835\udc5f\ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc4e\ud835\udc54\ud835\udc52 =\n\ud835\udc37\ud835\udc52\ud835\udc4e\ud835\udc61\u210e \ud835\udc5b\ud835\udc4e\ud835\udc62\ud835\udc5d\ud835\udc59\ud835\udc56\ud835\udc56\n\n\ud835\udc3c\ud835\udc5b\ud835\udc56\ud835\udc61\ud835\udc56\ud835\udc4e\ud835\udc59  \ud835\udc5b\ud835\udc4e\ud835\udc62\ud835\udc5d\ud835\udc59\ud835\udc56\ud835\udc56\n\u00d7 100. \n\n% death corrected vs. log extract conc (ppm) was plotted \n\nand the Lethal Dose 50 (LD50) from the graph from the \n\nequation of straight-line. Lethal concentration was \n\ndetermined from the equation of the straight line. \n\nResults  \nPhytochemical Screening \nThe term \"phytochemical investigation\" refers to the \n\nprocess of identifying and characterizing crude \n\npharmaceuticals in terms of their phytochemical \n\ningredients. Chemical elements of the plant were \n\nassessed. The results for the different types of \n\nphytochemicals presence are shown in Table 1. The \n\npresence of these phytochemicals explains why plants \n\nare used to treat ailments such as diabetes, anticancer \n\ndrugs, jaundice, etc. \n\nAntibacterial activity \nThe antibacterial susceptibility test showed that bacterial \n\nstrains are susceptible to standard antibiotics. The zone \n\nof inhibition (ZOI) shown by standard antibiotics CIP30: \n\nCiprofloxacin-30mcg/disc, T30: Tetracycline-\n\n30mcg/disc, and A10: Ampicillin-10mcg/disc are shown \n\nin Table 2. \n\nTriplicates of the experiment were carried out, and the \n\nZOI values are the average in millimeters (mm). The \n\nactivity was carried out against four disease-causing \n\nbacteria, including, Klebsiella pneumoniae, Bacillus subtilis, \n\nStaphylococcus aureus, and Streptococcus. In comparison to\n\nstandard antibiotics. Streptococcus was found to be a \n\nmore susceptible comparison to other bacterial strains \n\nshowing zone to inhibition 10.3mm, 8.5mm, 6.5mm, and \n\n6mm at 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml \n\nTable 1: Qualitative phytochemical screening of T. cordifolia  from methanol extract shows the presence of alkaloids, coumarin, \nsaponins, glycosides, reducing sugar and triterpenes \n\nPhytochemicals Alkaloids Coumarins Saponins Glycosides Reducing \n\nSugar \n\nSterols Triterpenes flavonoids Tannins and \n\npolyphenols \n\n+ + + + + - + - - \n\nPresent(+) Or Absent(-) \n\nTable 2: Antibacterial activity shown by the standard antibiotics, DMSO and plant extract against B. subtilis, K. pneumonia, S. aureus \nand Streptococcus species. Standard antibiotics Ampicillin, Ciprofloxacin and Tetracycline were taken as positive control while DMSO \nwas taken as negative control. Four different concentration of T. cordifolia sample (Tc-200mg/ml, 100mg/ml. 50mg/ml and 25mg/ml) \nwas tested for its bactericidal activity and corresponding values represent the respective zone of inhibition.", "start_char_idx": 9100, "end_char_idx": 12160, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "61bf99ab-5845-4cf3-9963-fd4b3f4b7d4d": {"__data__": {"id_": "61bf99ab-5845-4cf3-9963-fd4b3f4b7d4d", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f1baba6b-79b6-4a93-9b21-e44acb464b01", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "f96b00eb7a6f8af4828b19f05281250c67bb4f320626b7bbe7805c7fc5d9e10b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b619583e-d532-4308-943b-26339bb4b780", "node_type": "1", "metadata": {}, "hash": "908b9f2cb4b11a323e53f16686610439c61de110413ad7e17f9dc1c5d709ba8d", "class_name": "RelatedNodeInfo"}}, "text": "Bacterial Strain \nTest sample \n\nZone of Inhibition(mm) \n\nBacillus Subtilis Klebsiella pneumoniae Staphylococcus aureus Streptococcus \n\nAmpicillin(10\u00b5g) 11\u00b11 14\u00b10.5 12 10\u00b11 \nCiproflaxin(30\u00b5g) 26\u00b11 27 26 24 \nTetracycline(30\u00b5g) 19\u00b11 22\u00b11 19\u00b11.08 19 \nDMSO 0 0 0 0 \nTc - 200mg/ml 8 6.5 6 10.3 \nTc- 100mg/ml 7 6.5 6 8.5 \nTc - 50mg/ml 6.5 6 6 6.5 \nTc - 25mg/ml 6 6 6 6 \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 18-23   Shrestha & Lamichhane .   \n\n\u00a9NJB, BSN 21 \n\nof concentration respectively. Similarly, T. cordifolia was \n\nfound to be mildly inhibitory Bacillus subtilis showing a \n\nzone of inhibition 8mm, 7mm, 6.6mm and 6mm at \n\n200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml \n\nrespectively.  T. cordifolia did not show a prominent zone \n\nof inhibition for Klebsiella pneumoniae and Staphylococcus \n\naureus. The zone of inhibition against all the \n\nmicroorganisms is shown in Figure 2, Figure 3, and \n\nTable 2. \n\nAssay for scavenging DPPH free radicals \nDPPH scavenging percentage was found to be \n\nconcentration-dependent. The absorbance was \n\ndetermined using a spectrophotometer at 517nm for the \n\ncalculation of %scavenging of T. cordifolia. Ascorbic acid \n\nwas taken as a standard analyte. The scavenging \n\npercentage of ascorbic acid and extract of   T. cordifolia is \n\ncompared in Figure 4. From, the linear regression \n\nanalysis the IC50 value of T. cordifolia and ascorbic acid \n\nwas observed to be 238\u00b5g/ml and 4.76\u00b5g/ml \n\nrespectively. \n\nFigure 4. The graph shows the DPPH scavenging activity of \nmethanol extract of T. cordifolia.  The percent scavenging \nactivity is compared with Ascorbic acid. \n\nFigure 5. Toxicity effects of T. cordifolia  Shown by Brine \nshrimp Bioassay. The trend line equation gives lethal \nconcentration (LC50) of 232.64 ppm. \n\nBrine shrimp bioassay \nThe methanol extract of T. cordifolia showed good brine \n\nshrimp larvicidal activity. The regression trend line \n\ngraph was plotted using the concentration and death \n\nFigure 2. Comparision of ZOI of T.Cordifolia with standard \nantbiotic discs Amp10, CIP30 and T30. \n\nFigure 3. T. cordifolia showing ZOI against Streptococcus spp. \nat 200mg/ml, 100mg/ml, 50mg/ml and 25mg/ml. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 18-23      Shrestha & Lamichhane .   \n\n\u00a9NJB, BSN  22 \n\npercentage. The lethality concentration (LC50) obtained \n\nfrom regression analysis (Figure 5)was found to be \n\n232.64 ppm. \n\nDiscussion \nThe presence of phytochemicals alkaloids, coumarin, \n\nsaponins, glycosides, reducing sugar and triterpenes \n\ngives evidence of its anti-diabetic, anticancer, anti-\n\ninflammatory infections, anti-viral infections. Many of \n\nthese active compounds have various \n\nimmunomodulatory and physiological functions [18]. \n\nThe presence of these compounds may be the reason for \n\nthe antibacterial, antioxidant, and cytotoxic activity of \n\nplants.", "start_char_idx": 12163, "end_char_idx": 15000, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b619583e-d532-4308-943b-26339bb4b780": {"__data__": {"id_": "b619583e-d532-4308-943b-26339bb4b780", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "61bf99ab-5845-4cf3-9963-fd4b3f4b7d4d", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "7150af2f372dcf84a42778ddc06ca574308263dd6bc59007e97c3a14cf23f3da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "79717e2f-9bce-4f55-ba3e-f38c79877b44", "node_type": "1", "metadata": {}, "hash": "b705e885fe975407305d72382abe0c8e59e9028dd2300727de133ff491c5c7e3", "class_name": "RelatedNodeInfo"}}, "text": "The antibacterial activity was found to be \n\nsusceptible mostly to gram-positive bacteria. The \n\npresence of triterpenes shows that it has antibacterial \n\nactivity. A previous study suggests that ethanol extract \n\nT. cordifolia showed good inhibition against both gram-\n\npositive and gram-negative bacteria.   Present findings \n\nsupport the applicability of T. cordifolia in traditional \n\nsystems for its claimed uses like fever inflammations, \n\nurinary and skin diseases [19]. Methanol was employed \n\nto extract antioxidant-rich fractions from the stem of T. \n\ncordifolia in this investigation. Methanol is a better \n\nsolvent for the extraction of chemicals than other \n\nsolvents, according to previous research. The presence of \n\npolyphenols and tannins in plant extracts can contribute \n\nto their antioxidant activity [20].  \n\nAccording to Meyer\u2019s toxicity index [21], extracts with \n\nLC50 < 1000 \u03bcg/ml are considered as toxic, while extracts \n\nwith LC50 > 1000 \u03bcg/ml are considered as non-toxic . The \n\nlethal concentration of the extract was found to be 232.64 \n\nwhich indicates the extract is toxic and worthy of further \n\ninvestigation. The early toxicity information acquired \n\nfrom the Brine Shrimp Lethality Assay provides LC50 \n\nvalues, which can be used as a starting point for further \n\ntoxicity research. [22]. \n\nConclusion \nHerbal medicines are critical in the restoration of modern \n\ncivilization's health. T. cordifolia has been utilized \n\npharmacologically in the treatment of a variety of \n\ndiseases, according to the literature review. The findings \n\nof the phytochemicals study back up the common use of \n\nT. cordifolia to treat several ailments like jaundice, \n\nrheumatism, urinary tract infections, dermal diseases, \n\nanemia, inflammation, diabetes, etc. [7].  The main \n\nconstituents of T. cordifolia are tinosporine, tinosporide, \n\ntinosporaside, cordifolide, cordifol, heptacosanol, \n\nclerodane furano diterpene, diterpenoid furanolactone \n\ntinosporidine, columbin, and b-sitosterol. Berberine, \n\nPalmatine, Tembertarine, Magniflorine, Choline, and \n\nTinosporin are reported from its stem. This chemical \n\nconstituent plays a major role in the antibacterial, \n\nantioxidant, and cytotoxicity role of T. cordifolia [23]. \n\nT. cordifolia has gain attention due to its immense \n\napplication in traditional as well as modern medicine. \n\nTinospora cordifolia is seasonal and geographically \n\ndependent. At the same time, the organic and aqueous \n\nextracts of Tinospora cordifolia could be used in the \n\npharmaceutical sector in the future as a source of \n\nimportant phytochemical substances. \n\nAuthor\u2019s contributions \n Janardan Lamichhane contributed to designing the \n\nexperiment. Tara Shrestha contributed to all the \n\nlaboratory works and data analysis work. JL and TS \n\ncontributed equally in drafting and reviewing the \n\nmanuscript.   \n\nCompeting Interests \n No competing interests were disclosed. \n\nFunding \n This research is funded by the KOICA 1-1 project \n\nentitled \u2018Establishment of KS B&W center for \n\nindustrialization of natural resources in Nepal\u2019. \n\nAcknowledgment \nTara Shrestha is thankful to Janardan Lamichhane, for \n\nthe guidance during the project and department of \n\nbiotechnology, Kathmandu University for the \n\nequipment and laboratory facilities. \n\nEthical Approval and Consent \nNot applicable. \n\nReferences \n1. Handa SS. An overview of extraction techniques for medicinal and \n\naromatic plants. Extraction technologies for medicinal and \naromatic plants. 2008; 1. \n\n2. Doughari JH. Phytochemicals: extraction methods, basic \nstructures, and mode of action as potential chemotherapeutic \nagents. Phytochemicals-A global perspective of their role in \nnutrition and health. 2012; 3:1-27. DOI: 10.5772/26052 \n\n3.", "start_char_idx": 15001, "end_char_idx": 18755, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "79717e2f-9bce-4f55-ba3e-f38c79877b44": {"__data__": {"id_": "79717e2f-9bce-4f55-ba3e-f38c79877b44", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b619583e-d532-4308-943b-26339bb4b780", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "bacce945204e1f895c78c73b790420eb492d1c590b810729740b9023488b0980", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5044d523-d211-4c3a-82d3-2bb1634910a6", "node_type": "1", "metadata": {}, "hash": "950b513d3c4e3b93c00f68ada52b58a39fd78aa4f8d2dc65a07cfe089ca5414c", "class_name": "RelatedNodeInfo"}}, "text": "DOI: 10.5772/26052 \n\n3. Ahvazi M, Khalighi-Sigaroodi F, Charkhchiyan MM, Mojab F, \nMozaffarian VA, Zakeri H. Introduction of medicinal plants species \nwith the most traditional usage in Alamut region. Iranian journal \nof pharmaceutical research: IJPR. 2012; 11(1):185. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 18-23      Shrestha & Lamichhane .   \n\n\u00a9NJB, BSN  23 \n\n4. Spandana U, Ali SL, Nirmala T, Santhi M, Babu SS. A review on \nTinospora cordifolia. International Journal of Current \nPharmaceutical Review and Research. 2013;4(2):61-8. \n\n5. Mittal J, Sharma MM, Batra A. Tinospora cordifolia: a multipurpose \nmedicinal plant-A. Journal of Medicinal Plants. 2014;2(2). \n\n6. Saha S, Ghosh S. Tinospora cordifolia: One plant, many roles. \nAncient science of life. 2012 Apr;31(4):151.0. Doi: 10.4103/0257-\n7941.107344 \n\n7. Joshi G, Kaur R. Tinospora cordifolia: a phytopharmacological \nreview. International journal of Pharmaceutical sciences and \nresearch. 2016 Mar 1;7(3):890. Doi:10.13040/0975-8232.7 (3).890-97.  \n\n8. Kumar AB, Basalingappa KM, Kumar JR, Karthikeyan M. \npreliminary phytochemical analysis of methanolic extract of \ntinospora cordifolia and its anti-bacterial action on E. coli cell \ndivision. Journa l of Drug and Medicine. 2017 Jul; 9:52-60. \n\n9. Shanthi V, Nelson R. Anitbacterial activity of Tinospora cordifolia \n(Willd) Hook. F. Thoms on urinary tract pathogens. Int J Curr \nMicrobiol App Sci. 2013;2(6):190-4. \n\n10. Bhawya D, Anilakumar KR. In vitro antioxidant potency of \nTinospora cordifolia (gulancha) in sequential extracts. \nInternational Journal of Pharmaceutical & Biological Archives. \n2010;1(5):448-56. \n\n11. Upadhyay N, Ganie SA, Agnihotri RK, Sharma R. Free radical \nscavenging activity of Tinospora cordifolia (Willd.) Miers. Journal \nof Pharmacognosy and Phytochemistry. 2014 Jul 1;3(2):63-9. \n\n12. Azwanida NN. A review on the extraction methods use in \nmedicinal plants, principle, strength and limitation. Med Aromat \nPlants. 2015 Jul;4(196):2167-0412. DOI: 10.4172/2167-0412.1000196 \n\n13. Yadav M, Chatterji S, Gupta SK, Watal G. Preliminary \nphytochemical screening of six medicinal plants used in traditional \nmedicine. Int J Pharm Pharm Sci. 2014; 6(5):539-42.  \n\n14. Karaman I, \u015eahin F, G\u00fcll\u00fcce M, \u00d6\u01e7\u00fct\u00e7\u00fc H, \u015eeng\u00fcl M, Ad\u0131g\u00fczel A. \nAntimicrobial activity of aqueous and methanol extracts of \nJuniperus oxycedrus L. Journal of ethnopharmacology. 2003 Apr 1; \n85(2-3):231-5. DOI: 10.1016/s0378-8741(03)00006-0 \n\n15. Sharma OP, Bhat TK. DPPH antioxidant assay revisited. Food \nchemistry.", "start_char_idx": 18732, "end_char_idx": 21270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5044d523-d211-4c3a-82d3-2bb1634910a6": {"__data__": {"id_": "5044d523-d211-4c3a-82d3-2bb1634910a6", "embedding": null, "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-114", "node_type": "4", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "c176419c65b9e91157b952e9d992a407217d5f3bc8aad8e8a6b6628125b35c89", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "79717e2f-9bce-4f55-ba3e-f38c79877b44", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "43667644f5faab514b271d9e4dfdc18b92edea33e0f9483dc2ee29bb74a278ee", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8d51f7be-874a-4f25-8644-38179015ee07", "node_type": "1", "metadata": {}, "hash": "c3b47157f7f9d632d4923d9e26fab7d813bb99691a435992969e1907eb3c220a", "class_name": "RelatedNodeInfo"}}, "text": "DPPH antioxidant assay revisited. Food \nchemistry. 2009 Apr 15; 113(4):1202-5. \nhttps://doi.org/10.1016/j.foodchem.2008.08.008 \n\n16. Carmona-Jim\u00e9nez Y, Garc\u00eda-Moreno MV, Igartuburu JM, Barroso \nCG. Simplification of the DPPH assay for estimating the \nantioxidant activity of wine and wine by-products. Food chemistry. \n2014 Dec 15; 165:198-204. \nhttps://doi.org/10.1016/j.foodchem.2014.05.106 \n\n17. Padmaja R, Arun PC, Prashanth D, Deepak M, Amit A, Anjana M. \nBrine shrimp lethality bioassay of selected Indian medicinal plants. \nFitoterapia. 2002 Oct 1; 73(6):508-10. \nhttps://doi.org/10.1016/S0367-326X (02)00182-X \n\n18. Saha S, Ghosh S. Tinospora cordifolia: One plant, many roles. Anc \nSci Life. 2012; 31(4):151-159. doi:10.4103/0257-7941.107344). \nhttps://dx.doi.org/10.4103%2F0257-7941.107344 \n\n19. Jeyachandran R, Xavier TF, Anand SP. Antibacterial activity of \nstem extracts of Tinospora cordifolia (Willd) Hook. f & Thomson. \nAncient science of life. 2003 Jul; 23(1):40. \n\n20. Sahoo S, Ghosh G, Das D, Nayak S. Phytochemical investigation \nand in vitro antioxidant activity of an indigenous medicinal plant \nAlpinia nigra BL Burtt. Asian Pacific journal of tropical \nbiomedicine. 2013 Nov 1;3(11):871-6. \nhttps://doi.org/10.1016/S2221-1691 (13)60171-9 \n\n21. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B.,Nichols, \nD.E., McLaughlin, J.L., 1982. Brine Shrimp: A convenient general \nbioassay for active plant constituents.Planta Medica. 45, 31-34. \n\n22. Apu AS, Muhit MA, Tareq SM, Pathan AH, Jamaluddin AT, \nAhmed M. Antimicrobial Activity and Brine Shrimp Lethality \nBioassay of the Leaves Extract of Dillenia indica Linn. Journal of \nYoung Pharmacists. 2010 Jan 1; 2(1):50-3. \nhttps://doi.org/10.4103/0975-1483.62213 \n\n23. Devprakash SK, Subburaju T, Gurav S, Singh S. Tinospora \nCordifolia:-a review on its ethnobotany, phytochemical & \npharmacological profile. Asian Journal of Biochemical and \nPharmaceutical Research. 2011 Sep;4(1):291-302. \n\n \n\nhttps://dx.doi.org/10.4103%2F0257-7941.107344\nhttps://dx.doi.org/10.4103%2F0257-7941.107344\nhttps://doi.org/10.1016/s0378-8741(03)00006-0", "start_char_idx": 21220, "end_char_idx": 23329, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8d51f7be-874a-4f25-8644-38179015ee07": {"__data__": {"id_": "8d51f7be-874a-4f25-8644-38179015ee07", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5044d523-d211-4c3a-82d3-2bb1634910a6", "node_type": "1", "metadata": {"identifier": "njb-114", "author": "Shrestha, Tara; Lamichhane, Janardan", "title": "Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)", "date": "2021-07-31", "file": "njb-114.pdf"}, "hash": "b49bcb56548e65a226c8120267be7b1c833f345b0b7c5a46ed04aa9b2b72fe20", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1187d335-8cc8-4ea8-935a-7be4d4b8de4d", "node_type": "1", "metadata": {}, "hash": "ac6871f83211faeba126f3f205e133d9b2ba128deb1eff074906a8dcbbc6722e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8-17 Research article DOI: https://doi.org/10.3126/njb.v9i1.38645 \n\n\u00a9NJB, BSN 8 \n\nAntibiotic Susceptibility Pattern of Staphylococcus aureus Isolated \nfrom Pus/Wound Swab from Children Attending International \nFriendship Children's Hospital \nBidhya Maharjan1 , Shovana Thapa Karki2, Roshani Maharjan3 \n\u00b9Department of Microbiology, St. Xavier's College, Tribhuvan University, Maitighar, Nepal \n\n\u00b2Department of Pathology, International Friendship Children's Hospital, Maharajgunj, Kathmandu, Nepal \n3Department of Microbiology, Tri-Chandra Multiple College, Tribhuvan University, Ghantaghar, Kathmandu, Nepal \n\nReceived: 04 Nov 2020; Revised: 05 Jul 2021; Accepted: 19 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract \nA wound gets infected when the organism gets invaded through the breached skin, proliferated and production of various \nenzymes, toxins, etc. In order to treat the wound infection, antibiotic susceptibility pattern of organism should be determined \nbefore the prescription of the medicine. The present study was conducted from September 2017 to March 2018 with an aim to \ndetermine antibiotic susceptibility pattern of Staphylococcus aureus identified from the pus/wound swab among the patients \nvisiting the International Friendship Children's Hospital, Kathmandu, Nepal. Total 270 sample were processed, isolated and \nidentified using standard microbiological procedure and biochemical test. Antibiotic susceptibility test was carried out by \nusing Modified Kirby Bauer's Disc Diffusion Method. Out of total sample, 51.48% (139) showed growth. The growth \ndistribution was found to be high in out-patient department 84.9% (118) than in-patient department 15.1% (21). Among 139 \npositive growth, 83.5% were gram positive and 16.5% were gram negative. All together 12 different organisms were identified, \namong which S. aureus was found to be predominant organism 105 (75.5%). S. aureus was found to be sensitive towards \nLinezolid followed by Doxycycline whereas it was found resistant towards Ciprofloxacin. Among S. aureus identified, 50% \nwere Multidrug resistant (MDR) S. aureus and 55% were Methicillin resistance S. aureus (MRSA). MRSA was found to be \nsensitive towards Linezolid followed by Doxycycline and resistant towards Ciprofloxacin. The association between MDR and \nMRSA was found positively significant (i.e. p-value = 0.000). All strains of S. aureus were found to be sensitive towards \nVancomycin.  22.86% were double disk diffusion test (D-test) positive. The prevalence of D-test was found to be high in MRSA \n(75%). The relationship between D-test and MRSA was found to be significantly correlated with each other (r = 0.39). \nLinezolid, Chloramphenicol, Vancomycin and Doxycycline is a drug of a choice for both S. aureus and MRSA infection. \nKeywords: Pus/wound swab, Staphylococcus aureus, Antibiotic susceptibility test (AST), Multidrug resistance (MDR), \n\nMethicillin resistance Staphylococcus aureus (MRSA), D-test \n\n Corresponding author, email: 23bidhya@gmail.com \n\nIntroduction \nHuman skin acts as an excellent barrier to infection, \n\nprotect underlying tissues, bones, organs, etc. and \n\nprevents the entry of microbes (i.e. potential pathogens) \n\ninto our body unless the mechanism is breached due to \n\ninjury, trauma or surgical intervention [1, 2, 3].", "start_char_idx": 48, "end_char_idx": 3398, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1187d335-8cc8-4ea8-935a-7be4d4b8de4d": {"__data__": {"id_": "1187d335-8cc8-4ea8-935a-7be4d4b8de4d", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8d51f7be-874a-4f25-8644-38179015ee07", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "6e04798c27954c847c600b1ad6a493bd8d83e368778c72ae8d05d007c85a79d5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "985c5a8c-72ab-4426-92af-0dacec206bf2", "node_type": "1", "metadata": {}, "hash": "b6200c24554086c63fc9a9941d80622f30cfb8421e310c9c919875a5b1e85866", "class_name": "RelatedNodeInfo"}}, "text": "A break \n\nin the integrity of the skin or tissues which may be \n\nassociated with disruption of the structure and \n\ncompromises its protective function is called a wound [4]. \n\nA wound gets infected when proliferating \n\nmicroorganisms invade to a level that invokes a local or \n\nsystemic response in the host [5]. During wound \n\ninfection, the bacteria multiplies, healing is disrupted and \n\nwound tissues get damage and also spread to nearby \n\ntissues. The consequences of any tissue damage, wound \n\ninfection or any internal tissue injury is pus [6]. Pus is \n\ndefined as the accumulation of dead cells and \n\nmicroorganisms, together with accumulated fluid and \n\nvarious proteins [7]. \n\nWound infection is a common problem during injury, \n\nmainly in the case of children [4, 8]. Injuries in the \n\nchildren may be due to falls followed by burns, cuts and \n\nanimal bites which causes both financial and \n\npsychological strain on the family because it drags the \n\npatient to the health care facilities [9, 10]. Wound \n\ninfection account for 70-80% mortality and also an \n\nimportant cause of morbidity among surgical patients \n\nand 75% of mortality following burn injuries [11, 12, 13]. \n\nThe common organism responsible for pus formation or \n\nwound infection are: Coagulase negative S. aureus \n\n(CONS). S. aureus, Bacillus spp., Clostridium spp., \n\nPeptostreptococcus spp., Actinomyces spp., E. coli, Proteus \n\nspp., Neisseria spp., Vibrio vulnificus, Candida spp., etc. \n\n[14]. \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-9769-1201\nmailto:23bidhya@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7    Maharjan et al.      \n\n\u00a9NJB, BSN 9 \n\nS. aureus is a versatile pathogen capable of causing a wide \n\nrange of human diseases [15]. It is a significant human \n\npathogen that causes wound infection, soft tissue \n\ninfection and produces the pus [16, 17]. It belongs to the \n\nfamily Micrococcaceae, gram positive cocci having grape \n\nlike cluster arrangement of 0.5-1.5 \u00b5m diameter, aerobic, \n\nfacultatively anaerobic, \ua7b5-hemolytic, fermentative, \n\noxidase negative, non-sporing, non-motile, non-\n\ncapsulated, yellow zone formation around the colonies \n\non MSA and oil paint appearance on NA slopes [18, 19]. \n\nThere has been a huge problem all over the world in the \n\ntreatment of infectious disease due to increase in \n\nantibiotic resistant cases [20]. Multi-drug resistant (MDR) \n\nis defined as the non-susceptibility of organism to at least \n\none agent in 3 or more antimicrobial categories, \n\nextremely drug resistance (XDR) is non-susceptibility to \n\nat least 1 agent in all but 2 or fewer antimicrobial \n\ncategories and pan drug resistance (PDR) is non-\n\nsusceptibility to all agents in all antimicrobial categories \n\n[21]. Methicillin resistant S. aureus (MRSA) has been \n\nidentified as one of the major risk pathogens associated \n\nwith the development of antimicrobial resistant [22]. \n\nMRSA is defined as a strain of S. aureus that is resistant to \n\na large group of antibiotics called \ua7b5-lactams, which \n\ninclude Penicillin and Cephalosporin [23]. In Nepal, \n\nvarious laboratories have reported the emergence of \n\nMRSA mainly community-associated MRSA (CAMRSA) \n\nwhich have been detected in the Lumbini medical college \n\nand teaching hospital while doing cross-sectional studies \n\nof prevalence of MRSA [24].", "start_char_idx": 3399, "end_char_idx": 6884, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "985c5a8c-72ab-4426-92af-0dacec206bf2": {"__data__": {"id_": "985c5a8c-72ab-4426-92af-0dacec206bf2", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1187d335-8cc8-4ea8-935a-7be4d4b8de4d", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "d6ddf3d15b4f140df7653c2b9c664c5c1e1281776506b9941272f103ea731bef", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "56e62e7d-5df0-4109-b753-c207cf47105c", "node_type": "1", "metadata": {}, "hash": "3fa625109785ebcba527f1357dcdb8119f95099d775b41bd121d2fed2fe799a3", "class_name": "RelatedNodeInfo"}}, "text": "In another study, study \n\ncarried out to assess the extent of MRSA in the \n\nKathmandu Model Hospital Kathmandu, MRSA were \n\nmore frequently isolated from pus samples and that too \n\nfrom hospitalized patients [23]. \n\nVancomycin is a glycopeptide antibiotic that inhibits cell \n\nwall biosynthesis, remains a drug of choice for treatment \n\nof severe MRSA infections. S. aureus isolates with \n\ncomplete resistance to Vancomycin (MIC\u226516\u00b5g/ml) are \n\ntermed as Vancomycin resistant S. aureus (VRSA). VRSA \n\nwas first reported in the U.S in 2002 [25]. In one of the \n\nstudies conducted in the Manmohan Memorial College \n\nand Teaching Hospital, Kathmandu, Nepal, there all the \n\nMRSA identified was found to be susceptible towards the \n\nVancomycin [26]. \n\nD-test is a simple disc diffusion test to study the \n\nmacrolide lincosamide streptogramin B resistance \n\n(MLSB), both constitutive and inducible as well as \n\nmacrolide streptogramin B resistance (MSB) in S. aureus. \n\nMacrolide group (Erythromycin, Azithromycin, \n\nRokitamycin) is a drug used to treat of S. aureus infection \n\nand also used for those allergic to the Penicillin [24]. After \n\na few years of drug's introduction in therapy, \n\nstaphylococci developed resistance to Erythromycin in \n\n1956. These resistant strains were found in France, U.K \n\nand in the U.S.A [27]. Lincosamide (Clindamycin) is used \n\nfor the treatment of MRSA infection [28]. Since these both \n\nantibiotics have the same site of drug target, there is a \n\nhigh chance of cross resistant among these antibiotics due \n\nto modification of drug target [29]. This study helps to \n\nperceive the current status in prevalence of S. aureus in \n\npus/wound swab, the antibiotic susceptibility pattern of \n\nthe isolated S. aureus and also any presence of multidrug \n\nresistant strain among the isolates. It also helps to know \n\nthe resistant towards commonly used antibiotic and \n\naware the practitioner from misusing the antibiotic. \n\nHence, the aim of the study was to assess the prevalence \n\nof S. aureus and the antibiotic susceptibility pattern of S. \n\naureus isolated from the pus/wound swab from children \n\nattending International Friendship Children's Hospital \n\n(IFCH), Maharajgunj, Kathmandu. \n\nMaterials and Methods \nSample collection and identification of \nisolates \nThe research was conducted at the Microbiology \n\nLaboratory of International Friendship Children's \n\nHospital, Maharajgunj, Kathmandu from September 2017 \n\nto March 2018. A hospital based cross-sectional study \n\nwas carried out among the patients visiting to the \n\nhospital having wound infection below 16 years, \n\nrequesting for culture and susceptibility testing.  \n\nIn total, 270 pus/wound swab samples were collected \n\nusing aseptic technique. Out of total sample, 228 samples \n\nwere collected from out-patient department (OPD) and \n\n42 samples were collected from in-patient department \n\n(IPD). Within IPD also, 12 samples were collected from \n\ngeneral ward (GW), 4 samples from special ward (Sp. \n\nward), 12 samples from surgical/burn ward (S/B ward), \n\n6 samples from infant ICU (IICU), 3 samples from \n\npediatric ICU (PICU), 4 samples surgical ICU (SICU) and \n\n1 sample from neonates ICU (NICU). Here, 130 samples \n\nwere of male and 140 samples were of female. 17 samples \n\nwere of age group 0-1 month, 54 of 1 month-1year, 75 of \n\n1-3 years, 54 of 4-6 years, 57 of 6-12 years and 13 of 12-15 \n\nyears.  \n\nThe specimens were well labelled and then transported \n\nto the laboratory and processed immediately. After \n\nmacroscopic and microscopic observation, it was \n\ncultured on Blood Agar (BA) and Mac-Conkey Agar \n\n(MA) and incubated at 37\u00b0C for 24 hrs.", "start_char_idx": 6885, "end_char_idx": 10537, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "56e62e7d-5df0-4109-b753-c207cf47105c": {"__data__": {"id_": "56e62e7d-5df0-4109-b753-c207cf47105c", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "985c5a8c-72ab-4426-92af-0dacec206bf2", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "a719ed6828aa553f1ccb2d7ae8368bc7c011f4fe81472ea5a6e73bd859635891", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5810258c-91ee-422e-b1eb-90ff193708f1", "node_type": "1", "metadata": {}, "hash": "d092adcaf1f1df06d1f08dce06cfc8d775adba17ac52b3a16e7718a8a07b9639", "class_name": "RelatedNodeInfo"}}, "text": "The isolates were \n\nidentified by colony morphology, gram staining and \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7    Maharjan et al.      \n\n\u00a9NJB, BSN 10 \n\nvarious biochemical tests [30]. The gram-positive cocci in \n\ncluster observed under microscope was considered as \n\nStaphylococcus species and was subjected under different \n\nbiochemical test for the confirmation of S. aureus. The \n\nStaphylococcus species showing catalase positive, oxidase \n\nnegative, fermentative, yellow colony on mannitol salt \n\nagar (MSA), coagulase positive and DNase positive were \n\nconfirmed as S. aureus [30]. For gram negative organism, \n\ndifferent biochemical tests such as: catalase test, oxidase \n\ntest, Sulphur Indole Motility (SIM) test, methyl red (MR) \n\ntest, Voges-Proskauer (VP) test, citrate test, \n\noxidative/fermentative (O/F) test, urease test and triple \n\nsugar iron (TSIA) test were performed for the \n\nidentification of the organism.  \n\nAntibiotic susceptibility test \nThe antibiotic susceptibility testing was done by using \n\nmodified Kirby-Bauer disc diffusion method [31] on \n\nMueller Hinton agar using antibiotic discs of Hi-Media \n\nLaboratories Pvt. Ltd. The antibiotic used was selected by \n\nfollowing Clinical and Laboratory Standards Institute \n\n(CLSI) 2017 guideline [31] for S. aureus. The antibiotics \n\nused were Cotrimoxazole (1.25/23 mcg), \n\nChloramphenicol (30 mcg), Ciprofloxacin (5 mcg), \n\nGentamycin (10 mcg), Doxycycline (30 mcg), Linezolid \n\n(30 mcg), Vancomycin (30 mcg), Azithromycin (15 mcg), \n\nMeropenem (10 mcg) and Piperacillin (100/10 mcg). \n\nNovobiocin (30 mcg) was used to identify S. epidermidis \n\nand S. saprophyticus. If the identified S. aureus was found \n\nto be resistant to at least one agent in three or more \n\nantimicrobial categories, then the organism was \n\nconsidered as multidrug resistant (MDR) and if the \n\nidentified S. aureus was found to be resistant to at least 1 \n\nagent in all but 2 or fewer antimicrobial categories, then \n\nit was considered as extremely drug resistant (XDR) [21]. \n\nAfter screening MDR, the identified S. aureus was then \n\nscreened for Methicillin resistant S. aureus (MRSA) using \n\nCefoxitin disc (30 mcg).The organisms resistant i.e. \u226421 \n\nmm Zone of inhibition (ZOI) towards the Cefoxitin were \n\nconfirmed as MRSA and those sensitive were confirmed \n\nas Methicillin sensitive S. aureus (MSSA) [25]. If the \n\norganism was found to be Vancomycin resistant in disc \n\ndiffusion method, it was further processed for the \n\nconfirmation of Vancomycin resistant S. aureus (VRSA) \n\nby using minimum inhibitory concentration (MIC) \n\nmethod [31]. S. aureus ATCC 25923 was used as control \n\nstrain. \n\nD-test \nD-test was performed by using Erythromycin disc (15 \n\nmcg) and Clindamycin disc (2 mcg). The antibiotic discs \n\nwere placed on a lawn cultured MHA plate at 15 mm \n\napart and was incubated at 37\u00b0 C at 18-24 hrs [24]. The \n\norganisms that showed flattening zone of Clindamycin \n\nadjacent to the Erythromycin disc were considered as D-\n\ntest positive (MLSBi resistant, i.e. Inducible macrolide-\n\nlincosamide-streptogramin B resistance). If the organism \n\nwas found to be resistant towards both discs then, it was \n\ntaken as Constitutive MLSB (MLSBc) and if organism \n\nshowed sensitive towards Clindamycin but resistant \n\ntowards Erythromycin, then it was taken as D-test \n\nnegative [24].", "start_char_idx": 10538, "end_char_idx": 13898, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5810258c-91ee-422e-b1eb-90ff193708f1": {"__data__": {"id_": "5810258c-91ee-422e-b1eb-90ff193708f1", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "56e62e7d-5df0-4109-b753-c207cf47105c", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "1d4170c4a4cbb1cb5c03b45f430ab8c23de1b67f7592e057824196f1296fad91", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2c043bbd-ec09-4263-9f05-3b1656dbc497", "node_type": "1", "metadata": {}, "hash": "de3df1d32677f8a86ca02a5384099a6cd19de99a283876d64805e2594e82c9b4", "class_name": "RelatedNodeInfo"}}, "text": "Data analysis \nAll the data was entered in Statistical Package for the \n\nSocial Science (SPSS) version 16. Most of the data was \n\nanalysed by using SPSS version 16 (SPSS for Windows, \n\nChicago, SPSS Inc). The association between MDR and \n\nMRSA was determined by performing chi-square test \n\nanalysed by SPSS version 16 whereas the correlation \n\ncoefficient between D-test and MRSA was calculated by \n\nusing statistical method, i.e. Karl Pearson's correlation \n\ncoefficient. In the study, we used Pearson's chi-square \n\ntest to test whether MRSA influences the increase in MDR \n\ncases or not whereas Karl Pearson's correlation \n\ncoefficient test was used to test whether there is \n\nsignificant correlation between MRSA and D-test.  \n\nResults \nGrowth pattern of culture and distribution of \nculture positive within the departments \nOut of 270 pus/wound swab samples, 139 (51.48%) were \n\nfound to be culture positive while remaining 131 (48.52%) \n\nshowed no growth. OPD showed highest positive culture \n\n118 (85%) compared to that of the IPD 21 (15%). Within \n\nthe hospital department, highest growth was seen in the \n\ndepartment of Neonates ICU (NICU) 100% (1/1) \n\nfollowed by Surgical ICU (SICU)75% (3/4), Infant ICU \n\n(IICU) 66.67% (4/6), OPD 51.75% (118/228), \n\nSurgical/Burn ward (S/B ward) 50% (6/12), General \n\nward (GW) 41.67% (5/12), Pediatric ICU (PICU) 33.33% \n\n(1/3) and lowest in Special ward (Sp. ward) 25% (1/4) \n\n(Figure 1).  \n\nBacteriological profile of pus/wound swab \nIn the study, 116 (83.5%) out of 139 were gram positive \n\nand 23 (16.5%) were gram negative. Out of total 139 \n\nculture positive cases, S. aureus 105 (75.5%) was found to \n\nbe common isolates followed by Escherichia coli 7 (5.04%) \n\nand Staphylococcus epidermidis 7 (5.04%); Pseudomonas \n\naeruginosa 6 (4.3%); unidentified organism 4 (2.9%); \n\nEnterococcus 2 (1.45%), Proteus mirabilis 2 (1.45%) and \n\nStaphylococcus saprophyticus 2 (1.45%); Salmonella Typhi 1 \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7                Maharjan et al.      \n\n\u00a9NJB, BSN  11 \n\n(0.72%), Klebsiella oxytoca 1 (0.72%), Klebsiella pneumoniae \n\n1 (0.72%) and Citrobacter species 1 (0.72%) (Table 1).  \n\nFigure 1. Distribution of culture positive cases within the \n\ndepartments. \n\nTable 1. Bacteriological profile of pus/wound swab \n\nMicroorganism identified Number Percentage \n\nS. aureus 105 75.5% \n\nE. coli 7 5.04% \nCitrobacter spp.", "start_char_idx": 13901, "end_char_idx": 16306, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2c043bbd-ec09-4263-9f05-3b1656dbc497": {"__data__": {"id_": "2c043bbd-ec09-4263-9f05-3b1656dbc497", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5810258c-91ee-422e-b1eb-90ff193708f1", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "c930c2d325ddf6db48dde353b36f3f221c30dba37edba509bce812f4d986f618", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6b854fce-9366-4bdc-8aea-d1515ce982f0", "node_type": "1", "metadata": {}, "hash": "408ff162156e54681e38cf28936ff579d079398403ccd33628f818e6644e27ef", "class_name": "RelatedNodeInfo"}}, "text": "1 0.72% \n\nP. aeruginosa 6 4.3% \n\nEnterococcus 2 1.45% \n\nS. saprophyticus 2 1.45% \n\nS. epidermidis 7 5.04% \n\nS. Typhi 1 0.72% \n\nP. mirabilis 2 1.45% \nK. oxytoca 1 0.72% \nK. pneumoniae 1 0.72% \n\nUnidentified 4 2.8% \n\nTotal 139 100.00% \n\nDistribution of S. aureus according to the \ngender, age and within the hospital \ndepartments \nAmong 105 positive sample showing S. aureus, 51% (54) \n\nwere found to be female patient and 49% (51/105) were \n\nmale patient. The highest prevalence of S. aureus was \n\nfound among age group 12-15 yrs. 87.5% (7/8) followed \n\nby the age group 1-3 yrs. 84.21% (32/38), age group 1 \n\nmonth-1 yrs. 76.92% (20/26), age group 6-12 yrs. 75% \n\n(24/32), 0-1 month 71.42% (10/14) and age group 4-6 yrs. \n\n57.14% (12/21) (Figure 2). Most of the S. aureus was \n\nhighly isolated from IICU department 100% (4/4), PICU \n\n100% (1/1), NICU 100% (1/1) followed by GW 80% (4/5), \n\nOPD 78.81% (93/118), S/B ward 33.33% (2/6) and no S. \n\naureus were isolated from Sp. ward) 0% (0/1) and SICU \n\n0% (0/3) (Table  2). \n\n \nFigure 2. Distribution of S. aureus within the age group of the \npatient \n\nTable 2. Distribution of S. aureus within hospital departments \n\nDepartments S. aureus \n\n Total Number Percentage \n\nOPD 118 93 78.81% \n\nGeneral ward 5 4 80% \n\nSpecial ward 1 0 0% \nSurgical / burn ward 6 2 33.33% \n\nInfant ICU 4 4 100% \n\nPediatric ICU 1 1 100% \n\nSurgical ICU 3 0 0% \n\nNeonates ICU 1 1 100% \n\nTotal 139 105 75.5% \n\nTable 3.", "start_char_idx": 16307, "end_char_idx": 17745, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6b854fce-9366-4bdc-8aea-d1515ce982f0": {"__data__": {"id_": "6b854fce-9366-4bdc-8aea-d1515ce982f0", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2c043bbd-ec09-4263-9f05-3b1656dbc497", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "8e01d1b0ac3e673b1a68b0b06efbaf20068bcc7880b8ca6435a56d5278193e43", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "49d3bd2d-07aa-483d-8451-2f3dbc2ef546", "node_type": "1", "metadata": {}, "hash": "b852931bed8251a79ba76c9e8aa89507541a820653febdb55c36289e7927badf", "class_name": "RelatedNodeInfo"}}, "text": "Antibiotic susceptible pattern of S. aureus (N= 105) \n\nAntibiotic used \nAntibiotic Susceptible Pattern \n\nResistant Intermediate Sensitive \n\nGentamycin 11(10%) 9(9%) 85(81%) \n\nCiprofloxacin  71(68%) 10(10%) 24(23%) \n\nChloramphenicol  4(4%) 4(4%) 97(92%) \n\nCotrimoxazole 26(25%) 8(8%) 71(68%) \n\nCefoxitin  58(55%) 0(0%) 47(45%) \n\nErythromycin  58(55%) 18(17%) 29(28%) \n\nClindamycin  28(27%) 2(2%) 75(71%) \n\nPiperacillin  4(4%) 5(5%) 96(91%) \n\nMeropenem  1(1%) 2(2%) 102(97%) \n\nAzithromycin  54(51%) 7(7%) 44(42%) \n\nDoxycycline  0(0%) 2(2%) 103(98%) \n\nLinezolid  1(1%) 0(0%) 104(99%) \n\nVancomycin  9(9%) 0(0%) 96(91%) \n\nAntibiotic susceptibility pattern of S. aureus \nand Multidrug resistant (MDR) S. aureus  \nWhile performing antibiotic susceptibility test (Figure 3), \n\nout of 105 S. aureus, 104 (99%) were found to be sensitive \n\ntowards Linezolid followed by Doxycycline 103 (98%), \n\nMeropenem 102 (97%), Chloramphenicol  97 (92%) and \n\nVancomycin  96 (91%). The organism was found to be  \n\n14 26\n38 21 32\n\n8\n\n139\n\n10\n20\n\n32\n12\n\n24\n7\n\n105\n\n71.42%\n76.92%\n\n84.21%\n\n57.14%\n75%\n\n87.50%\n\n100.00%\n\n0\n\n50\n\n100\n\n150\n\n200\n\n250\n\n300\n\nTotal S. aureus Number S. aureus Percentage\n\n228 12\n4\n\n12\n6\n\n3\n4\n\n1\n\n118 5 1 6\n4\n\n1\n3\n\n1\n\n51.75%\n41.67%\n\n25%\n50%\n\n66.67%\n33.33%\n\n75% 100%\n\n0%\n10%\n20%\n30%\n40%\n50%\n60%\n70%\n80%\n90%\n\n100%\n\nGrowth Percentage Growth Number Departments Total\n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7                Maharjan et al.      \n\n\u00a9NJB, BSN  12 \n\nresistant towards Ciprofloxacin  71 (68%) followed by \n\nCefoxitin 58 (55%) and Erythromycin  58 (55%) \n\nrespectively (Table 3). In our study, 50% (52) were found \n\nto be multidrug resistant. Among multidrug resistant \n\nalso, one strain was found to be resistant to all the \n\nantimicrobial agent used to be tested, i.e. extremely drug \n\nresistant (XDR).  \n\nFigure 3. Antibiotic susceptibility test of Staphylococcus aureus \non MHA. (VA = Vancomycin, AZM = Azithromycin, PTZ = \n\nPiperacillin, DOX = Doxycycline and LZ = Linezolid). \n\nDistribution of MRSA among S. aureus positive \nsample and its antibiotic susceptibility \npattern \nIn the study, 55% (58) were found to be Cefoxitin resistant \n\nshowing Methicillin resistant strains (MRSA) whereas \n\n45% (47) were found to be Cefoxitin sensitive showing \n\nMethicillin sensitive strains (MSSA). All the resistant \n\nstrains were further tested for Vancomycin susceptible \n\ntest.  \n\nTable 4.", "start_char_idx": 17746, "end_char_idx": 20149, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "49d3bd2d-07aa-483d-8451-2f3dbc2ef546": {"__data__": {"id_": "49d3bd2d-07aa-483d-8451-2f3dbc2ef546", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6b854fce-9366-4bdc-8aea-d1515ce982f0", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "8d238b8f078e2e67932122ca31104c9f0fd73dc2733be40bc54946dbd012603b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e70ea337-e2ed-4b0d-8ad3-7053e6949089", "node_type": "1", "metadata": {}, "hash": "56f8c93f1cd60ddcd403319eafef0f992b20b264bb63f7a69c06eaef09f3ad12", "class_name": "RelatedNodeInfo"}}, "text": "Table 4. Antibiotic Susceptibility Pattern of MRSA (N=58) \n\nAntibiotics Resistant Intermediate Sensitive \n\nCotrimoxazole 18(31.04%) 3(5.17%) 37(63.79%) \nChloramphenicol 3(5.17%) 3(5.17%) 52(89.66%) \nGentamycin 6(10.35%) 7(12.07%) 45(77.58%) \nCiprofloxacin 51(87.93%) 6(10.35%) 1(1.72%) \nClindamycin 21(36.21%) 0(0%) 37(63.79%) \nErythromycin 38(65.53%) 8(13.79%) 12(20.68%) \nPiperacillin 4(6.9%) 4(6.9%) 50(86.20%) \nMeropenem 1(1.72%) 1(1.72%) 56(96.56%) \nAzithromycin 43(74.13%) 5(8.62%) 10(17.25%) \nLinezolid 1(1.72%) 0(0%) 57(98.28%) \nDoxycycline 0(0%) 2(3.44%) 56(96.56%) \nVancomycin 9(15.52%) - 49(84.48%) \n\nHere, MRSA was found sensitive towards Linezolid \n\n98.28% (57) followed by Doxycycline 96.56% (56), \n\nMeropenem 96.56% (56), Chloramphenicol 89.66% (52), \n\nPiperacillin 86.20% (50), Vancomycin 84.48% (49), \n\nGentamycin 77.58% (45), Cotrimoxazole 63.79% (37) and \n\nClindamycin 63.79% (37). MRSA was found to be \n\nresistant towards Ciprofloxacin 87.93% (51) followed by \n\nAzithromycin 74.13% (43), Erythromycin 65.53% (38) and \n\nwas found to be zero resistant towards Doxycycline. \n\n(Table 4).  \n\nVRSA and MIC \nAmong isolated S. aureus, 9 were found to be resistant \n\ntowards the Vancomycin disc. While performing \n\nminimum inhibitory concentration test, all positive \n\nstrains were found to be sensitive towards Vancomycin \n\nin a very low concentration, i.e. 0.25 \u00b5g/ml and minimum \n\nbactericidal concentration was found to be 0.25 \u00b5g/ml. \n\nAssociation between MDR and MRSA \nIn the study, 44 (84.61%) MRSA were found to be MDR \n\nand 14 (26.42%) MRSA were found to be MDR negative. \n\nBy analyzing the data of MDR and MRSA using chi-\n\nsquare test, the value was found to be chi-square (1, \n\nN=105) =35.958, p\u02c2.01. Therefore, MDR was found to be \n\nstatistically significant associated with MRSA.  \n\nD-test of S. aureus and Correlation between D-\ntest positive and MRSA  \nIn D-test (Figure 4), out of total 105 S. aureus identified, \n\n24 (22.86%) were found to be D-test positive, 21 (20.0%) \n\nwere D-test negative, 31 (29.52%) were sensitive to both \n\nErythromycin and Clindamycin and 29 (27.62%) were \n\nconstitutive resistant (Table 5).  \n\nFigure 4. Double disk diffusion test (D-test) on MHA medium \nshowing sensitive (K) and positive result (L). CD = Clindamycin \n\nand E = Erythromycin) \n\nTable 5.", "start_char_idx": 20141, "end_char_idx": 22449, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e70ea337-e2ed-4b0d-8ad3-7053e6949089": {"__data__": {"id_": "e70ea337-e2ed-4b0d-8ad3-7053e6949089", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "49d3bd2d-07aa-483d-8451-2f3dbc2ef546", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "c124c448d9faa2d5c07d9af0e6d996ba64ec8dd7605cd31182695f54f5277a6e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0074bfb0-528e-447e-9a6e-6b2cdd9d284f", "node_type": "1", "metadata": {}, "hash": "a2f1d1dcaf63e482a6b179e453dfad142b87f8441f03c93cb14a11eff99669d8", "class_name": "RelatedNodeInfo"}}, "text": "Correlation between MRSA and D-test \n\n D-test positive D-test negative Constitutive resistant Sensitive Total r  value \n\nMRSA 18  (75%) 8(38.1%) 20 (69%) 12 (38.71%) 58 0.39 \nMSSA 6 (25%) 13 (61.9%) 9 (31%) 19 (61.29%) 47  \n\nTotal 24 21 29 31 105 (100%)  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7                Maharjan et al.      \n\n\u00a9NJB, BSN  13 \n\nHere, 18 (75%) MRSA were D-test positive, 8 (38.1%) \n\nMRSA were D-test negative, 20 (69%) MRSA were \n\nconstitutive resistant and 12 (38.71%) MRSA were \n\nsensitive as shown in Table 5. The correlation coefficient \n\n(r) between D-test and MRSA was found to be 0.39 (r = \n\n.313, p\u02c2.01), i.e. D-test and MRSA was found to be \n\npositive but lowly correlated with each other.   \n\nDiscussion \nOut of total sample, 139 (51.48%) showed growth and 131 \n\n(48.52%) showed no growth. The growth result was \n\nfound nearly similar to the study conducted by \n\nHanumanthappa et al, where they found 56% growth \n\nrate [32]. The result was lower to the study conducted by \n\nRai et al [10] 58.6%; Khan et al [33] 65.2% and Patil et al \n\n[34] 86%. The lower growth might be due to difficult-to-\n\ngrow fastidious organisms, inappropriate methods of \n\ncollection and transportation of specimens or the \n\nadministration of antibiotics prior to specimen collection. \n\nAmong 139 positive growth result, 118 (85.65%) were \n\nfound to be positive from out-patient department and 21 \n\n(14.4 %) from in-patient department. High prevalence of \n\ngrowth in OPD might be due to increase in community \n\nacquired infection. Higher positive growth in OPD was \n\nalso found in the study carried by KC et al [35] and found \n\ncontrary to the study carried out by Pant et al, where they \n\nfound 63.1% from IPD and 56.2% from OPD [36].  \n\nOut of total growth 139, 116 (83.5%) were found to be \n\nGram-positive and 23 (16.5%) were found to be gram \n\nnegative. The high prevalence of Gram-positive \n\norganism might be due to the presence of Gram-positive \n\nbacteria as a normal flora of the human body. The result \n\nwas found to be similar to the research conducted by KC \n\net al [35]; Devi et al [37] and Pant et al [36]. The result was \n\nfound contrast to the study conducted by Patil et al \n\n(2019)78% gram negative and 22% Gram-positive \n\nbacteria [34].  \n\nThe predominance of S. aureus (75.5%) in the study might \n\nbe due to S. aureus being normal flora of skin, glands, \n\nnails, etc. and having various virulence factors. The result \n\nwas seems to be related to the study conducted by \n\nSultana et al (2015) 40.45% S. aureus followed by E. coli \n\n28.18% [38]; Barakoti et al (2017) 41.45% S. aureus \n\nfollowed by E. coli 22.79% [39]; Bankar et al (2018) 34.21% \n\nS. aureus followed by E. coli 23.02%[40] and Shahi et al \n\n(2018)70.6% S. aureus [41]. However Mahat et al [42] and \n\nPatil et al [34] Pseudomonas spp. as predominant \n\norganism.  \n\nThe infection in the age group 12-15 yrs. might be due to \n\nthe various activities performed in the school, \n\nenvironment, involved in fight, their playmates and \n\ncontact with various object.", "start_char_idx": 22450, "end_char_idx": 25510, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0074bfb0-528e-447e-9a6e-6b2cdd9d284f": {"__data__": {"id_": "0074bfb0-528e-447e-9a6e-6b2cdd9d284f", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e70ea337-e2ed-4b0d-8ad3-7053e6949089", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "fcb0beb26cc970c052aecfa99f8af85eafe326a7e810db116809e183b57f43a1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ebd69c89-3a9e-402f-9234-49fd7d5a36d0", "node_type": "1", "metadata": {}, "hash": "63018e4f9c15156354165360e5135f46a77dd6ee3c210d0ab5ce53ab30ede374", "class_name": "RelatedNodeInfo"}}, "text": "The children under 5 years \n\nare also prone to get pus/wound infection because of \n\nunintentional falls, burns, etc. and not only that these \n\ngroup also has low immune power to overcome any kind \n\nof infection, therefore it is likely to get infected. The \n\noutcomes were found to be contrast with the study \n\nconducted in 2017 by Pokhrel et al, where they got higher \n\nprevalence of S. aureus in age group 1-3yrs [43]; Rai et al \n\nin age less than 1 year [10] and Pant et al in age group 1-\n\n5yrs [36]. \n\nThe distribution of S. aureus among gender was found to \n\nbe high in female patient 54 (51%) than the male patient \n\n51 (49%). The finding resembled with the research \n\nconducted by Muluye et al [20] and Bhatt et al [44]. The \n\nresult obtained from our study was contrast to the \n\nresearch conducted by Shrestha et al [28] and Garoy et al \n\n[45]. \n\nThe high prevalence of S. aureus in ICU departments \n\nmight be due to the colonization of S. aureus from \n\npatient's own flora, transmission through staff hands, air, \n\nprocedure of surgery, inanimate object, longer period of \n\nhospital stays, etc. Similar study was carried out by \n\nBhatta et al. (2014) who had reported higher prevalence \n\nof S. aureus in hospital setting accounting [44].  \n\nS. aureus was found to be highly sensitive towards \n\nLinezolid (99%) followed by Doxycycline (98%). The \n\noutcome was found similar to the study conducted by \n\nNirmala et al [2] of 100% sensitive towards Linezolid and \n\nVancomycin and by Khan et al [33]. It was found to be a \n\nbit different from the research carried out in 2018 by \n\nTadesse et al [46] in which they found 100% sensitive \n\ntowards Ampicillin. \n\nFrom the study, out of 105 isolates, 52 (50%) were found \n\nto be MDR. Among MDR also one strain was found to be \n\nresistant to all the antimicrobial agents to be tested \n\n(Extremely drug-resistant). MDR cases may be due to \n\naccumulation of multiple genes, expression of genes that \n\ncode for multidrug efflux pumps, extruding a wide range \n\nof drugs, mutational alteration of the target protein, \n\nenzymatic inactivation of drugs, etc. [47].  \n\nHere, 44 (84.61%) MDR were found to be MRSA and 8 \n\n(15.39%) MDR were found be MSSA. The increase in \n\nMDR in MRSA may be due to a distinctive feature of \n\nMRSA, i.e. their resistance to \u03b2-lactam antibiotics. \n\nTherefore, once the S. aureus is resistant to Methicillin, it \n\nmay also show resistance towards other antibiotic classes \n\nlike: aminoglycosides, macrolides, tetracycline, \n\nchloramphenicol and lincosamide. Our result was lower \n\nin comparison to Upreti et al [48] with 68.2% MDR, \n\nPahadi et al [49] with 86.41% were MDR; Tadesse et al \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7                Maharjan et al.      \n\n\u00a9NJB, BSN  14 \n\n[46] 82.3% MDR; whereas higher than he study \n\nconducted by Kadariya et al [50] with 44.2% were MDR \n\nand Mama et al with 27.8% were MDR [51]. \n\nThe strong association between the MDR and MRSA was \n\nfound (p\u02c2.01) while performing Pearson chi-square test. \n\nHence, we can say that the prevalence of MDR increases \n\nas the prevalence of MRSA increased.", "start_char_idx": 25511, "end_char_idx": 28628, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ebd69c89-3a9e-402f-9234-49fd7d5a36d0": {"__data__": {"id_": "ebd69c89-3a9e-402f-9234-49fd7d5a36d0", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0074bfb0-528e-447e-9a6e-6b2cdd9d284f", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "63ec98c1658c0726a32550bee419529d2c15e5e2cdd785b354f98495311a0c91", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "929b7afa-f057-43a8-bc3b-a65cd0b7c735", "node_type": "1", "metadata": {}, "hash": "f4cf0ba176ce085174065ca601ea4ceff0c667c48b7b3c203b1f327b28e8a714", "class_name": "RelatedNodeInfo"}}, "text": "The data obtained \n\nfrom the research was found to be similar to the study \n\nconducted by Joachim et al in which 21.3% were MDR, \n\nout of which 72.7% of MRSA strains were MDR showing \n\nstatistically significant association between MRSA and \n\nMDR among S. aureus isolates (p=0.001) [52].  \n\nThe prevalence of MRSA was found to be 58 (55%) and \n\nmethicillin sensitive S. aureus (MSSA) was found to be 47 \n\n(45%). The study resembles to the study carried out by \n\nDevi et al (2017), where 50.79% were MRSA and 49.21% \n\nwere MSSA [37]. However, the study was in \n\ncontradiction to the study carried by Kayastha et al (2010) \n\n8.92% MRSA [23]; Ansari et al (2014) 43.1% MRSA [53]; \n\nJaiswal et al (2016) 72% MRSA [54] and Adhikari et al \n\n(2017) 35.50% MRSA [55].  \n\nSince our research was conducted from September 2017 \n\nto March 2018, the prevalence of MRSA seems to be \n\nincreasing in Nepal as well [56, 53, 55, 57, 48, 45, 41]. The \n\ndevelopment of resistance of S. aureus towards \n\nMethicillin may be due to the acquisition of \n\nstaphylococcal chromosome cassette mec (SCC mec) in its \n\nchromosome, which carries a mec A gene facilitating \n\nresistance to Methicillin via Penicillin binding protein \n\n(PBP-2a). Although the acquisition of the mecA gene, the \n\norganism cannot exhibit resistant towards Methicillin \n\nunless the gene is activated. \n\nMRSA was found to be sensitive towards Linezolid \n\n98.28% (57) followed by Doxycycline 96.56% (56) and \n\nresistant towards Ciprofloxacin 87.93% (51) followed by \n\nAzithromycin 74.13%. Similar sensitive pattern in MRSA \n\nwas found in the study carried out by Choudhury et al \n\n(2016) in which organism was found sensitive towards \n\nLinezolid (99.3%), Vancomycin (99.3%) and resistant \n\ntowards Cefuroxime (59.50%) [58].  \n\nIn our study, Vancomycin resistant was found to be 9% \n\n(9/105) from disc diffusion method but while performing \n\nthe MIC, S. aureus was found to be 100% sensitive \n\ntowards Vancomycin, i.e. 0.25 \u03bcg/ml and MBC was \n\nfound to be 0.25 \u03bcg/ml. Hence, isolated S. aureus was \n\nfound to be 100% susceptibility towards Vancomycin. \n\nTherefore, we need to perform MIC for the confirmation \n\nof Vancomycin resistant strain. The cause of Vancomycin \n\nresistance may be due to the activation of van A and van \n\nB gene. The finding was found to be similar to the \n\nresearch conducted by Kshetry et al, where organism was \n\nfound sensitive towards Vancomycin while performing \n\nMIC test [59] and study by Bamigboye et al showed 1.4% \n\nVRSA but found to be van A and van B gene negative \n\n[25].  \n\nFrom the study, only 22.86% (24) were found to be D-test \n\npositive, 20% (21) were found to be   D-test negative, \n\n29.52% (31) were found to be susceptible to both \n\nErythromycin and Clindamycin and 27.62% (29) were \n\nfound to be constitutive resistant. The resistance of the \n\nErythromycin and Clindamycin may be due to the \n\nresistance encoded in Erythromycin methylase (erm) \n\ngenes. The constitutive expression may be due to the \n\norganism being resistant to all macrolides, lincosamides \n\nand type B streptogramin antibiotics.", "start_char_idx": 28629, "end_char_idx": 31714, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "929b7afa-f057-43a8-bc3b-a65cd0b7c735": {"__data__": {"id_": "929b7afa-f057-43a8-bc3b-a65cd0b7c735", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ebd69c89-3a9e-402f-9234-49fd7d5a36d0", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "9c247ec797b01fa4d3a77bb1b3cd17d248e0fa115291c0d2b792459a698165ad", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "11c70eeb-b1dd-4b18-b171-f0cdd05cedfb", "node_type": "1", "metadata": {}, "hash": "496402d3c1d8754573d5005276dc1e199ab8db99259cce03e3c09bfdb8ceda68", "class_name": "RelatedNodeInfo"}}, "text": "The study \n\nresembled to the study carried out by Mama et al [51] \n\nwith 24.1% D-test positive, 1% D-test negative, 2% \n\nconstitutive D-test and 60.85% sensitive towards \n\nErythromycin and Clindamycin. \n\nIn this study, D-test positive was also seen high in MRSA \n\n75% (18/24) compare to the MSSA 25% (6/24). Similar \n\nresult was obtained in research conducted by Pal et al \n\n[60]. The correlation (r) between D-test and MRSA was \n\nfound to be 0.39 which means D-test and MRSA are \n\npositively but lowly correlated, i.e. D-test cases may \n\nincrease as increase in MRSA cases. The result obtained \n\nwas contrast with the study carried out by Gosh et al [61]. \n\nThe increase in reported inducible Clindamycin resistant \n\nshows the increase in prevalence of inducible \n\nClindamycin resistance along with constitutive resistant \n\namong the clinical isolates of S. aureus. Hence, the \n\nscreening of inducible Clindamycin resistant should be \n\ndone in every clinical laboratory.  \n\nConclusion \nPrevalence of wound infection was found to be high \n\n(51.48%) in our study. The growth rate was found high in \n\nOPD patient than IPD. S. aureus was predominant \n\norganism followed by E. coli and S. epidermidis. The \n\nprevalence of S. aureus was seen high in the age group of \n\n12-15 years. The cases were also seen high in the \n\ndepartment of IICU, PICU, and NICU. High prevalence \n\nof MRSA was observed in this study. The isolates were \n\nsensitive mainly towards Linezolid, Doxycycline, \n\nMeropenem, and Chloramphenicol, Vancomycin. The \n\norganism was found highly resistant towards \n\nCiprofloxacin. 50% of isolates were found to be MDR. \n\nAmong MDR, one strain was found to be XDR. MDR was \n\nmainly found in MRSA than MSSA strain. Hence, all \n\nMRSA are considered as MDR. D-test positive cases was \n\nfound higher in MRSA cases. Since, inducible D-test has \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7                Maharjan et al.      \n\n\u00a9NJB, BSN  15 \n\nbeen reported, it is necessary to screen the inducible \n\nClindamycin resistance before the prescription of the \n\nmedication for the effective treatment of infection. \n\nVancomycin, Linezolid, Doxycycline, Meropenem, and \n\nChloramphenicol were effective drug for S. aureus and \n\nMRSA. \n\nAuthor's contribution \nBM conducted laboratory experiments, data analysis, \n\ninterpretation and manuscript writing; STK designed the \n\nresearch conception, reviewed the manuscript; RM \n\ndesigned the research, contributed in data analysis, \n\nmanuscript writing, reviewing and editing. All authors \n\nread and approved the final manuscript. \n\nCompeting interests \nWe have read Nepal journal of biotechnology policy on \n\ndeclaration of competing interest and declare that we \n\nhave no competing interests. \n\nFunding \nThe author(s) declared that no grants were involved in \n\nsupporting this work. \n\nAcknowledgements \nWe are very beholden for the support provided by \n\nInternational Friendship Children's Hospital and the St. \n\nXavier's College, Kathmandu, Nepal. \n\nEthical approval and Consent \nThis research was approved by Nepal Health Research \n\nCouncil (NHRC), Kathmandu, Nepal (Ref. no.-2610), \n\nInternational Friendship Children's Hospital, \n\nMaharajgunj, Nepal and the St. Xavier's College, \n\nKathmandu, Nepal. Informed consent was obtained from \n\nparents of participants before their participation. \n\nReferences \n1. Mohammed A, Adeshina GO, Ibrahim YKE. Retrospective \n\nincidence of wound infections and antibiotic sensitivity pattern: A \nstudy conducted at the Aminu Kano Teaching Hospital, Kano, \nNigeria.", "start_char_idx": 31715, "end_char_idx": 35264, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "11c70eeb-b1dd-4b18-b171-f0cdd05cedfb": {"__data__": {"id_": "11c70eeb-b1dd-4b18-b171-f0cdd05cedfb", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "929b7afa-f057-43a8-bc3b-a65cd0b7c735", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "7b1dac83e790c6610ecec46d0fffdb3b13efeaa3df8b658585bd1f0b66ab2943", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ee2e6c92-9c19-45e4-a11c-46284ac9f6b2", "node_type": "1", "metadata": {}, "hash": "cbeb879799e305fd1c39f31263c76324a76f431af732282c2f1867f502f911a7", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of Medicine and Medical Sciences. \n2013;5(2):60-66. doi: 10.5897/IJMMS12.114 \n\n2. Nirmala S, Sengodan R. Aerobic bacterial isolates and their \nantibiotic susceptibility pattern from pus samples in a tertiary care \ngovernment hospital in Tamilnadu, India. Int J Curr Microbiol \nApp Sci. 2017 Jun 10;6(6):423-442. \nhttps://doi.org/10.20546/ijcmas.2017.606.050 \n\n3. Paul W, Sharma CP. Advanced in Wound Healing Materials: \nScience and Skin Engineering. 1st ed. United Kingdom: A Smithers \nGroup Company; 2015.25 p. \n\n4. Aftab S, Yusuf MA, Siddique MA, Tarik MM. Clinical and \nmicrobiological aspect of wound infection: a review update. \nBangladesh Journal of Infectious Diseases. 2015;1(2): 32-\n37.https://doi.org/10.3329/bjid.v1i2.24903 \n\n5. Swanson T, Angel D, Sussman G, Cooper R et al. International \nwound infection institute. Wound infection in clinical practice: \nPrinciples of best practice. London: A Wound International \nPublication. International consensus update 2016; 2016. 7p. \n\nhttp://eprints.hud.ac.uk/id/eprint/30637/1/iwii-\nconsensus_final-web.pdf  \n\n6. Medical News Today [Internet]. Pus: What is it and why does it \nhappen? 2017 (updated Wed 21 June \n2017).https://www.medicalnewstoday.com/articles/249182.php\n.  \n\n7. Kindt TJ, Goldsby RA, Osborne BA. Kuby Immunology. 6th ed. \nUnited State of America:Sara Tenney; 2007. 340p. \n\n8. Sethi D, Towner E, Vincenten J, Segui-Gomez M and Racioppi F. \nEuropean report on child injury prevention. World Health \nOrganization. 2008. \n\n9. Shriyan P, Prabhu V, Aithal K, Yadav UN, OrgochukwuMJ. Profile \nof unintentional injury among under five children in coastal \nKarnataka, India: a cross-sectional study. Int J Med Sci Public \nHealth. 2014 Jan;3(11):1317-\n1319.https://www.researchgate.net/publication/271186756. \n\n10. Rai S, Yadav UN, Pant ND, Yakha JK, Tripathi PP, Poudel A, et al. \nBacteriological profile and antimicrobial susceptibility patterns of \nbacteria isolated from pus/wound swab samples from children \nattending a tertiary care hospital in Kathmandu, Nepal. IntJ \nMicrobiol.2017 Mar 6;2017:2529085. doi: 10.1155/2017/2529085. \n\n11. Manikandan C, Amsath A. Antibiotic susceptibility of bacterial \nstrains isolated from wound infection patients in Pattukkottai, \nTamilnadu, India. Int J CurrMicrobiol App Sci. 2013;2(6):195-203. \nhttp://www.ijcmas.com/ \n\n12. Alebachew T, Yismaw G, Derabe A, Sisay Z. Staphylococcus aureus \nburn wound infectionamong patients attending Yekatit 12 hospital \nburn unit, Addis Ababa, Ethiopia. Ethiop J Health Sci. 2012 \nNov;22(3):209-213. \nhttps://www.ncbi.nlm.nih.gov/pubmed/23209356  \n\n13. Thanni LOA, Osinupebi OA, Deji M. Prevalence of bacterial \npathogens in infectedwounds in a Tertiary Hospital, 1995-2001: \nany change in trend? JNatilMed Assoc.", "start_char_idx": 35265, "end_char_idx": 38042, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ee2e6c92-9c19-45e4-a11c-46284ac9f6b2": {"__data__": {"id_": "ee2e6c92-9c19-45e4-a11c-46284ac9f6b2", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "11c70eeb-b1dd-4b18-b171-f0cdd05cedfb", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "eed2bfca959936effa70f16f097eeebbfefb2f198670d77b9f85198d7e766479", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5333db6d-02e5-4e71-aaa4-1c84fb7d009e", "node_type": "1", "metadata": {}, "hash": "664b9fee95e72f88f5007b6cab3cfad78b0fca8770ad0b98515b100df83a5ab5", "class_name": "RelatedNodeInfo"}}, "text": "JNatilMed Assoc. 2003 Dec; 95(12):1189-1195. \nhttp://www.ncbi.nlm.nih.gov/pmc/articles/pmc2594861/  \n\n14. Carson JA. Wound cultures: In Leber A (ed). Clinical microbiology \nprocedures handbook. 4th ed. Washington, DC: ASM; 2016.  \n3.13.1.1-3.13.2.4 p. \n\n15. Gordon RJ, Lowy FD. Pathogenesis of methicillin-resistant \nStaphylococcus aureus infection. Clin Infect Dis. 2008 Jun 1;46Suppl \n5(Suppl5):S350-S359. doi: 10.1086/533591. \n\n16. Yasmeen F, Sarwar MI, Hakeem A, Sherwani SK, Hussain MS, Zeb \nM, Irfan S, Khan MM. Identification of Staphylococcus aureus in pus \nsamples and its anti-microbial susceptibility against Imipenem, \nTobramycin and Linezolid. International IJBMSP. 2014 Jun;4(1):9-\n12. \nhttps://www.ijbmsp.org/index.php/IJBMSP/article/view/56/4\n3  \n\n17. Kumar AR. Antimicrobial sensitivity pattern of Klebsiella \npneumonia isolated from pus from tertiary care hospital and issues \nrelated to the rational selection of antimicrobials. JChem \nPharmRes. 2013;5(11):326-331. http://www.jocpr.com/  \n\n18. Gillespie SH, Hawkey PM. Principle and practice of clinical \nbacteriology. 2nd ed. England: John Wiley and Sons Ltd; 2006. 73-\n98 p. \n\n19. Parija SC. Textbook of Microbiology and Immunology. 2nd ed. \nIndia: Elsevier, a division of Reed Elsevier India Private Limited; \n2013. 173-181 p. \n\n20. Muluye D, Wondimeneh Y, Ferede G, Nega T, Adane K, Biadgo B, \net al. Bacterial isolates and their antibiotic susceptibility patterns \namong patients with pus and/or wound discharge at Gondar \nuniversity hospital. BMC ResNotes. 2014 Sep 9;7:619. \ndoi:10.1186/1756-0500-7-619. \n\n21. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, \nGiske CG, et al. Multidrug-resistant, extensively drug-resistant \nand pandrug-resistant bacteria: an international expert proposal \nfor interim standard definitions for acquired resistance. \nClinMicrobiol Infect. 2012;18(3):268-281. \nhttps://doi.org/10.1111/j.1469-0691.2011.03570.x  \n\n22. Harkins CP, Pichon B, Doumith M, Parkhill J, Westh H, Tomasz A, \net al. Methicillin-resistant Staphylococcus aureus emerged long \nbefore the introduction of methicillin into clinical practice. \nGenome Biol. 2017Jul 20;18(1):130.doi: 10.1186/s13059-017-1252-9.", "start_char_idx": 38026, "end_char_idx": 40225, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5333db6d-02e5-4e71-aaa4-1c84fb7d009e": {"__data__": {"id_": "5333db6d-02e5-4e71-aaa4-1c84fb7d009e", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ee2e6c92-9c19-45e4-a11c-46284ac9f6b2", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "e2f6fdf37643bd544b96a0ad5ecc791ba2f459d5f2c568e4046aa9a4b3d4abe4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b73aaad6-7d50-437e-b49a-e002044209c8", "node_type": "1", "metadata": {}, "hash": "ac89bbe3c96764fe4391004f9660e39cd4c2b79cb631c66e297ae0e639c0198a", "class_name": "RelatedNodeInfo"}}, "text": "https://doi.org/10.20546/ijcmas.2017.606.050\nhttps://doi.org/10.3329/bjid.v1i2.24903\nhttp://eprints.hud.ac.uk/id/eprint/30637/1/iwii-consensus_final-web.pdf\nhttp://eprints.hud.ac.uk/id/eprint/30637/1/iwii-consensus_final-web.pdf\nhttps://www.researchgate.net/publication/271186756\nhttp://www.ijcmas.com/\nhttps://www.ncbi.nlm.nih.gov/pubmed/23209356\nhttp://www.ncbi.nlm.nih.gov/pmc/articles/pmc2594861/\nhttps://www.ijbmsp.org/index.php/IJBMSP/article/view/56/43\nhttps://www.ijbmsp.org/index.php/IJBMSP/article/view/56/43\nhttp://www.jocpr.com/\nhttps://doi.org/10.1111/j.1469-0691.2011.03570.x\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7    Maharjan et al.      \n\n\u00a9NJB, BSN 16 \n\n23. Kayastha BB, Manandhar S and Shrestha B. Methicillin resistant \nStaphylococcus aureus (MRSA) in different clinical samples from \npatients presenting at Kathmandu Model Hospital. Research Gate. \n2010 Jan \n1.https://www.researchgate.net/publication/216834367_\n\n24. Raut S, Bajracharya K, Adhikari J, Pant SS,Adhikari B. Prevalence \nof methicillin resistant Staphylococcus aureus in Lumbini Medical \nCollege and Teaching Hospital, Palpa, Western Nepal. BMC Res \nNotes 10. 2017 Jun 2;187(2017):187. \nhttps://doi.org/10.1186/s13104-017-2515-y \n\n25. Bamigboye BT, Olowe OA, Taiwo SS. Phenotypic and molecular \nidentification of Vancomycin resistance in clinical Staphylococcus \naureus isolates in Osogbo, Nigeria. Eur J MicrobiolImmunol (Bp). \n2018;8(1):25\u201330.https://doi.org/10.1556/1886.2018.00003\n\n26. Lama U, Shah D, Shrestha U. Vancomycin resistant Staphylococcus \naureus reported from Tertiary Care Hospital in Nepal. Tribhuvan \nUniversity Journal of Microbiology. 2018 Nov 6;4:63-72. \nhttps://doi.org/10.3126/tujm.v4i0.21679\n\n27. Shrestha B, Pokhrel BM, Mohapatra T. Phenotypic \ncharacterization of nosocomial isolates of Staphylococcus aureus \nwith reference to MRSA. J Infect Dev Ctries.2009 Aug 30;3(7):554-\n560. doi: 10.3855/jidc.474 \n\n28. Shrestha J, Khanal S, Poudel P, Panta OP, Prajapati KG. Methicillin \nresistant Staphylococcus aureus isolated from wound infections. \nTribhuvan University Journal of Microbiology. 2018 Sep 26;5:19-\n24. https://doi.org/10.3126/tujm.v5i0.22297\n\n29. Shrestha B, Rana SS. D test: a simple test with big implication for \nStaphylococcusaureus macrolide-lincosamide-streptogramin B \nresistance pattern. Nepal MedCollJ. 2014 Sep;16(1):88-94. PMID: \n25799821 \n\n30. Cheesbrough M. District laboratory practice in tropical countries. \n2nded. New York: Cambridge University Press; 2016.80-85, 132-\n143, 62-70, 385-400p. \n\n31.", "start_char_idx": 40228, "end_char_idx": 42774, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b73aaad6-7d50-437e-b49a-e002044209c8": {"__data__": {"id_": "b73aaad6-7d50-437e-b49a-e002044209c8", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5333db6d-02e5-4e71-aaa4-1c84fb7d009e", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "116eb8efc29283cbcfe9b8068b40e43d52395361476381715adf95aafbf7d94f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d44106f6-4bde-4838-9e19-250bfe086099", "node_type": "1", "metadata": {}, "hash": "b16236a59650e65b6bdd02984365acc17e5235269d37cceeb85009a6aa48dacb", "class_name": "RelatedNodeInfo"}}, "text": "31. CLSI (Clinical and laboratory standards institute) Guidelines. \nM100 Performance standards for antimicrobial susceptibility \ntesting. 27th ed. 950 West Valley Road, Suite 2500, Wayne, PA \n19087 USA; 2017 \n\n32. Hanumanthappa P, Vishalakshi B, Krishna S. A study on aerobic \nbacteriological profile and drug sensitivity pattern of pus samples \nin a tertiary care hospital. Int J CurrMicrobiol App Sci. 2016 \nJan:5(1):95-102.http://dx.doi.org/10.20546/ijcmas.2016\n\n33. Khan RA, Jawaid M, Khaleel M. Bacteriological profile and \nantibiogram of isolates from pus samples in a tertiary care centre. \nInt J CurrMicrobiol App Sci. 2018 Jan 10;7(01): 387-394. doi: \nhttps://doi.org/10.20546/ijcmas.2018.701.044\n\n34. Patil SB, Paramne A, Harsh S. Antibiotic susceptibility of wound \nisolates in plastic surgery patients at a tertiary care centre. Indian \nJ Plast Surg. 2016 May;49(2):198-205. doi: 10.4103/0970-\n0358.191324. \n\n35. KC R, Shrestha A, Sharma VK. Bacteriological study of wound \ninfection and antibiotic susceptibility pattern of the isolates. Nepal \nJournal of Science and Technology. 2013 May 15;14(2):143-150. \nhttps://doi.org/10.3126/njst.v14i2.10428\n\n36. Pant M, Shrestha D, Thapa S. Antibiogram of bacterial species \ncausing skin wound infections. Novel Research in Microbiology \nJournal. 2018 Jun 25;2(3):53-60. doi: 10.21608/NRMJ.2018.8153\n\n37. Devi PN, Saikumar C. Prevalence and antimicrobial susceptibility \nof methicillin resistant Staphylococcus aureus in wound Infections \nin a Tertiary Care Hospital. Int J CurrMicrobiol App Sci. 2017 Sep \n28;6(10):3472-3479 https://doi.org/10.20546/ijcmas.2017.610.409\n\n38. Sultana S, MawlaN, Kawser S, Akhtar N, Ali MK. Current \nmicrobial isolates from wound swab and their susceptibility \npattern in a Private Medical College Hospital in Dhaka city. Delta \nMedical College Journal. 2015 Feb 14;3(1):25-30. \nhttps://doi.org/10.3329/dmcj.v3i1.22236\n\n39. Barakoti A, Guragain A, Adhikari RP, Amatya R.Profile and \nantimicrobial susceptibility pattern of aerobic bacterial isolates \nfrom pus/wound swab samples in a tertiary care hospital, \nKathmandu. Nepal Med Coll J. 2017 Jan;19(4):179-\n183.https://www.researchgate.net/publication/329266887\n\n40. Bankar N, Wankhade A, Bramhane RB, Hathiwala R, Chandi DH. \nBacteriological profile of pus / wound swab and antimicrobial \nsusceptibility of Staphylococcusaureus isolated from of pus & \n\nwound swab of indoor patients of tertiary care hospital in Durg, \nChhattisgarh India. ijirms [Internet]. 2018 Apr 9;3(04):1976 to \n1980.https://ijirms.in/index.php/ijirms/article/view/356 \n\n41. Shahi K, Rijal K, Adhikari N, Shrestha U, Banjara M, Sharma V, et \nal. Methicillin Resistant Staphylococcus aureus: Prevalence and \nantibiogram in various clinical specimens at Alka Hospital. tujm.", "start_char_idx": 42771, "end_char_idx": 45554, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d44106f6-4bde-4838-9e19-250bfe086099": {"__data__": {"id_": "d44106f6-4bde-4838-9e19-250bfe086099", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b73aaad6-7d50-437e-b49a-e002044209c8", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "ad2664af3416d7e07c9bb49b5dab12a03976c8f2df2f1b18000db9e8a9f3b76e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4a26f8eb-381d-42e3-a531-503194d70f04", "node_type": "1", "metadata": {}, "hash": "f3b775e02fd019471327257fd57f598a599016e442dc0c69cf1749e4b60a8246", "class_name": "RelatedNodeInfo"}}, "text": "tujm. \n2018;5(1):77-82.https://doi.org/10.3126/tujm.v5i0.22316\n\n42. Mahat P, Manandhar S, Baidya R. Bacteriological profile of wound \ninfection and antibiotic susceptibility pattern of the isolates. J \nMicrobiol Exp. 2017 Apr 17;4(5):00126. \ndoi: 10.15406/jmen.2017.04.00126\n\n43. Pokhrel P, Shrestha A, Panthi P, Manandhar S, Chaudhary DK. \nBacteriological profile and antibiotic susceptibility pattern of \nwound infection in children. EC Microbiology. 2017 Jan \n18;5.3(2017):93-100. \nhttps://www.researchgate.net/publication/312497749\n\n44. Bhatt C, Karki B, Baral B, Gautam S, Shah A, Chaudhary A. \nAntibiotic susceptibility pattern of Staphylococcus aureus and \nmethicillin-resistant Staphylococcus aureus in a tertiary care \nhospital.  Journal of Pathology of Nepal. 2014;4(7):548-\n551.https://doi.org/10.3126/jpn.v4i7.10297\n\n45. Garoy EY, Gebreab YB, Achila OO, Tekeste DG, Kesete R, Ghirmay \nR, et al. Methicillin-resistant Staphylococcus aureus (MRSA): \nPrevalence and antimicrobial sensitivity pattern among patients_ \na multicenter study in Asmara, Eritrea.  Can J Infect Dis Med \nMicrobiol. 2019 Feb 6;2019:8321834. doi: 10.1155/2019/8321834.\n\n46. Tadesse S, Alemayehu H, Tenna A, Tadesse G, Tessema TS, \nShibeshi W et al. Antimicrobial resistance profile of Staphylococcus \naureus isolated from patients with infection at Tikuranbessa \nspecialized hospital, Addis Ababa, Ethiopia. BMC \nPharmacolToxicol. 2018 May 21;19(1):24. doi: 10.1186/s40360-018-\n0210-9. \n\n47. Nikaido H. Multidrug resistance in bacteria. Annu Rev Biochem. \n2009;78:119\u2013146. doi: 10.1146/annurev.biochem.78.082907.145923\n\n48. Upreti N, Rayamajhee B, Sherchan SP, Choudhari MK, Banjara \nMR. Prevalence of methicillin resistant Staphylococcus aureus, \nmultidrug resistant and extended spectrum beta-lactamase \nproducing gram negative bacilli causing wound infections at a \ntertiary care hospital of Nepal. Antimicrob Resist Infect Control. \n2018 Oct 8;7:121.                  doi: 10.1186/s13756-018-0408-z. \n\n49. Pahadi P, Shrestha U, Adhikari N, Shah P, Amatya R. Growing \nresistance to vancomycin among methicillin resistant \nStaphylococcus aureus isolates from different clinical samples. \nJournal of Nepal Medical Association. 2014 Dec 31;52(196):977-\n981.https://doi.org/10.31729/jnma.2797\n\n50. Kadariya J, Thapaliya D, Bhatta S, Mahatara RL, Bempah S, Dhakal \nN, et al. Multidrug-resistant Staphylococcus aureus colonization in \nhealthy adults is more common in Bhutanese refugees in Nepal \nthan those resettled in Ohio. Biomed Res Int. 2019 Jul \n1;2019:5739247. https://doi: 10.1155/2019/5739247\n\n51.", "start_char_idx": 45549, "end_char_idx": 48136, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4a26f8eb-381d-42e3-a531-503194d70f04": {"__data__": {"id_": "4a26f8eb-381d-42e3-a531-503194d70f04", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d44106f6-4bde-4838-9e19-250bfe086099", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "ddb8c99f2591ce6c0d9dfd98db182e3c02e790d48ad8a87b6dc3337c2b91da40", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d0a73a6f-c0ed-465d-a3d2-6868af51c375", "node_type": "1", "metadata": {}, "hash": "0fc8d0f79e2e825e8cc85af91643297c7b5519d58cef91d63ea8b5191baf44ca", "class_name": "RelatedNodeInfo"}}, "text": "Mama M, Aklilu A, Misgna K, Tadesse M, Alemayehu E. \nMethicillin- and inducible clindamycin-resistant Staphylococcus \naureus among patients with wound infection attending Arba \nMinch Hospital, South Ethiopia. International Journal of \nMicrobiology. 2019 Apr 1;2019:2965490. \nhttps://doi.org/10.1155/2019/2965490\n\n52. Joachim A, Moyo SJ, Nkinda L, Majigo M, Mmbaga E, Mbembati \nN, et al. Prevalence of methicillin-resistant Staphylococcus aureus \ncarriage on admission among patients attending regional hospitals \nin Dar es Salaam, Tanzania. BMC Research Notes. 2017 \nAug;10(1):417. doi.10.1186/s13104-017-2668-8 \n\n53. Ansari S, Nepal HP, Gautam R, Rayamajhi N, Shrestha S, \nUpadhyay G, et al. Threat of drug resistant Staphylococcus aureus to \nhealth in Nepal. BMC Infect Dis. 2014 Mar 22;157 \n(2014).https://doi.org/10.1186/1471-2334-14-157 \n\n54. Jaiswal S, Thapa A, Mali G, Magar S, Gurung S, Shakya S, et al. A \nstudy of methicillin resistant pattern on clinical isolates of \nStaphylococcus aureus in tertiary care hospitals of Pokhara. BMR \nMicrobiology. 2016 Sep \n24;2(1).https://www.researchgate.net/publication/310331234\n\n55. Adhikari R, Pant ND, Neupane S, Neupane M, Bhattarai R, Bhatta \nS,et al. Detection of methicillin resistant Staphylococcus aureus and \ndetermination of minimum inhibitory concentration of \n\nhttps://www.researchgate.net/publication/216834367_METHICILLIN_RESISTANT_Staphylococcus_aureus_MRSA_IN_DIFFERENT_CLINICAL_SAMPLES_FROM_PATIENTS_PRESENTING_AT_KATHMANDU_MODEL_HOSPITAL\nhttps://doi.org/10.1186/s13104-017-2515-y\nhttps://doi.org/10.1556/1886.2018.00003\nhttps://doi.org/10.3126/tujm.v4i0.21679\nhttps://doi.org/10.3126/tujm.v5i0.22297\nhttp://dx.doi.org/10.20546/ijcmas.2016\nhttps://doi.org/10.20546/ijcmas.2018.701.044\nhttps://doi.org/10.3126/njst.v14i2.10428\nhttps://doi.org/10.20546/ijcmas.2017.610.409\nhttps://doi.org/10.3329/dmcj.v3i1.22236\nhttps://www.researchgate.net/publication/329266887\nhttps://ijirms.in/index.php/ijirms/article/view/356\nhttps://doi.org/10.3126/tujm.v5i0.22316\nhttps://doi.org/10.15406/jmen.2017.04.00126\nhttps://www.researchgate.net/publication/312497749\nhttps://doi.org/10.3126/jpn.v4i7.10297\nhttps://doi.org/10.31729/jnma.2797\nhttps://doi.org/10.1155/2019/2965490\nhttps://doi.org/10.1186/s13104-017-2668-8\nhttps://doi.org/10.1186/1471-2334-14-157\nhttps://www.researchgate.net/publication/310331234", "start_char_idx": 48137, "end_char_idx": 50497, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d0a73a6f-c0ed-465d-a3d2-6868af51c375": {"__data__": {"id_": "d0a73a6f-c0ed-465d-a3d2-6868af51c375", "embedding": null, "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-115", "node_type": "4", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "f815e4ff1ce6c2ea154bb153ba8f963ebca20ed4d9e3d57067c33a274b6b2c3b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4a26f8eb-381d-42e3-a531-503194d70f04", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "fbbcf489dcb1024477c47e69fbc48f65c0ca9e1264d874811f93b6be88269f6e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "56404414-01af-4425-bed9-4c1016e8c43d", "node_type": "1", "metadata": {}, "hash": "c64ac0303d2d79480fbe55d82f35d7e5b1681efa94cb986f37c385334deaec2b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):8 - 1 7    Maharjan et al.      \n\n\u00a9NJB, BSN 17 \n\nvancomycin for Staphylococcus aureus isolated from pus/wound \nSwab samples of the patients attending a tertiary care hospital in \nKathmandu, Nepal. Can J Infect Dis Med Microbiol. 2017 Jan \n5;2017:2191532. https://doi.org/10.1155/2017/2191532 \n\n56. Khanal LK, Jha BK. Prevalence of methicillin resistant \nStaphylococcus aureus (MRSA) among skin infection cases at a \nhospital in Chitwan, Nepal. Nepal Medical College Journal: \nNMCJ. 2010 Dec;12(4):224-228. PMID: 21744763\n\n57. Neopane P, Nepal HP, Shrestha R, Uehara O, Abiko Y. In vitro \nbiofilm formation by Staphylococcus aureus isolated from wounds \nof hospital-admitted patients and their association with \nantimicrobial resistance. Int J Gen Med. 2018 Jan 18;11:25-32. \ndoi: 10.2147/IJGM.S153268.\n\n58. Choudhury D, Chakravarty P. Prevalence and antimicrobial \nsusceptibility pattern of methicillin resistant Staphylococcus \naureusin Silchar Medical College and hospital, Assam, India. Int J \nBasic ClinPharmacol. 2016 Oct;5(5): 2174-2177. \nhttps://dx.doi.org/10.18203/2319-2003.ijbcp20163257\n\n59. Kshetry AO, Pant ND, Bhandari R, Khatri S, Shrestha KL, \nUpadhaya SK, et al. Minimum inhibitory concentration of \nVancomycin to methicillin resistant Staphylococcus aureus isolated \nfrom different clinical samples at a tertiary care hospital in Nepal. \nAntimicrobial Resistance and Infection Control. 2016;5:27. \nhttps://doi.org/10.1186/s13756-016-0126-3 \n\n60. Pal N, Sharma B, Sharma R, Vyas L. Detection of inducible \nclindamycin resistance among staphylococcal isolates from \ndifferent clinical specimens in western India. J Postgrad Med. \n2010;56(3):182-185.  http://www.jpgmonline.com/text.asp?2010/\n56/3/182/68637\n\n61. Ghosh S, Banerjee M. Methicillin resistance & inducible \nclindamycin resistance in Staphylococcus aureus. The Indian J Med \nResearch. 2016 Mar;143(3):362-364. doi: 10.4103/0971-5916.182628. \n\nhttps://doi.org/10.1155/2017/2191532\nhttps://dx.doi.org/10.18203/2319-2003.ijbcp20163257\nhttps://doi.org/10.1186/s13756-016-0126-3", "start_char_idx": 50500, "end_char_idx": 52592, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "56404414-01af-4425-bed9-4c1016e8c43d": {"__data__": {"id_": "56404414-01af-4425-bed9-4c1016e8c43d", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d0a73a6f-c0ed-465d-a3d2-6868af51c375", "node_type": "1", "metadata": {"identifier": "njb-115", "author": "Maharjan, Bidhya; Karki, Shovana Thapa; Maharjan, Roshani", "title": "Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital", "date": "2021-07-31", "file": "njb-115.pdf"}, "hash": "0f62c33d882f769a52196e27ba6bf5c9060abb2d6ed5eaded46df6cb57dd18b9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "de3841b5-2367-4b5c-aff3-c9da7c11f55e", "node_type": "1", "metadata": {}, "hash": "c02bbabca97013c705299b67c6ee51e2fa55493b6b3fc91917ed7a0ec7b2d32d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ;  9 (1): 1-7 Research article DOI: https://doi.org/10.3126/njb.v9i1.38644 \n\n\u00a9NJB, BSN 1 \n\nAntibiogram and Phytochemical Analysis Of Cinnamon, Clove, and \nSichuan Pepper Extracts. \nBibek Adhikari 1,2 , Pradeep Kumar Shah1, Roman Karki2\n\n1 Department of Microbiology, Tri-Chandra Multiple Campus, Ghantaghar, Kathmandu, Nepal \n2 National Food Research Centre (NFRC), Nepal Agricultural Research Council (NARC), Khumaltar, Lalitpur, Nepal \n\nReceived: 07 Aug 2020; Revised: 08 Apr 2021; Accepted: 16 Apr 2021; Published online: 31 Dec 2021 \n\nABSTRACT \nA wide range of medicinal plant extracts has phytochemicals that possess antimicrobial properties and these plants are used \nto treat several infections. The study aimed to assess the antimicrobial activities of some spices extracts and to evaluate the \nphytochemicals present in them. The extracts of spices were prepared using Soxhlet apparatus refluxing with methanol and \nethanol. The well diffusion technique was implemented for the evaluation of antimicrobial activities of the extracts and the \nzone of inhibitions was recorded in millimeters. The antimicrobial test was done against five bacterial isolates: Escherichia coli, \nProteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica serotype Typhi, and Staphylococcus aureus and a fungal isolate: \nCandida albicans. The extracts were concentrated by Rotary Vacuum Evaporator and a stock solution of 200 mg/mL was \nprepared by dissolving in 10 % DMSO. Concentrations of 40, 60, 80 and 100 mg/mL extracts were used for antimicrobial \nactivity. The result of this study showed that clove extracts had the highest antimicrobial property against all the test \nmicroorganisms. Methanolic extract of clove had the highest inhibitory effect against Proteus mirabilis (24.21\u00b10.15 mm), \nPseudomonas aeruginosa (19.78\u00b10.23 mm), and Candida albicans (20.07\u00b10.08 mm) whereas ethanolic extract was effective against \nEscherichia coli (20.44\u00b10.16 mm), Salmonella Typhi (21.66\u00b10.31 mm) and Candida albicans (21.11\u00b10.09 mm). Cinnamon and \npepper extracts, leaving some exceptions, also had antimicrobial properties. The presence of phytochemicals: polyphenols, \nflavonoids, and tannins are the major components responsible for antimicrobial activity. Thereby, this study successfully \ndemonstrated the possibilities of using spices extracts in the treatment of microbial infections. \n\nKeywords: Antimicrobial Activity, DMSO, Ethanol, Methanol, Phytochemicals. \n\n Corresponding Author, Email: a.bibek52@gmail.com \n\nIntroduction \nHerbal medicine or phytomedicine is the use of plants for \n\nmedicinal and therapeutic purposes for the curing of \n\ndiseases and improving human health [1]. A large \n\nportion of the world population, especially in developing \n\ncountries, relies on the traditional systems of medicine to \n\ntreat a variety of diseases [2]. Presently, more than two-\n\nthirds of the world\u2019s population leans on plant-based \n\nmedicines relying on the fact that they are harmless and \n\nefficient against various afflictions [3, 4]. \n\nAbundant molecules with antimicrobial properties are \n\npresent in medicinal herbs. Several infectious diseases are \n\ntreated using a wide range of plant extracts since they \n\npossess antimicrobial potentials. Noticing the side effects \n\non human health due to synthetic drugs, professionals \n\nare on the way to getting advantages from medicinal \n\nplants. A wide variety of screened plant molecules are \n\ntraded as raw materials for several herbal preparations in \n\nthe market.", "start_char_idx": 48, "end_char_idx": 3595, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "de3841b5-2367-4b5c-aff3-c9da7c11f55e": {"__data__": {"id_": "de3841b5-2367-4b5c-aff3-c9da7c11f55e", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "56404414-01af-4425-bed9-4c1016e8c43d", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "95317ecb21ff412bead936a81bdfc13f2dee87fab216636dabc7d616ffda9444", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "748332bb-ab9a-4082-abfb-94fb31656b1e", "node_type": "1", "metadata": {}, "hash": "6a15950509d4e129f3eafe59a3e378940b3338142109c6d67ffa032509d51ccc", "class_name": "RelatedNodeInfo"}}, "text": "Out of reported 422,127 worldwide plant \n\nspecies, approximately 35,000 to 70,000 plant genera are \n\nutilized for medicinal purposes [5, 6].  \n\nPlants have secondary metabolites called phytochemicals \n\n(Phyto from Greek - meaning plant) that protect plants \n\nagainst microbial infections or pests infestations. \n\nPhytochemicals are active ingredients that possess \n\ntherapeutic properties that are considered as a medicine \n\nor drug [1]. Alkaloids, flavonoids, phenolic compounds, \n\nand tannins are the essential phytochemicals present in \n\nthe plants with possible therapeutic activities and these \n\nphytochemicals obtained in plant extracts have \n\ndemonstrated antimicrobial potential against a wide \n\nrange of infectious microorganisms [7, 8]. \n\nMicrobial infection is a prevailing health problem around \n\nthe world. Plants remain one of the potential sources of \n\neffective agents against microbes, including the deadly \n\ninfection like tuberculosis (Mycobacterium tuberculosis), \n\nsyphilis (Treponema pallidum), gonorrhea (Neisseria \n\ngonorrhoeae), skin and wound infections [9], diarrhea [10], \n\ntyphoid fever (Salmonella Typhi), and Pseudomonas \n\naeruginosa which directly infects the urinary tract, the \n\npulmonary tract, wounds, burns and also causes other \n\nblood infections [8]. \n\nMicrobial infection has been a major cause of death \n\nglobally. The rapid increase in the development of \n\nresistance to antimicrobial agents by the microorganisms \n\nhas led to the new incidence and re-exposure of disease \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0003-1622-1783\nmailto:a.bibek52@gmail.com\nhttps://orcid.org/0000-0002-9897-2486\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7   Adhikari et al.  \n\n\u00a9NJB, BSN 2 \n\nmaking them difficult and expensive to treat. To \n\novercome these problems Pharmaceutical industries are \n\nin search of alternative antimicrobial agents. Spices \n\npossessing a wide range of bioactive compounds such as \n\nalkaloids, polyphenols, flavonoids, tannins, saponins, \n\nand various antioxidants have great potential as \n\nantimicrobial agents that can counteract pathogenic \n\nmicroorganisms. The spices extract solely or in \n\ncombination with other antibiotics have the potential to \n\nwork effectively against several infectious \n\nmicroorganisms [9]. \n\nCinnamomum verum (Syn. C. zeylanicum) also known as \n\nCinnamon is a small evergreen tree belonging to the \n\nfamily Lauraceae. The volatile oil produced from its leaf \n\nand barks are used as a flavoring agent in the food and \n\nbeverage industry [11,12]. It is also used to treat \n\nabdominal pain, impotence, frigidity, dyspnoea, \n\ninflammation of the eye, leukorrhoea, vaginitis, \n\nrheumatism, neuralgia, wounds, toothache, and diabetes \n\n[11]. The principal constituents of leaf, bark and root oils \n\nare eugenol, cinnamaldehyde, and camphor, respectively \n\n[12,13]. Eugenol has been reported to inhibit the growth \n\nof Escherichia coli O157:H7 and Listeria monocytogenes [14]. \n\nCinnamaldehyde has been reported to inhibit the growth \n\nof Staphylococcus aureus, E. coli O157:H7, and Salmonella \n\ntyphimurium [15,16]. \n\nSyzygium aromaticum also known as Clove is the aromatic \n\ndried flower buds of a perennial tree belonging to the \n\nfamily Myrtaceae. Essential oil of clove is used as \n\nanodyne for dental emergencies, and against acne, warts, \n\nscars, and parasites.", "start_char_idx": 3596, "end_char_idx": 7106, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "748332bb-ab9a-4082-abfb-94fb31656b1e": {"__data__": {"id_": "748332bb-ab9a-4082-abfb-94fb31656b1e", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "de3841b5-2367-4b5c-aff3-c9da7c11f55e", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "02ea7d90b6bda1a9ab2a29f4aad1e67d9ab01ebcf3ecf1c4a62a1499c2484076", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "812b48e7-ce33-4b48-aee3-834b2214d324", "node_type": "1", "metadata": {}, "hash": "08f2b470a9da77ab52d4c5b571844df2c9801cb13c6388d7d5f7b0cbbecf4c4d", "class_name": "RelatedNodeInfo"}}, "text": "Also, the clove has antimutagenic, \n\nanti-inflammatory, antioxidant, antiulcerogenic, \n\nantithrombotic, and antiparasitic, antibacterial, and anti-\n\ninflammatory properties [3]. Research has shown that \n\nclove oil is an effective mosquito repellent [17]. The major \n\nconstituents of clove\u2019s essential oil are carvacrol, thymol, \n\neugenol, and cinnamaldehyde. Several studies have \n\ndemonstrated potent antifungal, antiviral and \n\nantibacterial effects of clove [18]. Aqueous clove infusion \n\nwas found to inhibit the growth of germinated spores of \n\nBacillus subtilis, and inhibit the pathogens Campylobacter \n\njejuni, Salmonella enteritidis, and Escherichia coli [19]. Clove \n\noil exhibited antibacterial activity against S. epidermidis, \n\nSalmonella Typhi, Klebsiella pneumoniae, Listeria \n\nmonocytogenes, Staphylococcus aureus, and Bacillus cereus \n\n[20]. \n\nZanthoxylum armatum, commonly known as Sichuan \n\nPepper, belongs to the Rutaceae family. The bark, fruits, \n\nand seeds of Pepper are extensively used in the \n\nindigenous system of medicine as a carminative, \n\nstomachic, and anthelmintic. The seed and bark are also \n\nused as an aromatic tonic in fever, dyspepsia. Because of \n\ntheir deodorant, disinfectant, and antiseptic properties, \n\nthe fruits are used in dental troubles, their lotion for \n\nscabies, and also used to ward off houseflies. Besides this, \n\nit is also used as a flavoring agent in the confectionery \n\nindustry, and the manufacturing of soft drinks [21]. \n\nPepper consists of several phytochemicals such as \n\nchlorogenic acid, cinnamic acid, epicatechin, rutin, \n\ntrifolin, quercitrin, etc. which have anthelmintic, \n\nantifungal, and anti-insecticidal activities [22,23]. \n\nDifferent solvent extracts of pepper demonstrated \n\nantimicrobial properties against several pathogenic such \n\nas Escherichia coli, Staphylococcus aureus, Salmonella Typhi, \n\nProteus vulgaris, Pseudomonas aeruginosa, and Klebsiella \n\npneumoniae [24].  \n\nMaterials and Methods \nPlant Samples Collection \nPlant materials used in the study consisted Cinnamomum \n\nverum (Cinnamon), Syzygium aromaticum (Clove), and \n\nZanthoxylum armatum (Sichuan Pepper) which were \n\ncollected from different parts of Nepal (Table 1). Since \n\nthese three spices are most commonly found in the \n\nNepalese kitchen, the authors selected these spices for \n\nantimicrobial and phytochemical analyses. \n\nTable 1. Nomenclature of the spices and the locality from \nwhich they were obtained for this study. \n\nCommon Name Scientific \nName \n\nPart \nused \n\nCollected \nfrom \n\n Cinnamon Cinnamomum \nverum \n\nBark Kathmandu \n\n Clove Syzygium \naromaticum \n\nBud \n(fruit) \n\nKathmandu \n\n Sichuan Pepper Zanthoxylum \narmatum \n\nFruit Salyan \n\nChemicals \nBarium chloride, Dimethyl Sulfoxide, Folin-Ciocalteu \n\nreagent, Folin-Denis reagent, Methanol, and Sodium \n\nchloride were purchased from Thermo Fisher Scientific \n\nIndia Pvt. Ltd. Similarly, ethanol was purchased from \n\nJiangyin Tenghua Co. Ltd., China, and Gallic acid and \n\nTannic acid were purchased from Loba Chemie Pvt. Ltd., \n\nIndia. Additionally, Gentamicin, Muller Hinton agar \n\n(MHA), Nutrient agar (NA), Potato Dextrose agar (PDA), \n\nand sterile swabs were purchased from HiMedia \n\nLaboratories Pvt. Ltd., India.", "start_char_idx": 7107, "end_char_idx": 10340, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "812b48e7-ce33-4b48-aee3-834b2214d324": {"__data__": {"id_": "812b48e7-ce33-4b48-aee3-834b2214d324", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "748332bb-ab9a-4082-abfb-94fb31656b1e", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "578bf5d29e47938f02071b60bc5ab2b550fe5b5a0cd9afb24107542787b19937", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "141b2f38-886c-4bf3-a3b9-b36b557ac14d", "node_type": "1", "metadata": {}, "hash": "67555f481e4367be774d3fbde59286fd356102379789448adcdf187afaba184b", "class_name": "RelatedNodeInfo"}}, "text": "Ltd., India. \n\nMicroorganisms \nThe pure cultures of Candida albicans, Esherichia coli, \n\nPseudomonas aeruginosa, Salmonella enterica serotype \n\nTyphi, and Staphylococcus aureus isolated from patients \n\nwere obtained from the Department of Microbiology, \n\nTribhuvan University Teaching Hospital (TUTH), \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7   Adhikari et al.  \n\n\u00a9NJB, BSN 3 \n\nMaharajgunj, and the culture of Proteus mirabilis were \n\nobtained from Nepal Academy of Science and \n\nTechnology (NAST), Khumaltar. The bacterial samples \n\nwere maintained on NA and fungal samples on PDA at \n\n4\u00baC for further experiments. \n\nGentamicin \n10 \u00b5g disc of Gentamicin was used as a positive control \n\nagainst bacterial cultures. \n\nExtract Preparation \nAir-dried, 30 g spices powder was taken in 120 mL of \n\nabsolute methanol and absolute ethanol for 48 hours in \n\nSoxhlet apparatus (Borosil, India) separately and then \n\nWhatman no. 1 filter paper was used to filter the extract \n\nand allowed to evaporate using Rotary Vacuum \n\nEvaporator (Buchi Type) (Victo Lab, India) at 65\u00baC for \n\nmethanolic extract and 50\u00baC for ethanolic extract [7, 10]. \n\nThe concentrated extracts were stored in small containers \n\nin a refrigerator (4\u00baC) for further use. \n\nStandard concentration preparation \nTwo grams of each alcoholic extract were taken in a vial \n\nand 1mL, 100% Dimethyl Sulphoxide (DMSO) was added \n\nand dissolved in 9 mL of sterile double distilled water. \n\nThus, 200 mg/mL of stock was obtained as a standard \n\nconcentration. The extracts were then autoclaved (Victo \n\nLab, India) for 20 minutes at 121\u00baC and 15 pounds per \n\nsquare inch pressure [23]. \n\nInoculum preparation \nA colony of the organism from the stored agar plate was \n\ntaken, transferred to a test tube containing 2.5mL \n\nsterilized NB and incubated for about 4 hours at 37\u00baC in \n\nthe incubator (Sanjeev Scientific Udhyog, India) and \n\nstandardized the turbidity to 0.5 McFarland solution, \n\nmicrobial load equivalent to 1.5x108 CFU/mL [24]. \n\nAntimicrobial Assay by Well Diffusion Method \nMHA plates were prepared and test organisms were \n\ninoculated by spreading the bacterial inoculum on the \n\nsurface of the media with the help of a sterile swab. Wells \n\nof 6 mm diameter were punched in the agar by using a \n\ncork borer. 50 \u03bcL of extracts with the concentration of 40, \n\n60, 80, and 100 mg/mL were poured into the well. 50\u03bcL \n\nof absolute methanol, ethanol, and DMSO (10%) were \n\nused as controls. The plates were incubated at 37\u00b0C for 24 \n\nhours. Measurement of the diameter of the inhibition \n\nzone was done to evaluate the antimicrobial property \n\nwith the help of a Vernier Caliper and recorded in mm \n\n[25]. \n\nPhytochemical Analysis \nThe extracts of the spices were prepared as described by \n\nDimitrijevi\u0107 et al (2014) [26] and some modifications \n\nmade by Bhattarai et al (2019) [27]. 5 g powdered spice \n\nwas ground with 80 % methanol (30 mL) and was kept in \n\nan orbital shaker (Accumax, India) shaking continuously \n\nfor about 20 minutes, and filtered through Whatman No. \n\n1 filter paper in a 100 mL volumetric flask. The residue \n\nwas again subjected to two more extractions with 30 mL \n\neach of 80 % methanol for a total time frame of an hour. \n\nThe volume was made up to 100 mL using 80% methanol \n\nand the extracts were stored in a refrigerator at 4\u00baC until \n\nfurther use.", "start_char_idx": 10328, "end_char_idx": 13683, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "141b2f38-886c-4bf3-a3b9-b36b557ac14d": {"__data__": {"id_": "141b2f38-886c-4bf3-a3b9-b36b557ac14d", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "812b48e7-ce33-4b48-aee3-834b2214d324", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "801280c3df5b0258c4dacf640e4e121a2413b5b34f9293a6d4cd0123449f9c93", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f344dc9e-3648-4a2e-8d24-2fa51a0a13ea", "node_type": "1", "metadata": {}, "hash": "2e2cf9889b772fc794fbe993d2e40002392eef7a832923cd3885f7a09e188604", "class_name": "RelatedNodeInfo"}}, "text": "Polyphenol Content \nThe polyphenol content of sample extracts was measured \n\nby using the Folin-Ciocalteu method, as described by \n\nMahdavi et al (2010) [28]. 1 mL of extract was decanted in \n\na volumetric flask of 25 mL containing 9 mL of distilled \n\nwater. 1 mL of Folin-Ciocalteu reagent was added and \n\nshaken. After 5 minutes, 10 mL of 7 % Na2CO3 solution \n\nwas added and the volume was made up with distilled \n\nwater and mixed. The absorbance was measured at a \n\nwavelength of 765 nm using a Uv-Vis spectrophotometer \n\n(Genesys, USA) against a prepared reagent blank \n\n(distilled water), after incubation for 90 min at room \n\ntemperature. The polyphenol content was reported as mg \n\ngallic acid equivalent per 100g sample (mg GAE/100g). \n\nA calibration curve was created from the standard and \n\nused to determine the corresponding gallic acid \n\nconcentration of the samples. \n\nFlavonoid Content \nAluminum trichloride (AlCl3) assay as described by \n\nSamatha et al (2012) [29] with some modification done by \n\nFaleye et al (2018) [30] was used for the determination of \n\nflavonoid content. 0.5 mL of 80 % methanol extract was \n\ntaken in different test tubes followed by the addition of 2 \n\nmL of distilled water and 0.15 mL of sodium nitrite (5 % \n\nNaNO2, w/v). After 6 minutes, 0.15 mL of aluminum \n\ntrichloride (10 % AlCl3) was added and incubated for 6 \n\nmin, followed by the addition of 2 mL of sodium \n\nhydroxide (NaOH, 4 % w/v), and volume was made up \n\nto 5 mL with distilled water. After 15 min of incubation, \n\nabsorbance at 510 nm was measured against a reagent \n\nblank of distilled water. The calibration standard curve \n\nwas prepared by preparing gallic acid solutions and the \n\nresult was expressed as mg of gallic acid equivalents per \n\n100g (mg GAE/100g) of the sample. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7                 Adhikari et al.  \n\n\u00a9NJB, BSN  4 \n\nTannin Content \nThe Folin-Denis method was used for the determination \n\nof tannin content [31]. In a test tube containing 7.5 mL of \n\ndistilled water, 0.1 mL of the sample extract was added. \n\n0.5 mL of Folin-Denis phenol reagent and 1 mL of 0.5 % \n\nNa2CO3 solution were also added and diluted to 10 ml \n\nwith distilled water. The mixture was shaken well and \n\nkept at room temperature for 30 min. Absorbance at 775 \n\nnm was measured against the reagent blank and a set of \n\nreference standard solutions of gallic acid. The tannin \n\ncontent was expressed in terms of mg tannic acid \n\nequivalent per 100g (mg TAE/100g) of extract. \n\nStatistical Analysis \nExperimental analyses were performed in triplicates. \n\nIBM SPSS (Statistical Package for Social Sciences) \n\nStatistics version 20 was used for the statistical analysis. \n\nOne-way Analysis of variance (ANOVA) was analyzed \n\nand the significant differences among them were studied \n\nby using Tukey HSD at a 5 % level of significance. \n\nResults  \nAntimicrobial Activity by Well Diffusion \nMethod \nAgar well-diffusion method was employed to evaluate \n\nthe antibacterial and antifungal activities of different \n\nconcentrations (40, 60, 80, and 100 mg/mL) of the spice \n\nextracts. The result obtained by the well diffusion method \n\nhas been summarized in Table 2 and Table 3. The zone \n\nof inhibition greater than 6.5 mm has been taken into \n\naccount.", "start_char_idx": 13686, "end_char_idx": 16978, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f344dc9e-3648-4a2e-8d24-2fa51a0a13ea": {"__data__": {"id_": "f344dc9e-3648-4a2e-8d24-2fa51a0a13ea", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "141b2f38-886c-4bf3-a3b9-b36b557ac14d", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "55a698f4f4435607d1ab9d3199af2a86bba53cccd01273d23758889d48e5c02f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7ec507f3-e428-4d6f-8bc9-cf5da4b30dae", "node_type": "1", "metadata": {}, "hash": "c63d9870d5a0a7ba7f96bbe017fdf557217ee96500d5c6c2788a965906f5c739", "class_name": "RelatedNodeInfo"}}, "text": "The zone \n\nof inhibition greater than 6.5 mm has been taken into \n\naccount. From Table 2 the results obtained in this study \n\nrevealed that the bacterial strains: E. coli, P. mirabilis, P. \n\naeruginosa, S. Typhi, and S. aureus, and a fungal strain: C. \n\nalbicans to be susceptible against the methanolic and \n\nethanolic extracts of the three distinct spices. Both the \n\nmethanolic and ethanolic extracts of Cinnamon had a \n\nTable 2. Antibiogram of Methanolic Extracts \n\nSpices Conc (mg/mL) \nZone of Inhibition (ZOI) mm \n\nE. coli P. mirabilis P. aeruginosa S. Typhi S. aureus C. albicans \n\nCinnamon \n\n40 0 0 0 0 0 0 \n\n60 0 0 0 9.02\u00b10.08b 0 7.16\u00b10.07a \n\n80 6.60\u00b10.10a 7.89\u00b10.10a 0 10.19\u00b10.07c 6.52\u00b10.11a 8.02\u00b10.06b \n\n100 7.20\u00b10.11ab 9.02\u00b10.06b 0 11.12\u00b10.05d 7.87\u00b10.09b 10.00\u00b10.07c \n\nClove \n\n40 10.62\u00b10.18d 14.90\u00b10.42d 11.61\u00b10.22d 10.22\u00b10.05c 11.69\u00b10.16c 16.50\u00b10.21d \n\n60 14.53\u00b10.23f 18.63\u00b10.30e 13.99\u00b10.14e 11.60\u00b10.26de 13.60\u00b10.23d 18.48\u00b10.12e \n\n80 17.19\u00b10.34g 21.60\u00b10.26f 16.15\u00b10.22f 15.66\u00b10.11g 16.68\u00b10.11e 19.52\u00b10.07f \n\n100 18.43\u00b10.19h 24.21\u00b10.15g 19.78\u00b10.23g 18.95\u00b10.26h 17.41\u00b10.16f 20.07\u00b10.08g \n\nPepper \n\n40 7.54\u00b10.14b 7.99\u00b10.10a 0 9.35\u00b10.15b 10.99\u00b10.14c 0 \n\n60 8.73\u00b10.20c 12.36\u00b10.21c 7.07\u00b10.20a 12.06\u00b10.22e 11.43\u00b10.21c 0 \n\n80 12.97\u00b10.08e 14.4\u00b10.12d 8.46\u00b10.16b 13.46\u00b10.06f 16.65\u00b10.18e 0 \n\n100 15.29\u00b10.24f 15.06\u00b10.09d 9.74\u00b10.10c 15.12\u00b10.21g 16.99\u00b10.08ef 0 \n\nControls \n\nAbsolute \nMethanol \n\n7.15\u00b10.02bc 7.66\u00b10.24a 7.14\u00b10.27a 7.24\u00b10.07a 7.19\u00b10.14ab 7.45\u00b10.13a \n\nGentamicin 20.83\u00b10.02i 18.70\u00b10.08e 29.50\u00b10.03h 36.63\u00b10.11i 21.84\u00b10.08g ND \nND= Not Determined \nThe measurements are the mean values (n, 3) \u00b1 standard error of the mean, whereas different alphabets vertically indicate a significant \ndifference at 0.05 probability level. \nTable 3.", "start_char_idx": 16903, "end_char_idx": 18644, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7ec507f3-e428-4d6f-8bc9-cf5da4b30dae": {"__data__": {"id_": "7ec507f3-e428-4d6f-8bc9-cf5da4b30dae", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f344dc9e-3648-4a2e-8d24-2fa51a0a13ea", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "c3a65f7dc2fab7a7d8f777314e224509c26005cd2f170cbe01d7f38201ff469b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ec0dcfba-d147-47e0-9702-e371ce33bfe5", "node_type": "1", "metadata": {}, "hash": "3feb5fd90e3961a895bba9767b03aa8df65bc40e855aa5f0d4e13d482be0cae1", "class_name": "RelatedNodeInfo"}}, "text": "Table 3. Antibiogram of Ethanolic Extracts \n\nSpices Conc (mg/mL) \nZone of Inhibition (ZOI) mm \n\nE. coli P. mirabilis P. aeruginosa S. Typhi S. aureus C. albicans \n\nCinnamon \n\n40 7.60\u00b10.11abc 0 0 0 0 7.42\u00b10.03a \n\n60 7.94\u00b10.50bc 8.99\u00b10.10a 0 0 0 9.11\u00b10.45b \n\n80 8.12\u00b10.08c 10.06\u00b10.05abc 0 9.57\u00b10.30a 7.78\u00b10.08bc 11.14\u00b10.36c \n\n100 10.28\u00b10.08d 11.97\u00b10.09de 0 14.41\u00b10.30b 8.14\u00b10.15c 12.59\u00b10.18d \n\nClove \n\n40 10.25\u00b10.12d 10.41\u00b10.06bc 6.97\u00b10.13a 19.13\u00b10.08c 12.5\u00b10.06d 17.64\u00b10.48e \n\n60 13.12\u00b10.19e 11.15\u00b10.37cd 9.20\u00b10.10b 19.51\u00b10.18cd 14.42\u00b10.16e 18.75\u00b10.39ef \n\n80 15.45\u00b10.29f 12.38\u00b10.68e 10.84\u00b10.17c 20.25\u00b10.12d 15.10\u00b10.11f 19.28\u00b10.35f \n\n100 20.44\u00b10.16g 13.90\u00b10.05f 11.78\u00b10.08d 21.66\u00b10.31e 15.72\u00b10.08f 21.11\u00b10.09g \n\nPepper \n\n40 0 0 0 0 0 0 \n\n60 0 0 0 0 0 0 \n\n80 6.75\u00b10.02a 0 0 0 7.48\u00b10.12ab 0 \n\n100 7.08\u00b10.07ab 0 0 0 8.02\u00b10.12c 0 \n\nControls \n\nAbsolute \n\nEthanol \n8.19\u00b10.26c 9.98\u00b10.09ab 6.75\u00b10.13a 8.87\u00b10.27a 7.06\u00b10.01a 7.28\u00b10.14a \n\nGentamicin 20.83\u00b10.02g 18.70\u00b10.08g 29.50\u00b10.03e 36.63\u00b10.11f 21.84\u00b10.08h ND \n\nND= Not Determined \nThe measurements are the mean values (n, 3) \u00b1 standard error of the mean, whereas different alphabets vertically indicate \na significant difference at 0.05 probability level. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7                 Adhikari et al.  \n\n\u00a9NJB, BSN  5 \n\nsimilar effect against test microorganisms, with little to \n\nno effect at lower concentrations and distinctive effects at \n\nhigher doses. However, P. aeruginosa was found to be \n\nresistant to both extracts of Cinnamon. At 100 mg/ml \n\nconcentration, the ZOI measurement of methanolic \n\nextract of Cinnamon against E. coli, P. mirabilis, S. Typhi, \n\nS. aureus, and C. albicans were 7.20\u00b10.11, 9.02\u00b10.06, \n\n11.12\u00b10.05, 7.87\u00b10.09, and 10.00\u00b10.07 mm; whereas \n\n10.28\u00b10.08, 11.97\u00b10.09, 14.41\u00b10.30, 8.14\u00b10.15, and \n\n12.59\u00b10.18 mm by the ethanolic extracts of the same \n\nconcentration.", "start_char_idx": 18636, "end_char_idx": 20514, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ec0dcfba-d147-47e0-9702-e371ce33bfe5": {"__data__": {"id_": "ec0dcfba-d147-47e0-9702-e371ce33bfe5", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7ec507f3-e428-4d6f-8bc9-cf5da4b30dae", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "942a62f3b65222bc99847dddc74e91d9c1b9d54acf66ace5923109b4e060f80d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d7fdaa35-e536-4918-8204-ec6cc1467892", "node_type": "1", "metadata": {}, "hash": "0ce82b86ec0045089ec84bca862b9e6545d4e99ae9ffc8ab4cbb8215b45cd2ac", "class_name": "RelatedNodeInfo"}}, "text": "The methanolic extract of Clove demonstrated higher \n\nantimicrobial activity against P. mirabilis, P. aeruginosa, \n\nand S. aureus with the ZOI measurement of 24.21\u00b10.15 \n\nmm, 19.78\u00b10.23 mm, and 17.41\u00b10.16 mm at 100 mg/ml. \n\nWhereas ethanolic extract was comparatively found to be \n\nmost effective against E. coli, S. Typhi, and C. albicans with \n\n20.44\u00b10.16 mm, 21.66\u00b10.31 mm, and 21.11\u00b10.09 mm \n\ninhibition zone at the same concentration. \n\nThe methanolic extract of Pepper was effective against all \n\nthe bacterial strains: E. coli, P. mirabilis, P. aeruginosa, S. \n\nTyphi, and S. aureus with the ZOI of 15.29\u00b10.24 mm, \n\n15.06\u00b10.09 mm, 9.74\u00b10.10 mm, 15.12\u00b10.21 mm, and \n\n16.99\u00b10.08 mm at 100 mg/ml and had no any effect \n\nagainst fungal strain i.e., C. albicans. However, the \n\nethanolic extract had a selective activity against E. coli, \n\nand S. aureus with the ZOI measurement of 7.08\u00b10.07 \n\nmm, and 8.02\u00b10.12 mm respectively at the highest \n\nconcentration i.e., 100 mg/ml. \n\nAlso, absolute methanol and absolute ethanol used as \n\ncontrols demonstrated antimicrobial property to some \n\nextent. However, 10% DMSO (data not shown) had no \n\nsignificant actions against the tested microorganisms.  \n\nPhytochemical properties \nThe spices used for the experiment demonstrated a \n\ndecent amount of phytochemical properties which have \n\nbeen tabulated in Table 4. Among all the spices used, \n\nclove had the highest amount of phytochemical \n\nproperties and pepper showed the lowest. The \n\npolyphenol content in spices ranged from 188.48\u00b17.65 to \n\n455.86\u00b19.91 mg GAE/100g (y = 0.0178x, R\u00b2 = 0.9356; \n\nwhere x, y, and R2 represent concentration, absorbance, \n\nand correlation coefficient respectively). The flavonoids \n\nvaried from 117.52\u00b12.68 to 399.70\u00b15.34 mg GAE/100g (y \n\n= 0.0088x, R\u00b2 = 0.9924) whereas the range of tannin \n\ncontents were found to be in between 64.82\u00b11.89 to \n\n326.86\u00b17.96 mg TAE/100g (y = 0.0022x + 0.0055, R\u00b2 = \n\n0.9767). The phytochemicals followed a similar trend: \n\nPolyphenol> Flavonoids> Tannins, only the exception \n\nbeing Clove where Tannin contents were slightly higher \n\nthan the flavonoid content. \n\nDiscussions \nDose-reliant antimicrobial activity of extracts was \n\nobserved, which means with the increase in extract\u2019s \n\nconcentration (from 40 to 100 mg/mL) the lethal effect \n\nwas more pronounced.  \n\nThe methanolic and ethanolic extracts of cinnamon were \n\nfound to be equally effective against most of the \n\nexperimental microorganisms, the only exception being \n\nPseudomonas aeruginosa. Both extracts showed the highest \n\ninhibitory action against Salmonella Typhi. Vyas et al \n\n(2015) [12], recorded the antibacterial activity of \n\nmethanolic extract of cinnamon against S. aureus, and E. \n\ncoli and antifungal activity against C. albicans which is in \n\nharmony with the result obtained in this study. Similarly, \n\nTomar et al (2015) [32] reported ethanolic extract of \n\ncinnamon to be effective against E. coli, S. aureus, and C. \n\nalbicans, Al-Mariri et al (2014) [33] noted decent \n\nantibacterial property of the ethanolic extract and the \n\nessential oil of cinnamon against Proteus species.", "start_char_idx": 20517, "end_char_idx": 23644, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d7fdaa35-e536-4918-8204-ec6cc1467892": {"__data__": {"id_": "d7fdaa35-e536-4918-8204-ec6cc1467892", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ec0dcfba-d147-47e0-9702-e371ce33bfe5", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "538fe847277055992fbfa019e3024718a663a15648c24bd0c22713778b5e52a9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3b08dbe2-e9b2-4b1a-9494-6ebb65b88ed2", "node_type": "1", "metadata": {}, "hash": "2498f04a2092e8f592f14048b37b085cd101b83bbf3164c8b9a9c9a668f63d06", "class_name": "RelatedNodeInfo"}}, "text": "According to the study performed by Abdelfadel et al \n\n(2016) [34], water extract of cinnamon has potential \n\nantibacterial property against Salmonella species. \n\nSimilarly, the methanolic and ethanolic extracts of clove \n\nwere found to be effective against all of the \n\nmicroorganisms used in the experiment. Methanolic \n\nextract of clove showed higher inhibition against Proteus \n\nmirabilis, whereas the ethanolic extract was found to be \n\neffective against Salmonella Typhi. The one-way \n\nANOVA (p<0.05) of methanolic extract demonstrated it \n\nto be more efficient than Gentamicin against \n\nPseudomonas aeruginosa. ANOVA (p<0.05) argued that \n\nthere is a difference between the treatment but the post \n\nhoc test shows there is no difference between ethanolic \n\nextract and gentamicin. So, it has the same lethal effect as \n\ngentamicin. Lopez et al (2005) [35] reported clove oil to be \n\nefficacious against foodborne bacteria viz S. aureus, E. \n\ncoli, P. aeruginosa, and Salmonella species. Also, \n\nKaruppiah et al (2012) [36], found that the alcoholic \n\nextract of clove has an antimicrobial effect against P. \n\nmirabilis. The study performed by Pinto et al (2009) [37], \n\nTable 4. Phytochemicals present in Spices \n\nSpices \nPolyphenol \n(mg GAE/100g) \n\nFlavonoid \n(mgGAE/100g) \n\nTannin (mg \nTAE/100g) \n\nCinnamon 362.95\u00b119.49b 329.29\u00b11.90b 326.86\u00b17.96b \n\nClove 455.86\u00b19.91c 399.70\u00b15.34c 402.24\u00b14.25c \n\nSichuan \npepper \n\n188.48\u00b17.65a 117.52\u00b12.68a 64.82\u00b11.89a \n\nGAE= Gallic Acid Equivalent, TAE= Tannic Acid Equivalent \nThe measurements are the mean values (n, 3) \u00b1 standard error of the \nmean, whereas different alphabets vertically indicate a significant \n\ndifference at 0.05 probability level. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7   Adhikari et al.  \n\n\u00a9NJB, BSN 6 \n\nnoticed the essential oil of the clove has an antifungal \n\neffect against C. albicans. \n\nAdditionally, the methanolic extract of pepper was found \n\nto be inhibitory against all of the bacterial strains but not \n\nto the fungal strain (C. albicans), showing the highest \n\nactivity against S. aureus. On the contrary, the ethanolic \n\nextract was found to be effective only against E. coli and \n\nS. aureus at higher concentrations. The study performed \n\nby Joshi et al (2009) [38] and Dahal et al (2017) [24] \n\nreported the antimicrobial effect of pepper methanolic \n\nextracts against E. coli, S. aureus, S. Typhi, P. aeruginosa, \n\nand Proteus species which coincides with the result \n\nobtained by our study.  \n\nThe phytochemical analyses (Table 4) of the spices \n\nconfirm the presence of bioactive compounds as \n\nPolyphenols, Flavonoids, and Tannins which in terms are \n\nresponsible for antimicrobial, anti-inflammatory, and \n\nanti-cancer activities. The phytochemicals of the test \n\nspices correspond to that reported by Abdelfadel et al \n\n(2016) [34], Yang et al (2012) [39], Al-Numair et al (2007) \n\n[40], Gupta (2013) [41], Mishra et al (2014) [42], and Zhang \n\net al (2014) [23]. The phytochemicals and their derivatives \n\nare responsible for the antibacterial and antifungal \n\nactivities of the plant extracts.", "start_char_idx": 23647, "end_char_idx": 26747, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3b08dbe2-e9b2-4b1a-9494-6ebb65b88ed2": {"__data__": {"id_": "3b08dbe2-e9b2-4b1a-9494-6ebb65b88ed2", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d7fdaa35-e536-4918-8204-ec6cc1467892", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "f17884b7b6fcee535fad3a3c7618e53d6e65c21075b4415f372fa0f1f0862766", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3af4320b-a8db-485a-8585-4cbdff3e4fda", "node_type": "1", "metadata": {}, "hash": "d09cd416f21e171e15df5f4149cc1978a8ab5f90da0bf45e43bd8cf4fdbde042", "class_name": "RelatedNodeInfo"}}, "text": "Methanol (Polarity index = \n\n6.6) being highly polar than ethanol (Polarity index = 5.2) \n\nis responsible for a higher yield of phytochemicals from \n\nthe spices [43]. Therefore, the methanolic extracts of the \n\nspices demonstrated higher antimicrobial properties \n\nwith the experimental microorganisms than that of \n\nethanolic extracts. Cinnamaldehyde, eugenol, linalool, \n\neugenyl acetate, and cinnamyl acetate present in \n\ncinnamon is responsible for its antimicrobial property \n\n[35]. According to Gupta et al (2015) [44], a higher \n\nconcentration of eugenol, \u03b2-Caryophyllene, and eugenyl \n\nacetate are responsible for antibacterial and antifungal \n\nactivities of clove. Vashist et al (2016) [45] have \n\ndocumented, volatile components as linalool-8-mono \n\nterpentriol-3, 7-dimethyl 1-octane-3,6,7-triol, trans-\n\ncinnamic acid, etc are the major chemicals responsible for \n\nthe antimicrobial effect of pepper. \n\nConclusion \nThe spices sample used for investigation constituted \n\nphytochemicals. The spices with a high amount of \n\nphytochemicals were found to be more effective against \n\nvarious pathogenic microorganisms whereas those spices \n\nwith little phytochemicals were not as effective as those \n\nwith higher phytochemicals. This result reveals that \n\nphytochemicals are the major constituents of medicinal \n\nplants that are responsible for their antimicrobial \n\nproperty.  \n\nAuthor\u2019s contribution \nBA conceptualized the research proposal, performed lab \n\nworks, scoring, and data analyses. PKS and RK \n\nsupervised the research activities and supported data \n\nanalyses and interpretations. All authors read and \n\napproved the final manuscript. \n\nCompeting interests \nThe authors declare that there is no conflict of interest \n\nregarding the publication of this paper. \n\nFunding \nNational Food Research Centre (NFRC) partially funded \n\nthis research works. \n\nAcknowledgements \nThe authors would like to express high lexis of thanks to \n\nMr. Pravin Ojha, Scientist and Mr. Ujjwol Subedi, \n\nTechnical Officer, and other supportive staffs of National \n\nFood Research Centre (NFRC), Nepal Agricultural \n\nResearch Council (NARC) for their invaluable support \n\nand cooperation. \n\nEthical approval and consent \nNot Applicable\n\nReferences \n1. Shakya AK. Medicinal plants: Future source of new drugs. Int. J. \n\nHerb. Med. 2016; 4 (4): 59-64. \nhttps://doi.org/10.13140/RG.2.1.1395.6085\n\n2. McGaw LJ, J\u00e4ger AK, van Staden J. Antibacterial, anthelmintic and \nanti-amoebic activity in South African medicinal plants. J \nEthnopharmacol. 2000;72(1-2): 247-263. doi:10.1016/s0378-\n8741(00)00269-5 \n\n3. Kumar Y, Agarwal S, Srivastava A, Kumar S,  Agarwal G, Khan \nMZA. Antibacterial activity of Clove (Syzygium aromaticum) and \nGarlic (Allium sativum) on different pathogenic bacteria. Ind. J. Pure\nApp. Biosci. 2014; 2(3): 305-311.\n\n4. Singh G, Kapoor IP, Singh P, de Heluani CS, de Lampasona MP, \nCatalan CA. Chemistry, antioxidant and antimicrobial \ninvestigations on essential oil and oleoresins of Zingiber \nofficinale. Food Chem Toxicol. 2008;46(10): 3295-3302. \ndoi:10.1016/j.fct.2008.07.017\n\n5. Javid T, Adnan M, Tariq A, Akhtar B, Ullah R, AbdElsalam NM. \nAntimicrobial activity of three medicinal plants (Artemisia indica, \nMedicago falcata, and Tecoma stans).  Afr. J. Tradit. Complement.", "start_char_idx": 26748, "end_char_idx": 30039, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3af4320b-a8db-485a-8585-4cbdff3e4fda": {"__data__": {"id_": "3af4320b-a8db-485a-8585-4cbdff3e4fda", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3b08dbe2-e9b2-4b1a-9494-6ebb65b88ed2", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "6869a7d33e3bbb4b0ebc66ce057eb762e706ce30cb3aa1dc0bc46956fc73f696", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "717215a4-fe73-44c3-80d1-12a08e759e32", "node_type": "1", "metadata": {}, "hash": "a34308993cb1a33e5a5bd857ccfa05875b1a2bbdb8560fe49ed0f30e26280066", "class_name": "RelatedNodeInfo"}}, "text": "Afr. J. Tradit. Complement. \nAltern. Med. 2015; 12(3): 91-96. \nhttps://doi.org/10.4314/ajtcam.v12i3.11\n\n6. Venkatachalam D, Rahman A, Sunny B, Jacob J, Kuriyan N, Raman \nR, Vaniapurackal R. Screening of Antimicrobial Activity of Various \nExtracts of the Stem Justicia gendarussa. Asian Journal of Research in \nMedical and Pharmaceutical Sciences. 2019; 6(4): 1-7. \n\n7. Shihabudeen MSS, Priscilla H, Thirumurugan K. Antimicrobial \nactivity and phytochemical analysis of selected Indian folk \nmedicinal plants. 2010; 1(10): 430-434. \n\n8. Elnourani EA. Antimicrobial and Phytochemical Analyses of \nSelected Medicinal Plant from Kenya. Master's Degree University \nof Nairobi, Kenya. 2016.\n\n9. Opinde HR. Antimicrobial Activity And Qualitative Phytochemical \nComposition of Crude Extracts From Medicinal Plants Against \nSelected Enteric Bacterial Pathogens and Candida albicans. \n[dissertation]. Kenya , Kenyatta University; 2016.\n\n10. Nguta JM, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, \nKiama SG. Biological screening of Kenyan medicinal plants using \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 1 - 7   Adhikari et al.  \n\n\u00a9NJB, BSN 7 \n\nArtemia Salina L. (Artemiidae). Pharmacologyonline. 2011; 2: 458-\n478. https://doi.org/10.3389/fphar.2011.00030 \n\n11. Das M, Mandal S, Mallick B and Hazra J. Ethnobotany, \nPhytochemical and Pharmacological Aspects of Cinnamomum \nzeylanicum Blume. Int Res J Pharm. 2013; 4 (4): 58-63. \n\n12. Vyas P, Suthar A, Patel D, Dayma P, Raval J, Joshi D. Antimicrobial \nactivity of extracts of Cinnamomum zeylanicum bark and its \ncombination with antibiotics against various microorganisms. J \nChem Pharm Res. 2015; 7 (4): 68-70. \n\n13. Nabavi SF, Lorenzo AD, Izadi M, Sanchez ES, Daglia M, Nabavi SM. \nAntibacterial Effects of Cinnamon: From Farm to Food, Cosmetic \nand Pharmaceutical Industries. Nutrients. 2015; 7: 7729-7748. \nhttps://doi.org/10.3390/nu7095359\n\n14. Blaszyk and Holley (1998): Blaszyk M, Holley RA. Interaction of \nmonolaurin, eugenol, and sodium citrate on the growth of common \nmeat spoilage and pathogenic organisms. Int J Food Microbiol. 1998 \nFeb 17; 39(3):175-83. doi: 10.1016/s0168-1605(97)00134-7. PMID: \n9553796. \n\n15. Bowles BL, Sackitey SK, and Williams AC. Inhibitory effects of \nflavor compounds on Staphylococcus aureus, Staphylococcus \naureus WRRC B124. J Food Saf. 1995; 15: 337-347. \n\n16. Helander IM, Alakomi HL, Kala KL, Sandholm TM, Pol I, Smid EJ, \nGorris LGM and Wright AV. Characterization of the Action of \nSelected Essential Oil Components on Gram-Negative Bacteria. J \nAgric Food Chem. 1998; 46: 3590-3595. \n\n17.", "start_char_idx": 30012, "end_char_idx": 32598, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "717215a4-fe73-44c3-80d1-12a08e759e32": {"__data__": {"id_": "717215a4-fe73-44c3-80d1-12a08e759e32", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3af4320b-a8db-485a-8585-4cbdff3e4fda", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "d6ec69ca18132a2e527d191551f06911fd63c02fe68dd566ee2fa1908cbe4291", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e12f1259-a339-4041-a2ab-a08a703fb1bb", "node_type": "1", "metadata": {}, "hash": "de5bb9350bb295a19506bbffdf143342895e7be30bd3b7dc0bff57aa8dc91ef8", "class_name": "RelatedNodeInfo"}}, "text": "1998; 46: 3590-3595. \n\n17. (Trongtokit et al 2005): Trongtokit Y, Rongsriyam Y, Komalamisra \nN, Apiwathnasorn C. Comparative repellency of 38 essential oils \nagainst mosquito bites. Phytother Res. 2005 Apr; 19(4): 303-9. doi: \n10.1002/ptr.1637. PMID: 16041723.\n\n18. Saeed S and Tariq P (2008). In Vitro Antibacterial Activity of Clove \nagainst Gram-Negative Bacteria. Pak J Bot. 40 (5): 2157-2160. \n\n19. Banerjee S, Panda CK, Das S. Clove (Syzygium aromaticum L.), a \npotential chemopreventive agent for lung cancer. Carcinogenesis. \n2006 Aug; 27(8): 1645-54. doi: 10.1093/carcin/bgi372. Epub 2006 \nFeb 25. PMID: 16501250.\n\n20. Pulikottil SJ and Nath S. Potential of clove of Syzygium aromaticum \nin development of a therapeutic agent for periodontal disease. A \nreview. SADJ. 2015; 70 (3): 108-115.\n\n21. Joshi S, Gyawali A. Phytochemical and Biological Studies on \nZanthoxylum Armatum of Nepal. Journal of Nepal Chemical \nSociety. 2013; 30: 71-77. https://doi.org/10.3126/jncs.v30i0.9339\n\n22. Vashist H, Sharma RB, Sharma D, Upmanyu N. Pharmacological \nactivities on Zanthoxylum aramatum: A Review. World J Pharm \nPharm Sci. 2016; 5 (12), 408-423. \n\n23. Zhang Y, Luo Z, Wang D, He F, Li D. Phytochemical profiles and \nantioxidant and antimicrobial activities of the leaves of Zanthoxylum \nbungeanum. The Scientific World Journal. 2014; 1-13. \nhttps://doi.org/10.1155/2014/181072\n\n24. Dahal T, Kandel S, Pandeya A, Pokhrel B. Study of antibacterial and \nantioxidant activities of four common Nepalese kitchen spices. Afr \nJ Pharm Pharmacol. 2017; 11 (6): 103-107. \nhttps://doi.org/10.5897/AJPP2016.4615\n\n23. Jouda MM. The Antibacterial Effect of Some Medicinal Plant \nExtracts and their Synergistic Effect with Antibiotic and Non-\nantibiotic Drugs. Master's Islamic University-Gaza, Palestine. 2013. \n\n24. Mahesh B, Satish S. Antimicrobial activity of some important \nmedicinal plant against plant and human pathogens. \nWorld Journal of Agricultural Sciences: WJAS. 2008; 4 (S): 839-843. \n\n25. Ganesh P, Kumar RS, Saranraj P. Phytochemical analysis and \nantibacterial activity of pepper (Piper nigrum L.) against some \nhuman pathogens. Cent. Euro. J. Exp. Bio. 2014; 3 (2): 36-41. \n\n26. Dimitrijevi\u0107 DS, Kostic DA, Stojanovic GS, Mitic SS, Mitic MN, \nDordevic AS. Phenolic composition, antioxidant activity, mineral \ncontent, and antimicrobial activity of fresh fruit extracts of Morus \nalba L. J. Food and Nutr. Res. 2014; 53: 22-30. \n\n27. Bhattarai S, Subedi U, Bhattarai U, Karki R, Ojha P. Study on \nChemical and Bioactive Components of Different Floral Sources\u2019 \nHoney in Nepal. Journal of Food Science and Technology Nepal.", "start_char_idx": 32572, "end_char_idx": 35201, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e12f1259-a339-4041-a2ab-a08a703fb1bb": {"__data__": {"id_": "e12f1259-a339-4041-a2ab-a08a703fb1bb", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "717215a4-fe73-44c3-80d1-12a08e759e32", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "e978cfcf703c187bc69fa485fa45892bdc3c65ad85d8f7023d128550843a75c8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5e0a7e06-368f-46e8-83cf-dc21549bdd12", "node_type": "1", "metadata": {}, "hash": "78bc6ba6c0edeb018b8dcef605586239c65a347039997b3df6c1ea2904f172df", "class_name": "RelatedNodeInfo"}}, "text": "Journal of Food Science and Technology Nepal. \n2019; 11: 51-9. https://doi.org/10.3126/jfstn.v11i0.29690\n\n28. Mahdavi R, Nikniaz Z, Rafraf M, Jouyban A. Determination and \ncomparison of total polyphenol and vitamin C contents of natural \nfresh and commercial fruit juices. Pak J Nutr. 2010; 9 (10): 968-972. \nhttps://doi.org/10.3923/pjn.2010.968.972\n\n29. Samatha T, Shyamsundacharya R, Srinivas P, Swamy NR. \nQuantification of total phenolic and total flavonoid contents in \nextracts of Oroxylum indicum L. Kurz. Asian J Pharm Clin Res. 2012; \n5 (4): 177-179. \n\n30. Faleye OS, Dada EO. Studies on the Phytochemical and Proximate \nProperties of the Extract of Unripe Annona muricata (Linn.) Fruit. \nJournal of Scientific Research and Reports. 2018; 20(5): 1-9. \nhttps://doi.org/10.9734/JSRR/2018/21584\n\n31. Polshettiwar SA, Ganjiwale RO, Wadher SA, Yeole PG. \nSpectrophotometric estimation of total tannins in some ayurvedic \neyedrops. Ind. J. Pharm. Sci. 2007; 69(4): 574-576. \nhttps://doi.org/10.4103/0250-474X.36949\n\n32. Tomar RS, Shrivastava V. In-vitro efficacy evaluation of ethanolic \nextract of Cinnamomum zeylanicum (Cinnamon) as potential \nantimicrobial agent against clinical isolates. Int J Pharm Sci Res. \n2015; 6 (5): 2201-2204. \n\n33. Al-Mariri A, Safi M. In-vitro antibacterial activity of several plant \nextracts and oils against some gram-negative bacteria. Iran J Med \nSci. 2014; 39 (1): 36-43. \n\n34. Abdelfadel, MM; Khalaf, HH; Sharoba, AM; Assous, MTM. Effect \nof extraction methods on antioxidant and antimicrobial activities of \nsome spices and herbs extracts. Journal of Food Technology and \nNutritional Sciences. 2016; 1: 1-14 \n\n35. Lopez P, Sanchez C, Batlle R, Nerin C. Solid- and vapor-phase \nantimicrobial activities of six essential oils: susceptibility of selected \nfoodborne bacterial and fungal strains. J Agric Food Chem. 2005; 53 \n(17): 6939-6946. https://doi.org/10.1021/jf050709v\n\n36. Karuppiah P, Rajaram SK. Antibacterial effect of Allium sativum, \ncloves, and Zingiber officinale rhizomes against multiple-drug \nresistant clinical pathogens. Asian Pac J Trop Biomed. 2012; 2 (8): \n597-601. https://doi.org/10.1016/S2221-1691(12)60104-X\n\n37. Pinto E, Silva LV, Cavaleiro C, Salgueiro L. Antifungal activity of \nthe clove essential oil from Syzygium aromaticum on Candida, \nAspergillus, and dermatophyte species. J Med Microbiol. 2009; 58: \n1454-1462. https://doi.org/10.1099/jmm.0.010538-0\n\n38. Joshi B, Lekhak S, Sharma A. Antibacterial property of different \nmedicinal plants: Ocimum sanctum, Cinnamomum zeylanicum, \nXanthoxylum armatum, and Origanum ajorana. J Sci Eng Technol. \n2009; 5 (1): 143-150. https://doi.org/10.3126/kuset.v5i1.2854\n\n39.", "start_char_idx": 35156, "end_char_idx": 37841, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5e0a7e06-368f-46e8-83cf-dc21549bdd12": {"__data__": {"id_": "5e0a7e06-368f-46e8-83cf-dc21549bdd12", "embedding": null, "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-116", "node_type": "4", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "2c998fe0e3d60c1f4e172c21ddad8ca621353b0b426a13b96a8fc114c96431b3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e12f1259-a339-4041-a2ab-a08a703fb1bb", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "5c44e33b13ebea9ad47ea722adc3ff9ffb0a0ab3827917c7926c6e097a895cc0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6b457671-cc67-4783-8363-ff77ce12619e", "node_type": "1", "metadata": {}, "hash": "c1a0bba23c25472315153712e15375b2b4e2667924805547c99773f9b801b59a", "class_name": "RelatedNodeInfo"}}, "text": "Yang CH; Li RX; Chuang LY. Antioxidant activity of various parts \nof Cinnamomum cassi. Molecules. 2012; 17: 7294-7304. \nhttps://doi.org/10.3390/molecules17067294\n\n40. Al-Numair KS., Ahmad D, Ahmad SB, Abdullah, Al-Assaf H. \nNutritive value, levels of polyphenols and anti-nutritional factors \nin Sri Lankan cinnamon (Cinnamomum zeyalnicum) and Chinese \ncinnamon (Cinnamomum cassia). Res. Bult., 2007; 154: 5-21. \n\n41. Gupta D. Comparative analysis of spices for their phenolic content, \nflavonoid content, and antioxidant capacity. American \nInternational Journal of Research in Formal, Applied & Natural \nSciences. 2013; 4: 38-42. \n\n42. Mishra RP, Sharma K. Antimicrobial activities of Syzigium \naromaticum L. (Clove). Int Res JBiological Sci. 2014; 3(8), 22-25. \n\n43. Vargas RA, Malacara CFP, Petricevich VL. Characterization of \nchemical compounds with antioxidant and cytotoxic activities in \nBougainvillea x buttiana Holttum and Standl, (var. Rose) Extracts. \nAntioxidants. 2016; 5 (45), 1-11. \nhttps://doi.org/10.3390/antiox5040045\n\n44. Gupta D, Girija. Evaluation of in vitro antioxidant and \nantimicrobial activities of various spices of Indian origin. \nInternational Journal of Pharmacy and Pharmaceutical Sciences. \n2015; 7(8); 137-141. \n\n45. Vashist H, Sharma RB, Sharma D, Upmanyu N. Pharmacological \nactivities on Zanthoxylum aramatum: A Review. World J Pharm \nPharm Sci. 2016; 5 (12), 408-423.\n\nhttps://doi.org/10.3923/pjn.2010.968.972", "start_char_idx": 37842, "end_char_idx": 39291, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6b457671-cc67-4783-8363-ff77ce12619e": {"__data__": {"id_": "6b457671-cc67-4783-8363-ff77ce12619e", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5e0a7e06-368f-46e8-83cf-dc21549bdd12", "node_type": "1", "metadata": {"identifier": "njb-116", "author": "Adhikari, Bibek; Shah, Pradeep Kumar; Karki, Roman", "title": "Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts", "date": "2021-07-31", "file": "njb-116.pdf"}, "hash": "79b251f82b22afeebd3139fb56189f667995856a8e83f2778a072142df9c797d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ef154172-65aa-4a99-94a5-615488cdbe84", "node_type": "1", "metadata": {}, "hash": "a157c4d2615ac6839024a3def153ec772cd22a4c7230a37c72b2201dcec7837b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2  Review article DOI: https://doi.org/10.3126/njb.v9i1.38643 \n\n\u00a9NJB, BSN 85 \n\nAdvances In Agricultural Biotechnology \nMamata KC  , Anuj Lamichhane  \n\nAgriculture and Forestry University, Rampur, Chitwan; Nepal \n\nReceived: 04 Nov 2020; Revised: 20 Jul 2021; Accepted: 24 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract \nAgricultural biotechnol.ogy is becoming the major sector in crop improvement through the use of scientific techniques for the \nmodification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. With the evolvement \nfrom Mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. \nRecent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing \ncrop yield potential. With the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, \nembryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, \nvarious transgenic crops have been developed over the decades and have been approved for commercialization. This \ndevelopment and adoption of transgenic technology have been shown to increase crop yields, reduce CO2 emission, reduce \npesticide and insecticide use and decrease the costs of crop production.  Even though the biotechnological approach and \ntransgenic organisms have immense potential to contribute to the world\u2019s food security, several concerns of genetically \nmodified crops being a threat to the environment and human health have developed. This review will address applications \nand concerns of biotechnology in crop improvement considering health hazards and ecological risks. \n\nKeywords: Agricultural biotechnology, genetic engineering, transgenic organisms, benefits, concerns. \n\n Corresponding author, email: kcmamata24@gmail.com \n\nIntroduction \nBiotechnology refers to the implementation of \n\ncomprehensive scientific techniques to alter and enhance \n\nthe characteristics of different plants, animals, and \n\nmicroorganisms that are of economic importance [1]. \n\nBiotechnology is a broad term that includes applications \n\nof microorganisms and different foreign genes (gene of \n\ninterest) in the processing of food; agriculture and \n\nforestry; environmental protection, medical sector, etc. \n\n[2]. Agricultural biotechnology is the branch of \n\nbiotechnology that involves the exertion of scientific \n\ntechniques for the modification and improvement of \n\ncrops as well as livestock [3]. With the increasing \n\npopulation, traditional agriculture is not sufficient to \n\nmeet the demands of food worldwide, thus the \n\ncontinuous increase in agricultural productivity depends \n\non effective unification of biotechnology with classical \n\nbreeding to create an \"Evergreen Revolution\" [4].  \n\nCrop productivity has advanced largely during the 20th \n\ncentury based on applications of Mendelian genetics, but \n\nif farmers are to address the demands that will be laid on \n\nthem over the next half-century more effectively, \n\nresearch in biotechnology and molecular biology should \n\nbe aimed towards removing the physiological constraints \n\nof the crops and increasing crop yield potential [5]. \n\nRecent developments in plant molecular biology and \n\ngenomes not only has provided us the knowledge and \n\nunderstanding of plant genomes but also the possibility \n\nof modifying them [6]. Biotechnology provides series of \n\ntechniques that give access to a wider gene pool and also \n\npermits the accurate progress to produce new and useful \n\nplant and animal genotypes working along with \n\nconventional breeding techniques side by side  [7]. The \n\nuse of traditional techniques, without any question, has \n\nprofoundly improved important heritable characters \n\nsuch as yield, resistance to disease, etc. in crops, however, \n\nthere are certain restrictions to these techniques like it \n\nmay take a very long time to introduce, select and \n\nestablish a trait into a cultivar or it may be impossible to \n\nincorporate certain traits with these techniques. Genetic \n\nengineering overcomes these limitations by introducing \n\nthe desired trait in short time without altering other \n\ncharacters of the plant [8]. \n\nIn this technological era, agriculture faces a new stream \n\nof technological revolution associated with \n\nbiotechnology which could offer considerable assurance \n\nfor agricultural sustainability by quality enhancement of \n\nthe product, disease and insect pest resistance, \n\nenvironmental protection, and improving agricultural \n\nproductivity [9].", "start_char_idx": 48, "end_char_idx": 4724, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ef154172-65aa-4a99-94a5-615488cdbe84": {"__data__": {"id_": "ef154172-65aa-4a99-94a5-615488cdbe84", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6b457671-cc67-4783-8363-ff77ce12619e", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "be2c07fe2d36442190129b9f97ed82d5f960fa7d58ad03138cf441dbd7a7e169", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e91253a6-bb1a-4939-b93e-10016bfa9a45", "node_type": "1", "metadata": {}, "hash": "44e8d5486e5ae566a6b31cb8b6c958ab5e311341e7436610a6e732cb969f6174", "class_name": "RelatedNodeInfo"}}, "text": "With the advances in the field of \n\nmolecular biology, scientists can manipulate DNA to \n\nproduce transgenic organisms, the process is known as \n\n\u201cGenetic Engineering\u201d and offers a range of benefits \n\nalong with possible risks [3]. There are controversial \n\nsocial and regulatory consequences with genetic \n\nengineering and food made from transgenic crops [10]. \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-9302-6554\nmailto:kcmamata24@gmail.com\nhttps://orcid.org/0000-0002-3593-604X\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  86 \n\nSo, all of transgenic crops developed are not released for \n\ncommercial cultivation. This review tries to address the \n\nrecent advances of biotechnology in agriculture and its \n\nmajor concerns. \n\nBackground and History of biotechnology in \nAgriculture \nAgriculture is the backbone of the human food supply. \n\nAgriculture was practiced manually, in the beginning, \n\nusing primitive technologies based on plow and harrow. \n\nThe Industrial revolution (1875-1885) enabled accelerated \n\neconomic development which led to the movement of \n\npeople from rural areas to industrialized cities. It was \n\naround this time the chemical fertilizers were introduced \n\nfor protection against disease and attainment of higher \n\nyields [11]. The human population at present is 7.87 \n\nbillion increasing at 1.1%  average annual rate of \n\npopulation change in year 2015-2020 [12]. This \n\ncontinuous increase in population, estimated to reach 9 \n\nbillion by 2050, poses a serious challenge to global food \n\nsecurity. With the increasing world population, \n\nagricultural land has been utilized for settlement \n\npurpose. This has decreased the land under cultivation \n\nand ultimately the productivity. So, increasing food \n\ndemands of the world can be met by increasing the global \n\nagriculture productivity. But lower land under \n\nagriculture cultivation demanded a drastic innovation in \n\ntechnology which not only increase the agriculture \n\nproductivity but also sustain it for long time. This was \n\nprovided by the breakthrough of biotechnology field [13].  \n\nGregor Mendel\u2019s paper \u201cExperiments on plant \n\nhybridization\u201d, published in 1866; included how \n\ndifferent traits were passed from generation to \n\ngeneration which marked the beginning of new \n\ntechnologies designed for improvement in crop species \n\n[1].  But, gene modification in crops is supposed to have \n\nbegun around 10,000 years ago as a result of random or \n\nchance through the selection of novel crop types [14]. In \n\n1960, Green Revolution helped in increasing productivity \n\nof three main cereal crops viz. rice, maize, and wheat. A \n\nparticularly important finding was the discovery of the \n\nmolecular structure of deoxyribonucleic acid (DNA) and \n\nthe fact that DNA was involved in inheritance. The \n\ngenetic code was cracked in the 1960s and made a way \n\nfor the transfer of genetic material even easier. With the \n\ntransfer of genes from one organisms to another, different \n\nnovel organisms are created, often referred as \n\n\u2018Genetically modified organisms (GMOs)\u2019 [15]. With \n\ndevelopment of several GMOs; modern biotechnology \n\nhas focused on genetic manipulation for agriculture, \n\nhorticulture, environment, medicine, forensic science, \n\nand many other fields [16]. The major events in history of \n\nbiotechnological development is presented in Table 1 \n\n[11]. \n\nTable 1: Summary of the main events in the development \n\nof biotechnology [11]  \n\nClassical biotechnology \n\n1664 Discovery of microorganisms. \n\n1884 Discovery of bacteria. \n\n1857 Microbiology of lactic fermentation. \n\n1860 End of the spontaneous generation theory \n\n1866 Theory of Inheritance (Gregor John Mendel) \n\n1902 Chromosomal Theory of Inheritance  \n\n1910 Discovery of linkage  \n\n1928 Transformation in bacteria  \n\n1941 One gene-one enzyme hypothesis \n\n1946 Bacterial conjugation. \n\n1947 Chargaff\u2019s rule  \n\nModern Biotechnology \n\n1953 DNA structure.", "start_char_idx": 4725, "end_char_idx": 8853, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e91253a6-bb1a-4939-b93e-10016bfa9a45": {"__data__": {"id_": "e91253a6-bb1a-4939-b93e-10016bfa9a45", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ef154172-65aa-4a99-94a5-615488cdbe84", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "80b062eada13b281a81b8c767c52d8f5fe031e922565a51c64d292fbcbc61233", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "67e2b0a7-7857-4f1d-a16f-76b38370ec14", "node_type": "1", "metadata": {}, "hash": "6c5c61f62682567e2cd3894297d45ef610b73ffdb7cf903c63b2109a98256df4", "class_name": "RelatedNodeInfo"}}, "text": "1947 Chargaff\u2019s rule  \n\nModern Biotechnology \n\n1953 DNA structure. \n\n1958 Semi-conservative Replication of DNA \n\n1959 Gene regulation. \n\n1960 Green Revolution \n\n1966 Genetic code decoding \n\n1970 The high specificity of restriction enzymes. \n\nRise of phyto-genetics \n\nCIMMYT foundation 1973 Recombinant DNA replication in E.coli \n\n1978 Human proinsulin gene isolation \n\n1985 Polymerase chain reaction. \n\n1992 Beginning of the Golden Rice project \n\n1996 Full-fledged commercialization of GM crops \n\nAgricultural Biotechnology in Crop \nImprovement \nAgricultural biotechnology refers to the use of biological \n\norganisms or range of tools for the improvement of the \n\nplants, animals, microorganisms, or food derived from \n\nthem. Following are some biotechnology tools used in \n\nagriculture: \n\nTransgenesis \nTransgenesis also called genetic engineering or \n\nrecombinant DNA (rDNA) technology; includes multiple \n\ntechniques used for the desired manipulation of genetic \n\nmaterial (cutting and joining together) particularly DNA \n\nfrom various species, and subsequent introduction of the \n\nresulting hybrid DNA into a new organism to form new \n\ncombinations of heritable genetic material [17][18]. \n\nOrganisms resulting from transgenesis are called \n\nGenetically Modified Organisms (GMOs). \n\nAround 530 different transgenic events in 32 crops have \n\nbeen approved for cultivation in different parts of the \n\nworld [19]. Among them, Maize accounts for the \n\nmaximum number of events (240), followed by cotton \n\n(67), potato (50), Argentine canola (42), soybean (42), \n\ncarnation (19), and so on. Transgenesis has been applied \n\nto develop Herbicide-tolerant (HT) transgenic crops, \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  87 \n\nInsect-resistant (IR) transgenic crops, Abiotic stress-\n\ntolerant (AST) transgenic crops, disease-resistant \n\ntransgenic crops, and nutritionally improved transgenic \n\ncrops. \n\nHerbicide Tolerant transgenic crops \nThe first herbicide-tolerant transgenic crop to be \n\ncommercialized was Glyphosate-tolerant soybean \n\n(Roundup Ready soybean), which harbored EPSPS gene \n\nfrom CP4 strain of Agrobacterium tumefaciens.  Most of the \n\ncommercialized glyphosate-resistant crops harbor this \n\ngene [20]. Two different genes from Streptomyces spp., \n\nnamely pat and bar, were utilized for developing \n\nGlufosinate-resistant crops. Similarly, other HT \n\ntransgenic crops specific to other herbicides like 2,4-D, \n\nIsoxafutole, Oxynil, and Sulfonylurea, have been \n\ncommercialized recently [21]. A total of 351 herbicide \n\ntolerance events have been approved for cultivation [19]. \n\nOf these, the maximum number of HT events (212) has \n\nbeen commercialized in Maize, followed by Cotton (45), \n\nArgentine canola (34), and others.  \n\nInsect Resistant Transgenic Crops \nMost of the insect-resistant transgenic crops are \n\ndeveloped from cry genes from Bacillus thuringiensis (Bt); \n\nwhich provides resistance against a wide variety of insect \n\npests (Lepidopterons, Coleopterans, and Dipterans) [22]. \n\nCry genes not merely provide resistance against insect \n\npests but also is non-toxic to mammals. The first \n\ncommercially successful crop was Cotton in which cry \n\ngene was inserted that provided resistance against its \n\nlepidopteron insect pest. After the success of transgenic \n\ncotton, cry genes have been incorporated in many crops, \n\nviz., potato, rice, canola, soybean, maize, chickpea, \n\nalfalfa, and tomato [21]. Similarly, vip genes isolated \n\nfrom Bacillus species (B. thuringiensis and B. cereus) are \n\nincorporated in cotton and maize for insect resistance \n\n[23][19]. Genes encoding protease inhibitor (PI) from \n\ndifferent sources (plants, bacteria, and fungi) have been \n\nused to produce insect resistant plants.", "start_char_idx": 8787, "end_char_idx": 12587, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "67e2b0a7-7857-4f1d-a16f-76b38370ec14": {"__data__": {"id_": "67e2b0a7-7857-4f1d-a16f-76b38370ec14", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e91253a6-bb1a-4939-b93e-10016bfa9a45", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "867da07b989f59c5dc2f4123112f36e848c258e872057870492f2abb95d8ba7c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "47f54be0-0753-4a41-973e-ebd71a56ce41", "node_type": "1", "metadata": {}, "hash": "6eb4ab5f2ae967967701b4455a9b48684d49008d42d347aa923766771dd34a39", "class_name": "RelatedNodeInfo"}}, "text": "The cptII and \n\npotato protease inhibitor II genes have been introduced \n\nin tobacco, and rice, and cotton, respectively to provide \n\nresistance against insects [21][19]. To date, 305 insect \n\nresistance events have been approved for cultivation [19]. \n\nOf these, the maximum number of insect-resistant events \n\n(208) has been commercialized in Maize, followed by \n\nCotton (50), Potato (30), and others. \n\nAbiotic Stress Tolerant Transgenic Crops \nThe impact of abiotic stresses is increasing in crops with \n\nchanging climatic conditions. Certain plants adapt to \n\nthese abiotic stresses at the molecular level by altering the \n\nexpression of an array of genes. This helps to create near-\n\noptimal conditions for plant growth and development \n\n[21]. Due to the complexity of the abiotic stress \n\nadaptation trait (many genes are involved), a lesser \n\nnumber of abiotic stress tolerance events have been \n\ncommercialized as compared to traits like disease, insect, \n\nand herbicide tolerance.  A total of 12 abiotic stress \n\ntolerance events have been approved for cultivation in \n\nMaize(7), Sugarcane(3), and Soybean (2)  [19]. The use of \n\nbacterial cold shock proteins (csp) to mitigate the effects \n\nof abiotic stresses, like cold in Arabidopsis, cold, heat, and \n\nwater deficit in rice, and water deficit in maize, has been \n\ndemonstrated by Castiglioni et al. in 2008 [24]. Two \n\ngenes: the cspA gene from E. coli and the cspB gene from \n\nsoil bacterium B. subtilis were incorporated in maize, \n\nwhich not only showed better adaptation during water-\n\nscarce conditions but also did not lead to pleiotropic \n\neffects in maize. Recently, Hahb-4 gene from Helianthus \n\nannus (Sunflower) is introduced in Verdeca\u2019s drought \n\ntolerant transgenic Soybean commercialized as Verdeca \n\nHB4 Soybean. The gene produces isolated nucleic acid \n\nmolecule encoding the transcription factor Hahb-4 which \n\nbinds to a dehydration transcription regulating region of \n\nplant [19].  Similarly, using betA gene from E. coli and \n\nRhizobium meliloti drought-tolerant transgenic Sugarcane \n\nhas been made. These transgenic sugarcane crops \n\nwithstand drought conditions up to 36 days and produce \n\n10-30% higher sugar as compared the non-transgenic \n\nplants under drought conditions in field trial [25,26]. \n\nDisease Resistant Transgenic Crops \nDiseases are caused by pathogens (fungi, bacteria, \n\nviruses, and other micro-organisms), and cause huge loss \n\nin crop yield. Despite the environmental hazards caused \n\nby the use of agrochemicals, management of diseases in \n\nplants is usually done using agrochemicals, which pose \n\nthe challenge of the development of chemical-resistant \n\npests [21]. Scientists have been able to breed plants with \n\ndisease resistance traits using transgenesis. So far, 29 \n\ndisease resistance events have been approved for \n\ncultivation [19]. Of these, the maximum number of \n\ndisease-resistant events (19) has been commercialized in \n\nPotato, followed by Papaya (4), Squash (2), and others.  \n\nMost of the disease-resistant crops commercialized \n\nconfer resistance against viruses [21]. Using gene \n\nencoding the viral coat protein of tobacco mosaic virus \n\n(TMV), the first disease-resistant plant was found, which \n\nwas resistant to TMV infection [27]. Similarly, transgenic \n\npapaya conferring resistance to Papaya Ringspot Virus \n\n(PRSV) has been developed through a \u201cpathogen-\n\nderived resistance mechanism\u201d, where the \u2018prsv cp\u2019 gene \n\nis introduced by microparticle bombardment into papaya \n\n[28]. In bean (Phaseolus vulgaris L.), RNAi-mediated \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  88 \n\nresistance against Bean Golden Mosaic Virus (BGMV)  \n\nwas developed by silencing the sequence region of the \n\nAC1 viral gene which inhibited the synthesis of the viral \n\nreplication protein of the BGMV [29]. In potato (Solanum \n\ntuberosum L.", "start_char_idx": 12588, "end_char_idx": 16502, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "47f54be0-0753-4a41-973e-ebd71a56ce41": {"__data__": {"id_": "47f54be0-0753-4a41-973e-ebd71a56ce41", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "67e2b0a7-7857-4f1d-a16f-76b38370ec14", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "dd4c1128da53d9f6ddce5c8a82830efe90e553bb7e380b1947505766bc30c376", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7590a645-8412-4e3e-9c18-5b4773817ebd", "node_type": "1", "metadata": {}, "hash": "2a7c71979a9a989a9ca4a3ba33403046f141e08bd95f6aa97a956aa7c6cd2bc5", "class_name": "RelatedNodeInfo"}}, "text": "In potato (Solanum \n\ntuberosum L.), the Rpi-vnt1.1 gene from Solanum venturii \n\nis introduced using Agrobacterium-mediated gene \n\ntransfer, which produces late blight resistance protein \n\nand confers resistance to potato late blight [30]. The major \n\nconstituents of the fungal cell wall (chitin and \u03b1-1, 3 \n\nglucan) are degraded by the chitinase enzyme thus when \n\nthe chitinase gene was introduced in tobacco and rice, it \n\nhas been reported to enhance fungal resistance in the \n\nplant [31].  \n\nNutritionally Improved Transgenic Crops \nMany successful efforts have been made to improve \n\nnutritional qualities in crops using transgenesis. The \n\nmost recent example includes biofortified rice line GR2E \n\n(Golden Rice), developed by the introduction of gene \n\n\u2018crt1\u2019 from Pantoea ananatis and gene \u2018psy1\u2019 from Zea \n\nmays. Golden Rice is capable of synthesizing carotenoids \n\nin the endosperm. GR2E was approved for use as food in \n\nthe Philippines, Australia, New Zealand, Canada, and \n\nthe United States [19]. Similarly, to improve the nutritive \n\nvalue of potato, the transgenic potato tubers were \n\ndeveloped by expressing Amaranthus seed albumin gene \n\n\u2018AmA1\u2019, which is plentiful of all essential amino acids for \n\nhuman diet specification according to the WHO standard \n\n[32]. An effort was made to enhance the pro-vitamin A \n\ncontent in tomato by producing transgenic tomato and \n\nconverting phytoene to lycopene with the transference of \n\nbacterial gene for phytoene-desaturase enzyme.  And \n\nalso three times more \u03b2 carotene content was produced \n\nby these transgenic plants than normal plants [33]. \n\nAntisense fae1 gene transferred to Brassica napus and \n\nBrassica juncea has resulted in low erucic acid content \n\n[34]. In maize, the introduction of the \u2018cordapA\u2019 gene \n\nfrom Corynebacterium glutamicum has increased the \n\nproduction of amino acid lysine [19]. \n\nTissue Culture \nTissue culture is the culture of cells, tissues, organs, or \n\ntheir components in a nutrient medium under sterile \n\nconditions [35]. It usually involves the use of small pieces \n\nof plant tissue (explants) which are cultured in aseptic \n\nconditions [36]. Tissue culture manipulates and extends \n\nthe period of cells, anthers, pollen grains, or other tissues \n\nand develops a whole, living growing organisms. Using \n\ntissue culture, genetically engineered cells can be \n\ntransformed into genetically engineered organisms [37]. \n\nTissue culture has been used extensively to create genetic \n\nvariability through the in-vitro culture of protoplasts, \n\nanthers, microspores, ovules, and embryos, to improve \n\ncrop plants and to increase the number of desirable \n\ngermplasm available to the plant breeder. It is one of the \n\npivotal tools of biotechnology [38]. Tissue culture is used \n\nin the germination of seeds that are difficult to germinate \n\nlike Banana. Grand Naine (G9) variety of banana is \n\nprepared using tissue culture, which results in mass \n\npropagation of disease-free high yielding clones, and true \n\nto type plants [39]. Similarly, the Meristem tip culture of \n\nbanana plants produces plants devoid of banana bunchy \n\ntop virus (BBTV) and brome mosaic virus (BMV) [40]. In \n\nvitro cell and organ, culture can be used for the \n\nconservation of endangered germplasms. The plants that \n\ndo not produce seeds (sterile) or produce seeds that \n\ncannot be stored for a long period (recalcitrant seeds), can \n\nbe preserved using tissue culture techniques for the \n\nmaintenance of gene bank [36]. \n\nEmbryo rescue for wide hybridization  \nEmbryo resulting from inter-specific or inter-generic \n\ncrosses may fail to produce a hybrid because of pre or \n\npost-fertilization incompatibility barriers. These barriers \n\ncan be overcome by rescuing such embryos and culturing \n\nthem for producing a whole plant, which facilitates the \n\ntransfer of desirable genes from wild relatives into \n\ncultivated species [38][18][41]. This technique is known \n\nas embryo rescue or wide hybridization. Wide \n\nhybridization and Embryo rescue were done in \n\nCapsicum to transfer fruit rot-resistant traits by \n\nDebbarama et al.", "start_char_idx": 16469, "end_char_idx": 20567, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7590a645-8412-4e3e-9c18-5b4773817ebd": {"__data__": {"id_": "7590a645-8412-4e3e-9c18-5b4773817ebd", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "47f54be0-0753-4a41-973e-ebd71a56ce41", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "26d2f443adb11a60245f7346ce16724a8a57a663f40996661456883e73616eee", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "69a4ffd2-03af-4b78-b3f4-a68fb2190a19", "node_type": "1", "metadata": {}, "hash": "eab53ef355ce3c4428718b9546b88ef9547b8a3eb0c536a6b615779569ed894c", "class_name": "RelatedNodeInfo"}}, "text": "in 2013 [42].  \n\nSomatic hybridization \nSomatic hybridization is a technique that integrates \n\nsomatic cells from two different cultivars, species, or \n\ngenera of plants for the manipulation of cellular genomes \n\n[43]. Somatic hybridization by protoplast fusion helps in \n\nthe regeneration of novel germplasm and into whole \n\norganisms through tissue culture [44][45]. Similarly, \n\nincompatibility barriers at inter-specific or intergeneric \n\nlevels can be overcome by somatic hybridization. Fusion \n\nbetween protoplasts of Potato (Solanum tuberosum) and \n\nTomato (Lycopersicum esculentum) has created Pomato \n\n(Solanopersicon, a new genus). It not only overcomes \n\nbarriers of sexual incompatibility but also creates novel \n\ngenotypes [46] \n\nA salt-tolerant hybrid callus culture was developed by \n\nsomatic hybridization between Rice (Oryza sativa) and \n\nMangrove grass (Myriostachya wightiana), which is useful \n\nin the development of salt-tolerant rice varieties [47]. \n\nDisease resistance genes are also transferred using \n\nsomatic hybridization like asymmetric somatic \n\nhybridization was used to transfer bacterial blight \n\nresistance trait from wild Oryza meyeriana L. to Oryza \n\nsativa L. ssp. Japonica [48]. Similarly, those genetic traits \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  89 \n\nthat are cytoplasmically controlled like male sterility, \n\nresistance to certain antibiotics and herbicides, can be \n\neasily transferred using protoplast transformation \n\nfollowed by somatic hybridization [43]. Cybridization \n\nhas been used to transfer Cytoplasm Male Sterility (CMS) \n\nin rice [49].  \n\nMolecular marker aided genetic analysis and \nselection \nMolecular marker aided genetic analysis helps in gene \n\nidentification i.e. it studies DNA sequences particularly \n\nto identify the genes, QTL (Quantitative trait loci), and \n\nmolecular markers; as well as associate them with the \n\norganism. Molecular marker aided selection helps to \n\nidentify and trace the inheritance of previously identified \n\nDNA fragments through a series of generations [37]. \n\nMolecular marker-assisted breeding uses molecular \n\nmarkers along with linkage maps and genomics to alter \n\nand improve plants or animal traits based on genotypic \n\nassays [50]. Rice genotypes having resistance to Bacterial \n\nBlight(BB) and Basmati quality and desirable agronomic \n\ntraits were identified using phenotypic and molecular \n\nmarker-assisted selection, which can be either directly \n\nused in the development of commercial varieties or used \n\nas a donor of BB resistance in Basmati breeding programs \n\n[51]. Similarly, Marker-assisted selection allowed \n\nidentification of sources of Coffee Berry Disease and \n\nCoffee rust resistance for use in preventive breeding for \n\nresistance to these diseases. Several genes from other \n\nCoffea species were important sources for gene \n\npyramiding in breeding programs aimed at multiple and \n\ndurable resistance [52]. Genetic analysis of Fusarium \n\nHead Blight Resistance in CIMMYT bread wheat line \n\nC615 was done using traditional and conditional QTL \n\nmapping by Yi et al. in 2018 [53]. This study showed \n\ngenetic relationships between FHB response and related \n\ntraits at the QTL level providing useful information for \n\nmarker-assisted selection for the improvement of FHB \n\nresistance while breeding. \n\nDoubled Haploid/ Genome doubling \nA doubled haploid (DH) is a genotype formed when \n\nhaploid cells undergo chromosome/genome doubling. \n\nHaploid cells like pollen, egg cells, or other cells of \n\ngametophyte are subjected to spontaneous chromosome \n\ndoubling, giving a doubled haploid cells, which is then \n\ngrown into a doubled haploid plant [54]. It allows the \n\ndevelopment of pure line varieties or inbred parental \n\nlines quicker compared to traditional breeding [55].  \n\nDouble haploid technology in wheat accelerated time to \n\nmarket and faster genetic gains in yield and resistance \n\ngain, which helped in reducing varietal development \n\ntime [56][57]. Similarly, anther-culture followed by DH \n\noffers a great opportunity to accelerate breeding progress \n\nand improve grain quality.", "start_char_idx": 20568, "end_char_idx": 24738, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "69a4ffd2-03af-4b78-b3f4-a68fb2190a19": {"__data__": {"id_": "69a4ffd2-03af-4b78-b3f4-a68fb2190a19", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7590a645-8412-4e3e-9c18-5b4773817ebd", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "ab56b085845183319a416701f6a0b334f4094a98299ec6b5679443fa4fef8e2d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9294abc0-91dc-4a45-8b20-f239cbfd8c4f", "node_type": "1", "metadata": {}, "hash": "a3e94ac9c5132a4ccab7b93ba835577dfef05cc8078df810f042447200401d59", "class_name": "RelatedNodeInfo"}}, "text": "DH plants through anther-\n\nculture provide an efficient method for rapid production \n\nof homozygous lines of rice which are found to be more \n\nviable than other lines [58]. Similarly, in another study by \n\nBakhshi, Bozorgipour, and Shahriari-Ahmadi in 2017, \n\nchromosome elimination method was used to develop \n\ndouble haploid wheat lines via crosses with maize as the \n\nmale parent [59]. Further 3 wheat lines were selected to \n\ndevelop and adapt under heat stress conditions. \n\n\u2018Omics\u2019 technologies \n\u2018Omics\u2019 technologies are subcategories of bioinformatics \n\nwhich include genomics, proteomics, transcriptomics, \n\ngenome sequencing, and metabolomics [60]. Genomics is \n\nused to understand the structure, function, and evolution \n\nof genes; and identify DNA that confers to traits in the \n\norganisms. Proteomics helps to analyze the protein in \n\ntissue for identifying gene expression in that tissue as \n\nwell as decipher the specific function of proteins encoded \n\nby particular genes[61][37].   \n\nOmics based approach helps to decipher the entire \n\ngenome for gaining insights into plant molecular \n\nresponses, which provides specific strategies for crop \n\nimprovement. Using the omics approach, we can identify \n\nDNA (gene) encoding for a certain trait (genomics), RNA \n\ncoded by it (transcriptomics), proteins formed \n\n(proteomics), metabolites produced (metabolomics), and \n\nphenotype expressed (phenomics). Omics technology \n\nprovides valuable information on the structure and \n\nbehavior of crop genomics. Any gene responsible for a \n\nparticular trait can be used to enhance breeding in \n\ndifferent ways [62]. A herbicide-tolerant maize line was \n\ndeveloped by precise insertion of a target gene using site \n\ndirect mutagenesis [63]. \n\nConcerns of Agriculture Biotechnology \nBiotech crops were grown in 29 countries in 2019, \n\ncontributing significantly to food security, sustainability, \n\nclimate change mitigation, and upliftment in the lives of \n\nfarmers and families worldwide [64]. However, some \n\nconcerns regarding gene manipulation in crops being \n\necologically harmful and unsafe for human \n\nconsumptions. Major concerns of agriculture \n\nbiotechnology are briefly discussed below: \n\nAdverse effects on non-target organisms \nThe use of transgenic crops for a specific cause \n\n(disease/pest resistance) has caused unintended effects \n\non non-target organisms. Reduction in monarch butterfly \n\npopulation has been reported on the adoption of \n\nglyphosate-resistant transgenic crops in the USA and \n\nMexico [65]; and higher mortality was reported when its \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  90 \n\nlarva fed on milkweed leaves dusted with the genetically \n\nmodified Bt maize as compared to laboratory conditions \n\n[66]. Similarly, wide-scale adoption of Bt cotton in China \n\nincreased the population of minor pest (Mirid bug), \n\nwhich acquired the status of major pest later [67]. \n\nBiosafety issues \nThere have been concerns about the safety of transgenic \n\nfood being a threat to human health and the \n\nenvironment. Risks associated with human health \n\ninclude allergenicity, toxicity, horizontal gene transfer, \n\nand feed safety [68]. When introducing a gene into an \n\norganism, the level of allergens might increase in the \n\nmodified organism above the natural range or new \n\nallergen might be introduced. So, bean crops modified to \n\nincrease the level of cysteine and methionine content \n\nwere discarded after the discovery of the expressed \n\nprotein of transgene being highly allergenic [69]. So \n\ntesting of transgenic food may be required to avoid harm \n\nto the consumers. Similarly, WHO has claimed genetic \n\nmaterial can be transferred from transgenic food to cells \n\nof the human body or bacteria in the intestinal tract or to \n\nsoil microbes mainly because the DNA ingested from \n\ntransgenic food is not completely degraded by digestion \n\n[68]. The possibility of horizontal transfer of antibiotic-\n\nresistant marker genes from transgenic food to animal \n\nand human gut microbes may result in antibiotic \n\nresistance in the gut microflora, though its possibility is \n\nextremely low [21]. Similarly, the cultivation of \n\ngenetically modified crops could cause \u201cgenetic erosion\u201d \n\nas farmers restrict themselves to few popularly grown \n\nvarieties.", "start_char_idx": 24739, "end_char_idx": 29075, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9294abc0-91dc-4a45-8b20-f239cbfd8c4f": {"__data__": {"id_": "9294abc0-91dc-4a45-8b20-f239cbfd8c4f", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "69a4ffd2-03af-4b78-b3f4-a68fb2190a19", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "03570e0315d3ac1769b8b46a360765fd38427ddb3e48d97441eab8de4c989165", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d881b8de-bcf8-4b0f-a064-e8172a24f1c6", "node_type": "1", "metadata": {}, "hash": "5c5022a2c8ace2d1b390e41aa0018466dc2167cdbee270a37dd5f12b7eaef37a", "class_name": "RelatedNodeInfo"}}, "text": "GM crops are not part of the natural process, so \n\nthey could cause unpredictable changes in ecology and \n\nevolutionary response; the resurgence of pests and \n\nemergence of superweed are the results of these. \n\nResistance breakdown \nExtensive cultivation of insect-resistant and herbicide-\n\ntolerant crops increases the chances of the development \n\nof resistance in the targeted insect population through \n\nhigh selection pressure. New insect biotypes may evolve \n\nwith resistance against transgenic technology. Similarly, \n\nsuperweed having resistance against herbicides may \n\nemerge. The field evolved pest resistance to Bt maize has \n\nbeen reported in Spodoptera frugiperda (Fall armyworm) in \n\nBrazil to cry1F expressing corn and cry1Ac expressing \n\nsoybean [67]. In China, field evolved resistance to Bt \n\ncotton in Cotton bollworm (Helicoverpa armigera) to \n\ncry1Ac expressing cotton has been reported [70]. \n\nEconomic, Social and Political concerns \nThere are economic concerns about GM crops, as the \n\nprice of seeds will be so high that small farmers and \n\nfarmers in developing countries are unable to afford \n\nseeds for GM crops [71]. Concern about negative socio-\n\neconomic impacts of rapid technological change on-farm \n\nor rural structure is also present. In Muslim communities, \n\nthe use of GMOs is considered halal or haram [72]. The \n\nlabeling of genetically modified foods is one of the major \n\npolitical concerns. USA does not label GM foods, but \n\nthere must be a common consensus on labeling \n\ngenetically modified foods and their products in all \n\ncountries. Similarly, differences in biotechnology \n\nregulations differ in the US and EU, due to minor \n\ndifferences in consumers' preferences [68]. \n\nConclusion \nAgriculture has come a long way from the green \n\nrevolution to the gene revolution. It is being applied and \n\nupdated more and more daily. With the ability to know \n\nand modify the genetic makeup of organisms using \n\nbiotechnological tools, we can cope with the increasing \n\ndemand for food through the development of novel \n\nvarieties of crops with a higher yield, better resistance \n\nagainst biotic and abiotic factors, and ensure \n\nenvironmental sustainability. The use of biotechnology in \n\nagriculture has not only helped to increase the \n\nproductivity of crops but also reduced the cost of \n\nproduction by decreasing needs for inputs (pesticides) \n\nand improved the livelihood of the farmers. Similarly, \n\nnew varieties of plants with higher yields in fewer inputs \n\nhave wider environment adaptability; give better \n\nrotation to conserve natural resources has been \n\ndeveloped through biotechnology applications. Despite \n\nthese rapid developments, concerns regarding the safety \n\nissues of GM crops on human health, food/feed safety, \n\non the environment, social, economic, and political are \n\nraised continuously. Complete and transparent \n\nassessment of GM crops application and their effects \n\nshould be done, with strong regulatory implementation \n\nmechanism for use of GM crops. Alternatively, new \n\nmethods such as cisagenesis, intragenesis, and genome \n\nediting can be utilized for developing improved crops.  \n\nCompeting Interests \nThe authors declare that they have no competing \n\ninterests. \n\nAuthor\u2019s Contribution \nAll authors contributed equally. \n\nAcknowledgments \nNot applicable. \n\nReferences \n1.  Persley GJ, Siedow JN. Applications of Biotechnology to Crops: \n\nBenefits and Risks. 1999.  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  91 \n\n2.  Gavrilescu M, Chisti Y. Biotechnology - A sustainable alternative \nfor chemical industry. Vol. 23, Biotechnology Advances. Elsevier \nInc.; 2005. p. 471\u201399.  \n\n3.  Wieczorek A. Use of Biotechnology in Agriculture -- Benefits and \nRisks. 2003;  \n\n4.  Altman A, Hasegawa PM, editors. Plant Biotechnology and \nAgriculture: Prospects for the 21st Century - Google Books. \nElsevier Inc.; 2012.  \n\n5.  Ruttan VW. BIOTECHNOLOGY AND AGRICULTURE: A \nSKEPTICAL PERSPECTIVE. Vol.", "start_char_idx": 29076, "end_char_idx": 33100, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d881b8de-bcf8-4b0f-a064-e8172a24f1c6": {"__data__": {"id_": "d881b8de-bcf8-4b0f-a064-e8172a24f1c6", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9294abc0-91dc-4a45-8b20-f239cbfd8c4f", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "d06ea5e0c294cc2e4865ddf50d511a16055abf9c1da2f475c0cb26dd1a69d41d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aefb8845-80b6-4e1f-a3d5-b9e97abc1bc9", "node_type": "1", "metadata": {}, "hash": "114e21283869fc36a9dbf9cb46ef9365570bc097321c05a684567ed94da78be1", "class_name": "RelatedNodeInfo"}}, "text": "Vol. 2, \nhttp://www.agbioforum.missouri.edu/v2n1/v2n1a10-\nruttan.htm. AgBioForum; 1999.  \n\n6.  Job D. Plant biotechnology in agriculture. In: Biochimie. 2002. p. \n1105\u201310.  \n\n7.  Buckwell A, Moxey A. Biotechnology and agriculture. Food \nPolicy. 1990 Feb;15(1):44\u201356.  \n\n8.  Jaynes JM, Yang MS, Espinoza N, Dodds JH. Plant protein \nimprovement by genetic engineering: use of synthetic genes. \nTrends Biotechnol. 1986;4(12):314\u201320.  \n\n9.  Postlewait A, Parker DD, Zilberman D. The advent of \nbiotechnology and technology transfer in agriculture. Technol \nForecast Soc Change. 1993 May;43(3\u20134):271\u201387.  \n\n10.  Herdt RW. Biotechnology in Agriculture. Annu Rev Environ \nResour [Internet]. 2006 Oct 13 [cited 2021 Jul 6];31:265\u201395. \nAvailable from: \nhttps://www.annualreviews.org/doi/abs/10.1146/annurev.ene\nrgy.31.031405.091314 \n\n11.  Cano EA, Morgado C. The role of biotechnology in agricultural \nproduction and food supply. Cienc e Investig Agrar. 2017;44(1):1\u2013\n11.  \n\n12.  UNFPA. World Population Dashboard | UNFPA - United Nations \nPopulation Fund [Internet]. 2021 [cited 2021 Jul 6]. Available from: \nhttps://www.unfpa.org/data/world-population-dashboard \n\n13.  Y\u0131lmaz R. Modern biotechnology breakthroughs to food and \nagricultural research in developing countries. GM Crop Food \n[Internet]. 2019 Jan 2 [cited 2021 Jul 6];10(1):12\u20136. Available from: \nhttps://www.tandfonline.com/doi/abs/10.1080/21645698.2019.\n1600969 \n\n14.  Datta SK. Impact of plant biotechnology in agriculture. Biotechnol \nAgric For. 2007;59(January 2007):3\u201331.  \n\n15.  Catacora-Vargas G. Genetically Modified Organisms A Summary \nof Potential Adverse Effects Relevant to Sustainable Development. \n2011.  \n\n16.  Mannion AM, Morse S. Biotechnology in agriculture: Agronomic \nand environmental considerations and reflections based on 15 \nyears of GM crops. Prog Phys Geogr. 2012;36(6):747\u201363.  \n\n17.  Batt CA. Genetic Engineering [Internet]. Second Edi. Vol. 2, \nEncyclopedia of Food Microbiology: Second Edition. Elsevier; \n2014. 83\u201387 p. Available from: http://dx.doi.org/10.1016/B978-0-\n12-384730-0.00143-9 \n\n18.  Robert JS, Baylis F. Genetic engineering. In: International \nEncyclopedia of Public Health. Elsevier Inc.; 2008. p. 35\u20139.  \n\n19.  ISAAA database.  GM Approval Database [Internet]. 2021 [cited \n2021 Jun 12]. Available from: \nhttps://www.isaaa.org/gmapprovaldatabase/cropslist/default.\nasp \n\n20.  Dill GM, Cajacob CA, Padgette SR. Glyphosate-resistant crops: \nadoption, use and future considerations. Pest Manag Sci Pest \nManag Sci. 2008;64:326\u201331.  \n\n21.  Kumar K, Gambhir G, Dass A, Tripathi AK, Singh A, Jha AK, et al. \nGenetically modified crops: current status and future prospects. \nPlanta [Internet]. 2020;251(4):1\u201327. Available from: \nhttps://doi.org/10.1007/s00425-020-03372-8 \n\n22.  McPherson SA, Perlak FJ, Fuchs RL, Marrone PG, Lavrik PB, \nFischhoff DA.", "start_char_idx": 33096, "end_char_idx": 35945, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aefb8845-80b6-4e1f-a3d5-b9e97abc1bc9": {"__data__": {"id_": "aefb8845-80b6-4e1f-a3d5-b9e97abc1bc9", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d881b8de-bcf8-4b0f-a064-e8172a24f1c6", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "b122ee006ec4fd85d8d959eb3a50db1adb6a52e69d45d9727172aae0344231f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "250ed75d-d51a-4697-85cb-464654b7775b", "node_type": "1", "metadata": {}, "hash": "003ff9aba12abc6a1460d012fb8f7d771a7e3ce3c42eafca1c33a08f02d37dd3", "class_name": "RelatedNodeInfo"}}, "text": "Characterization of the coleopteran-specific protein \ngene of bacillus thuringiensis var. Tenebrionis. Bio/Technology \n[Internet]. 1988 [cited 2021 Jun 12];6(1):61\u20136. Available from: \nhttps://www.nature.com/articles/nbt0188-61 \n\n23.  Fang J, Xu X, Wang P, Zhao JZ, Shelton AM, Cheng J, et al. \nCharacterization of chimeric Bacillus thuringiensis Vip3 toxins. \nAppl Environ Microbiol [Internet]. 2007 Feb [cited 2021 Jun \n13];73(3):956\u201361. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/17122403/ \n\n24.  Castiglioni P, Warner D, Bensen RJ, Anstrom DC, Harrison J, \n\nStoecker M, et al. Bacterial RNA chaperones confer abiotic stress \ntolerance in plants and improved grain yield in maize under \nwater-limited conditions [Internet]. Vol. 147, Plant Physiology. \nPlant Physiol; 2008 [cited 2021 Jun 13]. p. 446\u201355. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/18524876/ \n\n25.  Waltz E. Beating the heat. Nat Biotechnol. 2014;32(7):610\u20133.  \n26.  James C. 20th Anniversary (1996 to 2015) of the Global \n\nCommercialization of Biotech Crops and Biotech Crop Highlights \nin 2015. ISAAA Br No 51 [Internet]. 2015; Available from: \nhttps://www.isaaa.org/resources/publications/briefs/51/ \n\n27.  Kavanagh TA, Spillane C. Strategies for engineering virus \nresistance in transgenic plants. Euphytica [Internet]. 1995 Feb \n[cited 2021 Jun 20];85(1\u20133):149\u201358. Available from: \nhttps://link.springer.com/article/10.1007/BF00023943 \n\n28.  Fitch MMM, Manshardt RM, Gonsalves D, Slightom JL, Sanford \nJC. Virus resistant papaya plants derived from tissues bombarded \nwith the coat protein gene of papaya ringspot virus. \nBio/Technology [Internet]. 1992 [cited 2021 Jun 20];10(11):1466\u201372. \nAvailable from: https://www.nature.com/articles/nbt1192-1466 \n\n29.  Bonfim K, Faria JC, Nogueira EOPL, Mendes \u00c9A, Arag\u00e3o FJL. \nRNAi-mediated resistance to Bean golden mosaic virus in \ngenetically engineered common bean (Phaseolus vulgaris). Mol \nPlant-Microbe Interact [Internet]. 2007 May 30 [cited 2021 Jun \n20];20(6):717\u201326. Available from: \nhttps://apsjournals.apsnet.org/doi/abs/10.1094/MPMI-20-6-\n0717 \n\n30.  Roman ML, Izarra M, Lindqvist-Kreuze H, Rivera C, Gamboa S, \nTovar JC, et al. R/Avr gene expression study of Rpi-vnt1.1 \ntransgenic potato resistant to the Phytophthora infestans clonal \nlineage EC-1. Plant Cell Tissue Organ Cult [Internet]. 2017 Nov 1 \n[cited 2021 Jun 20];131(2):259\u201368. Available from: \nhttps://link.springer.com/article/10.1007/s11240-017-1281-9 \n\n31.  Lee HI, Raikhel N V. Prohevein is poorly processed but shows \nenhanced resistance to a chitin-binding fungus in transgenic \ntomato plants - PubMed. 1995.  \n\n32.  Chakraborty S. Increased nutritive value of transgenic potato by \nexpressing a nonallergenic seed albumin gene from Amaranthus \nhypochondriacus. Proc Natl Acad Sci. 2000 Mar;97(7):3724\u20139.  \n\n33.", "start_char_idx": 35946, "end_char_idx": 38766, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "250ed75d-d51a-4697-85cb-464654b7775b": {"__data__": {"id_": "250ed75d-d51a-4697-85cb-464654b7775b", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aefb8845-80b6-4e1f-a3d5-b9e97abc1bc9", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "f6ceebe214628ccd4aed2b84f9d13757ead0eab8a685040471e238728a259915", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b08c2f32-ca35-4ab5-be4a-c073de3fb010", "node_type": "1", "metadata": {}, "hash": "139dbf3d7ea0e70485425b7727a52559da6079c95c0bdc7b9d4a375cd6d59fa6", "class_name": "RelatedNodeInfo"}}, "text": "2000 Mar;97(7):3724\u20139.  \n\n33.  R\u00f6mer S, Fraser PD, Kiano JW, Shipton CA, Misawa N, Schuch W, \net al. Elevation of the provitamin A content of transgenic tomato \nplants. Nat Biotechnol. 2000 Jun;18(6):666\u20139.  \n\n34.  Tian B, Wei F, Shu H, Zhang Q, Zang X, Lian Y. Decreasing erucic \nacid level by RNAi-mediated silencing of fatty acid elongase 1 \n(BnFAE1.1) in rapeseeds (Brassica napus L.). African J Biotechnol. \n2011;10(61):13194\u2013201.  \n\n35.  Thorpe TA. History of plant tissue culture. Mol Biotechnol. \n2007;37(2):169\u201380.  \n\n36.  Hussain A, Ahmed I, Nazir H, Ullah I. Plant Tissue Culture: \nCurrent Status and Opportunities. Recent Adv Plant Vitr Cult. \n2012;1\u201328.  \n\n37.  Kumar V, Poulose L. Agricultural Biotechnology: current status \nand future prospects. Biodiversity, Conserv Sustain Dev. \n2016;2(December).  \n\n38.  Brown DCW, Thorpe TA. Crop improvement through tissue \nculture [Internet]. Vol. 11, World Journal of Microbiology & \nBiotechnology. Kluwer Academic Publishers; 1995 [cited 2021 Jun \n20]. p. 409\u201315. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/24414749/ \n\n39.  Paudel RK. G9 banana more disease-resistant but less marketable, \nfarmers say. The Kathmandu Post [Internet]. 2020 Jul 24; Available \nfrom: https://kathmandupost.com/money/2020/06/29/g9-\nbanana-more-disease-resistant-but-less-marketable-farmers-say \n\n40.  El-Dougdoug KA, M. M. E-S. Management of viral disease in \nbanana using certified and virus tested plant material. African J \nMicrobiol Res [Internet]. 2011 [cited 2021 Jun 20];5(32):5923\u201332. \nAvailable from: http://www.academicjournals.org/AJMR \n\n41.  Sahijram L, Rao BM. Hybrid embryo rescue in crop improvement. \nIn: Plant Biology and Biotechnology: Volume II: Plant Genomics \nand Biotechnology. New Delhi: Springer India; 2015. p. 363\u201384.  \n\n42.  Debbarama C, Khanna V, Tyagi W, Rai M, Meeti N. Wide \nHybridization and Embryo-Rescue for Crop Improvement in \nCapsicum. Agrotechnology. 2013;11(003).  \n\n43.  Shuro AR. Review Paper on the Role of Somatic Hybridization in \nCrop Improvement. www.arcjournals.org Int J Res Stud Agric Sci \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 8 5 - 9 2               KC & Lamichhane   \n\n\u00a9NJB, BSN  92 \n\n[Internet]. 2018 [cited 2021 Jun 24];4(9):2454\u20136224. Available from: \nhttp://dx.doi.org/10.20431/2454-6224.0409001 \n\n44.  Glimelius K, Fahlesson J, Landgren M, Sj\u00f6din C, Sundberg E. Gene \ntransfer via somatic hybridization in plants. Trends Biotechnol. \n1991;9(1):24\u201330.  \n\n45.  Garcia LE, Edera AA, Marfil CF, Sanchez-Puerta MV. Male sterility \nand somatic hybridization in plant breeding. Rev la Fac Ciencias \nAgrar. 2019;51(2):475\u201386.  \n\n46.  Begna T. Review on Somatic Hybridization and Its Role in Crop \nImprovement. J Biol Agric Healthc [Internet]. 2020 [cited 2021 Jun \n24];10(11).", "start_char_idx": 38737, "end_char_idx": 41513, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b08c2f32-ca35-4ab5-be4a-c073de3fb010": {"__data__": {"id_": "b08c2f32-ca35-4ab5-be4a-c073de3fb010", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "250ed75d-d51a-4697-85cb-464654b7775b", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "e27040c79686c1eb593ac92dbe63fbe0fc530a3099e580e9ebf65b64e15d87f9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2c81da77-8250-4a9f-8d73-4cbd7bdc17a3", "node_type": "1", "metadata": {}, "hash": "5dc22022e93e17c1734f2d874dda543f09533324818c8ab20cda0d15eefcbf94", "class_name": "RelatedNodeInfo"}}, "text": "2020 [cited 2021 Jun \n24];10(11). Available from: www.iiste.org \n\n47.  Kiran Kumar M, Sandeep BV, Sudhakar Rao P. Development of \nsalt tolerant callus cultures by somatic hybridization between \nOryza sativa and mangrove grass Myriostachya wightiana. Ann \nAgrar Sci. 2018 Dec 1;16(4):396\u2013404.  \n\n48.  Yan CQ, Qian KX, Yan QS, Zhang XQ, Xue GP, Huangfu WG, et \nal. Use of asymmetric somatic hybridization for transfer of the \nbacterial blight resistance trait from Oryza meyeriana L. to O. \nsativa L. ssp. japonica. Plant Cell Rep [Internet]. 2004 Mar [cited \n2021 Jun 24];22(8):569\u201375. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/14595515/ \n\n49.  Eckardt NA. Cytoplasmic male sterility and fertility restoration \n[Internet]. Vol. 18, Plant Cell. Oxford University Press; 2006 [cited \n2021 Jun 24]. p. 515\u20137. Available from: \n/pmc/articles/PMC1383628/ \n\n50.  Jiang G-L. Molecular Markers and Marker-Assisted Breeding in \nPlants. In: Plant Breeding from Laboratories to Fields. InTech; \n2013.  \n\n51.  Joseph M, Gopalakrishnan S, Sharma RK, Singh VP, Singh AK, K. \nSingh N, et al. Combining bacterial blight resistance and Basmati \nquality characteristics by phenotypic and molecular marker-\nassisted selection in rice. Mol Breed [Internet]. 2004 May [cited \n2021 Jun 24];13(4):377\u201387. Available from: \nhttps://link.springer.com/article/10.1023/B:MOLB.0000034093.\n63593.4c \n\n52.  Alkimim ER, Caixeta ET, Sousa TV, Pereira AA, de Oliveira ACB, \nZambolim L, et al. Marker-assisted selection provides arabica \ncoffee with genes from other Coffea species targeting on multiple \nresistance to rust and coffee berry disease. Mol Breed [Internet]. \n2017 Jan 1 [cited 2021 Jun 24];37(1):1\u201310. Available from: \nhttps://link.springer.com/article/10.1007/s11032-016-0609-1 \n\n53.  Yi X, Cheng J, Jiang Z, Hu W, Bie T, Gao D, et al. Genetic analysis \nof fusarium head blight resistance in CIMMYT bread wheat line \nC615 using traditional and conditional QTL mapping. Front Plant \nSci [Internet]. 2018 May 1 [cited 2021 Jun 24];9:573. Available from: \nwww.frontiersin.org \n\n54.  Ren J, Wu P, Trampe B, Tian X, L\u00fcbberstedt T, Chen S. Novel \ntechnologies in doubled haploid line development. Plant \nBiotechnol J. 2017;15(11):1361\u201370.  \n\n55.  Heffer P. Biotechnology: a modern tool for food production \nimprovement  [Internet]. [cited 2021 Jun 24]. Available from: \nhttp://www.fao.org/3/y2722e/y2722e1f.htm \n\n56.  Barkley A, Chumley FG. A doubled haploid laboratory for kansas \nwheat breeding: An economic analysis of biotechnology adoption. \nInt Food Agribus Manag Rev. 2012;15(2):99\u2013120.  \n\n57.  Wessels E, Botes WC. Accelerating resistance breeding in wheat by \nintegrating marker-assisted selection and doubled haploid \ntechnology. South African J Plant Soil [Internet]. 2014 Jan 2 [cited \n2021 Jun 24];31(1):35\u201343. Available from: \nhttps://www.tandfonline.com/action/journalInformation?journ\nalCode=tjps20 \n\n58.", "start_char_idx": 41480, "end_char_idx": 44379, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2c81da77-8250-4a9f-8d73-4cbd7bdc17a3": {"__data__": {"id_": "2c81da77-8250-4a9f-8d73-4cbd7bdc17a3", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b08c2f32-ca35-4ab5-be4a-c073de3fb010", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "84796f448f0a801571315af97f29a9c999ba8cb8c0d5272fbf0477aa0a898ab2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bab5ff43-b894-4623-a216-c258d2b23592", "node_type": "1", "metadata": {}, "hash": "36eba494da5aede5df2beb82c1784c797b4d80c32ae37321b63f0fc8c4c7db8c", "class_name": "RelatedNodeInfo"}}, "text": "Siddique R. Impact of different media and genotypes in improving \nanther culture response in rice (Oryza sativa) in Bangladesh. Eur \nSci J. 2015;11(6):164\u20139.  \n\n59.  Bakhshi T, Bozorgipour R, Shahriari-Ahmadi F. Evaluating the \nProduction of Doubled Haploid Wheat Lines Using Various \nMethods of Wheat and Maize Crossing to Develop Heat-Tolerant \nWheat Varieties. Cumhur Univ Fac Sci Sci J [Internet]. 2017 Feb 17 \n[cited 2021 Jun 24];38(1):64\u201378. Available from: \nhttp://dx.doi.org/10.17776/csj.54969http://dergi.cumhuriyet.ed\nu.tr/cumuscij/index \n\n60.  Varshney RK, Dubey A. Novel genomic tools and modern genetic \nand breeding approaches for crop improvement. J Plant Biochem \nBiotechnol. 2009;18(2):127\u201338.  \n\n61.  Kaur R, Shilpa, Kumar K, Sharma N. Genomics in agriculture. \nCAB Rev Perspect Agric Vet Sci Nutr Nat Resour. \n2015;10(September).  \n\n62.  Jain D, Ashraf N, Khurana JP, Shiva Kameshwari MN. The \u2018Omics\u2019 \nApproach for Crop Improvement Against Drought Stress. In \nSpringer, Cham; 2019 [cited 2021 Jun 24]. p. 183\u2013204. Available \nfrom: https://link.springer.com/chapter/10.1007/978-3-319-\n91956-0_8 \n\n63.  Shukla VK, Doyon Y, Miller JC, Dekelver RC, Moehle EA, Worden \nSE, et al. Precise genome modification in the crop species Zea mays \nusing zinc-finger nucleases. Nature [Internet]. 2009 May 21 [cited \n2021 Jun 24];459(7245):437\u201341. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/19404259/ \n\n64.  ISAAA. Global Status of Commercialized Biotech/GM Crops: \n2019: Africa Leads Progress in Biotech Crop Adoption with \nDoubled Number of Planting Countries in 2019. In: ISAAA Brief \nNo 53 [Internet]. Ithaca, NY; 2019. Available from: \nhttps://www.isaaa.org/resources/publications/briefs/55/defa\nult.asp \n\n65.  Brower LP, Taylor OR, Williams EH, Slayback DA, Zubieta RR, \nRam\u00edrez MI. Decline of monarch butterflies overwintering in \nMexico: Is the migratory phenomenon at risk? Insect Conserv \nDivers [Internet]. 2012 Mar 1 [cited 2021 Jun 24];5(2):95\u2013100. \nAvailable from: \nhttps://onlinelibrary.wiley.com/doi/full/10.1111/j.1752-\n4598.2011.00142.x \n\n66.  Losey JE, Rayor LS, Carter ME. Transgenic pollen harms monarch \nlarvae [Internet]. Vol. 399, Nature. Macmillan Magazines Ltd; 1999 \n[cited 2021 Jun 24]. p. 214. Available from: www.nature.com \n\n67.  Lu Y, Wu K, Jiang Y, Xia B, Li P, Feng H, et al. Mirid bug outbreaks \nin multiple crops correlated with wide-scale adoption of Bt cotton \nin China. Science (80- ) [Internet]. 2010 May 28 [cited 2021 Jun \n24];328(5982):1151\u20134. Available from: \nhttps://pubmed.ncbi.nlm.nih.gov/20466880/ \n\n68.  Terefe M. Biosafety Issues of Genetically Modified Crops: \nAddressing the Potential Risks and the Status of GMO Crops in \nEthiopia. Cloning Transgenes. 2018;7(2).  \n\n69.  Butler D, Relchhardt T, Abbott A, Dickson D, Saegusa A. Long-\nterm effect of GM crops serves up food for thought. Nature \n[Internet].", "start_char_idx": 44381, "end_char_idx": 47247, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bab5ff43-b894-4623-a216-c258d2b23592": {"__data__": {"id_": "bab5ff43-b894-4623-a216-c258d2b23592", "embedding": null, "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-117", "node_type": "4", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "15e286beb1024a3ef2648a89c87ba221e3d0378ce0908000e55f10145e9391a7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2c81da77-8250-4a9f-8d73-4cbd7bdc17a3", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "210ae11ba2fd553defb4fc57577f832563a17e5cbe4dc3d61dc60c9acbff3116", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b2c14327-1418-4a2c-9fe4-e1a87b89f65d", "node_type": "1", "metadata": {}, "hash": "2f3408a974d1f2234c7a97bc27bbc51721153da74c0fe8d1e8be6efbb50acd9b", "class_name": "RelatedNodeInfo"}}, "text": "Nature \n[Internet]. 1999 Apr 22 [cited 2021 Jun 24];398(6729):651\u20136. \nAvailable from: https://pubmed.ncbi.nlm.nih.gov/10227281/ \n\n70.  Zhang H, Tian W, Zhao J, Jin L, Yang J, Liu C, et al. Diverse genetic \nbasis of field-evolved resistance to Bt cotton in cotton bollworm \nfrom China. Proc Natl Acad Sci U S A [Internet]. 2012 Jun 26 [cited \n2021 Jun 24];109(26):10275\u201380. Available from: \nwww.pnas.org/cgi/doi/10.1073/pnas.1200156109 \n\n71.  Raney T. Economic impact of transgenic crops in developing \ncountries. Curr Opin Biotechnol. 2006;17(2):174\u20138.  \n\n72.  Safian M, Hanani Y. Islam and Biotechnology: With Special \nReference to Genetically Modified Foods. Sci Relig Glob Perspect \nPhiladelphia, PA, USA. 2005;4\u20138.", "start_char_idx": 47228, "end_char_idx": 47946, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b2c14327-1418-4a2c-9fe4-e1a87b89f65d": {"__data__": {"id_": "b2c14327-1418-4a2c-9fe4-e1a87b89f65d", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bab5ff43-b894-4623-a216-c258d2b23592", "node_type": "1", "metadata": {"identifier": "njb-117", "author": "K.C., Mamata; Lamichhane, Anuj", "title": "Advances in Agricultural Biotechnology", "date": "2021-07-31", "file": "njb-117.pdf"}, "hash": "492ee3a08b1fc54b5ce01de090f359388d2772bed6316beb8d437bfaafc685a7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5da236f2-a5b2-4392-b107-5a9b120f18b1", "node_type": "1", "metadata": {}, "hash": "ae98477216e4112f663e0999d6f05c31d5cad0d254fcd9bad9b570f4320434c0", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  D e c ;  8 (3): 102-110 DOI: https://doi.org/10.3126/njb.v8i3.33664 Research article  \n\n\u00a9NJB, BSN 102 \n\nScreening of Actinomycetes From Soil for Antibacterial Activity \nShailesh Budhathoki1 and Anima Shrestha2\n\n1Department of Microbiology, St. Xavier\u2019s College, Maitighar, Kathmandu, Nepal. \n2Department of Microbiology, Tri-Chandra Multiple Campus, Ghantaghar, Kathmandu, Nepal. \n\nReceived: 30 Oct 2020; Revised: 21 Dec 2020; Accepted: 22 Dec 2020; Published online: 30 Dec 2020 \n\nAbstract \nActinomycetes are Gram positive, free living saprophytes which are distributed in soil as one of the major populations and \nare primary source of antibiotics. This study was carried out with a quest to isolate actinomycetes from soil samples of \ndifferent places and assess their antibacterial activity. Isolation of actinomycetes was carried out by serial dilution of soil \nsample followed by spread plate method. The antimicrobial extract was extracted using ethyl acetate. Assessment of \nantimicrobial activity was performed by using Agar cup plate assay against test organisms (Pseudomonas aeruginosa, \nEscherichia coli, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Bacillus subtilis, Staphylococcus aureus).\nAntibacterial activity was tested against Methicillin Sensitive Staphylococcus aureus and Methicillin Resistant Staphylococcus \naureus in the isolates having effective inhibitory activity against Staphylococcus aureus. From 15 soil samples of 12 different \nlocations, 121 actinomycetes isolates were isolated. Among them, 58 (47.9%) isolates were inhibitory against at least 1 test \norganism in primary screening, of which 22 isolates effective against more than 1 test organism was chosen for secondary \nscreening. Out of them, 8 were inhibitory against 2 test organisms while 14 were inhibitory against 3 test organisms. \nStaphylococcus aureus was found to be the most susceptible test organism with its susceptibility against 12 actinomycetes \nisolates. Among 12 isolates effective against Staphylococcus aureus, 10 were found to have an inhibitory effect against \nMethicillin Susceptible Staphylococcus aureus while 6 were found to have inhibitory effect against Methicillin Resistant \nStaphylococcus aureus strain. The findings of this study highlight the inhibitory potential of actinomycetes and the need \nforfurther investigation for obtaining novel antimicrobial agents from actinomycetes from various unexplored areas. \n\nKeywords: Actinomycetes, Inhibitory activity, Isolates, Antimicrobial, Antibiotic \n\n Corresponding author, email: animashrestha77@gmail.com \n\nIntroduction \nYear by year microbial diseases are increasing, which \n\nhave become the biggest threat to public health.  There \n\nare about 200 known diseases transmitted by \n\nmicroorganisms such as bacteria, fungi, viruses, etc. to \n\nhumans [1]. Antibiotic refers to the chemical compound \n\nderived from microorganisms or living cells that inhibit \n\nand/or stop the growth of a microorganism. They are \n\nused in the treatment of external or internal infections. \n\nWhile some antibiotics are produced by a \n\nmicroorganism, most are now manufactured \n\nsynthetically [2]. Bacteria, fungi, actinomycetes, algae, \n\nlichens and green plants produce antibiotics. \n\nActinomycetes are one of the most important \n\nmicroorganisms that hold the prime position in the \n\nproduction of bioactive metabolites. They are responsible \n\nfor the production of almost half of the discovered \n\nbioactive secondary metabolites notably antitumor \n\nagents, immunosuppressive agents, enzymes and \n\nespecially antibiotics [3]. In industrial microbiology, \n\nactinomycetes make its position at the top as a potential \n\nsource of antibiotics [4].", "start_char_idx": 48, "end_char_idx": 3787, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5da236f2-a5b2-4392-b107-5a9b120f18b1": {"__data__": {"id_": "5da236f2-a5b2-4392-b107-5a9b120f18b1", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b2c14327-1418-4a2c-9fe4-e1a87b89f65d", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "51ba012f5ee3639bfa8f933b6615cd0901f3dcf244225b659996f7649540f9b9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a477c4f5-33bf-43aa-b249-197b5884cd59", "node_type": "1", "metadata": {}, "hash": "918f948b5221e87523806ea2b51e019e79d04255424d2cc65da2d7c1884582ff", "class_name": "RelatedNodeInfo"}}, "text": "Actinomycetes are prokaryotic organisms with \n\nfilamentous nature, branching pattern, and conidia \n\nformation, which are like those of fungi. For this reason, \n\nthey are also known as Ray fungi. They are gram-\n\npositive, free-living, saprophytic bacteria [5, 6]. \n\nActinomycetes populations are identified as one of the \n\nmajor groups of soil population, which may vary with \n\nsoil type [7]. The number and types of actinomycetes \n\npresent in soil would be greatly influenced by \n\ngeographical location such as soil temperature, soil type, \n\nsoil pH, organic matter content, cultivation, aeration and \n\nmoisture content [8]. These are inhabitants of soil that \n\nalso play important role in recycling of organic matter, \n\nproduction of novel pharmaceuticals, nutritional \n\nmaterials, cosmetics, enzyme inhibitors, immune-\n\nmodifiers and vitamins [9, 10]. \n\nNumbers of antibiotics have now been isolated from \n\ncultures of actinomycetes, such as actinomycetin, \n\nmicromonosporin, mycetin, and actinomyces lysozyme \n\netc.  have been only partially purified, whereas others, \n\nincluding actinomycin, proactinomycin, streptothricin, \n\nand streptomycin, have been isolated and crystallized \n\nThese substances differ greatly chemical structure and \n\ntheir antimicrobial properties, toxicity to animals, and in \n\nvivo activity [11].  Compounds isolated from \n\nactinomycetes have numerous chemical structures such \n\nas macrolides, tetracyclines, aminoglycosides, \n\nglycopeptides and ansamicines which are used in \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nmailto:animashrestha77@gmail.com\nhttps://orcid.org/0000-0002-9919-476X\nmailto:animashrestha77@gmail.com\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN 103 \n\nantibacterial treatments whereas anthracyclines \n\nsupplement anticancer chemotherapy. Toxic polyether-\n\ntype antibiotics are used as anti-coccicidal agents [12]. \n\nMore than 70\u201380% of all known antibiotics have been \n\nisolated from actinomycetes, which are used in medicine \n\nand agriculture. The two major groups of soil \n\nactinomycetes that serve as important sources of \n\nantibiotics are Streptomyces and Micromonospora. \n\nStreptomyces spp. are the biggest producer of antibiotics \n\nthat account for about 80% of the total antibiotic \n\nproducts.  Micromonospora closely follows with less than \n\none tenth as much as Streptomyces [1]. Members of \n\nStreptomyces are a rich source of bioactive compounds, \n\nnotably antibiotics, enzymes, enzyme inhibitors and \n\npharmacologically active agents [13]. Streptomyces are \n\nvaluable resources for a novel products like antifungals, \n\nantivirals, antitumorals, anti-hypertensives, \n\nimmunosuppressants and especially antibiotics [14].  \n\nThe nature of the active agents or the antibiotics \n\nproduced by actinomycetes depends upon the species; \n\nfrequently upon the strain; the composition of the \n\nmedium in which it is grown, and the conditions of \n\ncultivation. The antimicrobial properties of a given \n\nactinomycetes culture also depend upon the composition \n\nof the medium in which it is grown [11]. Also, it is \n\nessential to maintain the optimum temperature, pH and \n\nsalinity to produce bioactive secondary metabolites. The \n\nabsence of optimum conditions can lead to failure in \n\nproduction or no growth could also be observed [15]. \n\nHowever, with the time of the discovery of antibiotics, \n\nthe emergence antibiotic resistance bacteria have been a \n\nmajor problem. Also, there is a rapid emergence of drug \n\nresistant strains of the pathogen than the rate of discovery \n\nof new drugs and antibiotics [16].", "start_char_idx": 3791, "end_char_idx": 7523, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a477c4f5-33bf-43aa-b249-197b5884cd59": {"__data__": {"id_": "a477c4f5-33bf-43aa-b249-197b5884cd59", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5da236f2-a5b2-4392-b107-5a9b120f18b1", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "4d763015af1d4f1f7d6be3dcbbc7c5ff89fa5ab54723b5a1683e75d365cc2412", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6769f4a9-ee13-4e2d-ad9d-22b02d48e61c", "node_type": "1", "metadata": {}, "hash": "7a14963021f40632b94c9ec656fc32c2526865e99b301f725e284c449d05f9db", "class_name": "RelatedNodeInfo"}}, "text": "The development of \n\nresistance by the pathogens as well as the emergence of \n\nnew pathogens has led to the necessity for the discovery \n\nof new antibiotics/antimicrobials for their infection. \n\nHence screening of antimicrobial activity of \n\nactinomycetes and study of their antimicrobial action \n\nagainst pathogens is an important process for the \n\ndiscovery of an antibiotic. \n\nIn Nepal, various infections are a major public health \n\nproblems. Antibiotics may be prescribed by the physician \n\nand other healthcare workers inappropriately or they \n\nmay be purchased directly by consumers without \n\nprescription to the healthcare system. Many patients self-\n\ntreat with antibiotics, including prior to hospital \n\nadmission, which can contribute to increased resistance \n\nrate [17]. Drug resistance in microorganisms has been \n\nincreasing and this has posed a serious threat for the \n\nmankind. Discovery of the novel antimicrobials that can \n\ncontrol the growth of these \n\nmicroorganisms is of global importance.  As, \n\nactinomycetes are the most important source of \n\nantibiotics this study is intended to find out the \n\nantimicrobial activity of actinomycetes and their \n\npotential to inhibit the microbial growth. This study will \n\nserve as baseline data for future studies of the \n\nantimicrobial potential of actinomycetes isolates and the \n\ndiscovery of novel metabolites from actinomycetes in \n\nNepal.  \n\nMaterials and Methods \nCollection of Sample \n15 soil samples from 12 different locations (Nuwakot, \n\nLagankhel, Katunje, Chovar, Lubhu, Budanilkantha, \n\nChapagaun, Godawari, Sundarijal, Sankhamul, Gokarna \n\nand Chandragiri) were collected for study (Figure 1). \n\nLaboratory processing of soil samples was carried out at \n\nResearch Laboratory of Kathmandu Centre for Genomics \n\n& Research Laboratory (KCGRL) and Microbiology \n\nLaboratory of St. Xavier\u2019s College. Duplicate soil samples \n\nfrom a depth of 5-10 cm from the surface were collected \n\nin two separate sealed plastic containers [4, 14].  \n\nFigure 1. Map of sample collection sites \n\nSoil treatment (before isolation) \nSoil samples so collected were left to air dry at room \n\ntemperature for (10-15) days, to reduce the number of \n\ncontaminant bacteria (especially Gram-negative bacteria) \n\nin soil samples as previously reported [15, 18]. \n\nIsolation of Actinomycetes \nThe soil sample was serially diluted to 10-2, using distilled \n\nwater as diluents. The dilutions were vigorously shaken \n\nin a vortex shaker to liberate actinomycetes spores from \n\nthe soil into the supernatant liquid. Aliquots of \n\nsupernatant liquid from 10-1 and 10-2 dilutions of soil \n\nsample was plated on selective, solidified, Starch Casein \n\nAgar (SCA) incorporated with Amoxicillin 20 mg/l as \n\nreported previously [9] and Ketoconazole 30 mg/l [4] so \n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN 104 \n\nas to eliminate contaminant gram positive soil dwelling \n\nbacteria and fungi respectively, using spread plate \n\nmethod. The inoculated SCA plates were left at room \n\ntemperature for 5 minutes to allow the agar to absorb the \n\nliquid and were incubated at 28\u00b0C for 7 days [19]. \n\nSub-culture of actinomycetes isolates \nAfter 7 days of incubation at 28\u00b0C, colonies exhibiting a \n\ndry chalky appearance were selected. Starch Casein Agar \n\n(SCA) plate was divided into a number of sections and \n\neach of characteristic actinomycetes colonies was sub-\n\ncultured in sections of SCA plate. The plates were \n\nincubated at 28\u00b0C for 7 days. After colonies had \n\ndeveloped, they were further sub-cultured in a test tube \n\ncontaining SCA slants, incubated at 28\u00b0C for 7 days and \n\nthen stored in the refrigerator as the stock [19]. The \n\nisolates were sub-cultured again at an interval of 2-3 \n\nweeks to prevent the cultures from dying out.", "start_char_idx": 7524, "end_char_idx": 11343, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6769f4a9-ee13-4e2d-ad9d-22b02d48e61c": {"__data__": {"id_": "6769f4a9-ee13-4e2d-ad9d-22b02d48e61c", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a477c4f5-33bf-43aa-b249-197b5884cd59", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "c0b04f0365c57b3313e55a36ae5c265a44729107412ff5a5691cd65c82738ebf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "53807cf7-1468-4dc3-bb25-4d8ab28a459a", "node_type": "1", "metadata": {}, "hash": "99cf0af6f391fb0be6a6d9ea51406c556b1241ea6837f5ba55d5ea407c28a9fc", "class_name": "RelatedNodeInfo"}}, "text": "Primary Screening of isolates \nThe duplicate perpendicular streak method was \n\nemployed for the primary screening of inhibitory action \n\nof isolates against bacterial species. Actinomycetes \n\nisolates were streaked vertically down the center of the \n\nNutrient agar plate and then incubated at 28\u00b0C until \n\nvisible growth of actinomycetes appear as a confluent \n\nvertical line of the colony (usually 7 days). After \n\nincubation, each of the test organisms (Pseudomonas \n\naeruginosa, Escherichia coli, Klebsiella pneumoniae, \n\nSalmonella typhi, Salmonella paratyphi, Bacillus subtilis, \n\nStaphylococcus aureus), isolated from hospital admitted \n\npatients and confirmed with microscopic, cultural and \n\nbiochemical characteristics [20], were streaked 1 cm apart \n\nand 2mm on the side of actinomycetes colony on nutrient \n\nagar, perpendicular to the colony and then incubated at \n\n37\u00b0C for 24 hours. After incubation, plates were observed \n\nfor any inhibition in the growth of test organisms [1, 6]. \n\nInhibition in growth was noted as a lack of growth of test \n\norganisms on the streak line.  \n\nSelection of isolates for Fermentation: \nIsolates inhibiting two or more test organisms \n\n(Pseudomonas aeruginosa, Escherichia coli, Klebsiella \n\npneumoniae, Salmonella typhi, Salmonella paratyphi, Bacillus \n\nsubtilis, Staphylococcus aureus) in primary screening were \n\nselected for further processing i.e. fermentation for \n\nproduction of antibiotic fractions.  \n\nFermentation \nAmong isolated actinomycetes, 22 isolates exhibiting the \n\ngreatest action against test organisms in primary \n\nscreening were selected based on comparative inhibition \n\nagainst one or more isolates and the ability to inhibit two \n\nor more test organisms. Yeast Extract- Malt Extract broth \n\n(ISP-2) was prepared in a conical flask. The colony of \n\nselected actinomycetes was cut from their pure culture \n\nand put into the broth. The conical flasks were placed into \n\na rotary shaker incubator at 28\u00b0C for 8 days. [13, 21] \n\nExtraction of antibiotic fraction: \nAfter incubation, the broth was collected and centrifuged \n\nat 4000 rpm for 30 minutes. The supernatant was \n\ncollected and an equal amount of ethyl acetate was added \n\nto it. The two phases were vigorously shaken for 1 hr. \n\nThen, the mixture was poured into a separating funnel \n\nand allowed to stand for 5 minutes during which \n\naqueous and organic phases separated. The lower \n\naqueous phase was discarded, while the upper organic \n\nphase was collected and heated in a water bath at 40\u00b0C to \n\nevaporate ethyl acetate [22]. The residue so left was \n\nweighed and dissolved in a small amount of phosphate \n\nbuffer of pH 7 to solubilize crude antibiotic extract [13]. \n\nThe mixture was transferred to an Eppendrof tube and \n\nstored at refrigerator temperature for further use. \n\nSecondary Screening: \nAgar well diffusion method was employed for secondary \n\nscreening of inhibitory action against test organisms. Test \n\norganisms (Pseudomonas aeruginosa, Escherichia coli, \n\nKlebsiella pneumoniae, Salmonella typhi, Salmonella \n\nparatyphi, Bacillus subtilis, Staphylococcus aureus) were \n\ninoculated into the nutrient broth and incubated at 37\u00b0C \n\nfor 4 hrs. After incubation, the turbidity of the broth was \n\ncompared with McFarland standard 0.5. Lawn/Carpet \n\nculture of test organisms was performed on Mueller \n\nHinton Agar plates from McFarland adjusted broth \n\ncultures. Wells of 8 mm was bored in Mueller Hinton \n\nAgar plates using sterile agar borer and 40 \u03bcl of ethyl \n\nacetate extracted antibiotic fractions were added into \n\nwells. Plates were left at room temperature for 20-30 mins \n\nto diffuse antibiotic fractions, and then incubated at 37\u00b0C \n\nfor 24 hrs. After 24 hrs, plates were examined and the \n\ndiameter of the zone of inhibition around each well was \n\nmeasured [13].", "start_char_idx": 11346, "end_char_idx": 15163, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "53807cf7-1468-4dc3-bb25-4d8ab28a459a": {"__data__": {"id_": "53807cf7-1468-4dc3-bb25-4d8ab28a459a", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6769f4a9-ee13-4e2d-ad9d-22b02d48e61c", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "171433707c8f9dce91819603eb86ca1446b24bf079dfaaa123a36333d2a52c1e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "035117e1-99e2-401b-a817-344a012dc3b2", "node_type": "1", "metadata": {}, "hash": "cf34d57a468bbd20cd561f9bf06737e9798316e21a77ff853a323b9cc612296a", "class_name": "RelatedNodeInfo"}}, "text": "Results \nSoil harvest large number of antibacterial \nactinomycetes \nFrom 15 soil samples, 121 isolates of actinomycetes were \n\nobtained. Among them, 58 isolates were found to have \n\neffective inhibitory activity against at least 1 test \n\norganism while 63 were ineffective against test \n\norganisms (Table 1). Twenty-two isolates produced \n\nobservable inhibitory effects against two or more test \n\norganisms during primary screening (Table 2).  \n\nAmong 58 isolates exhibiting inhibitory activity, 36 were \n\nactive against one test organism and 22 against more than \n\none test organism. Of those 22, 8 and 14 isolates were \n\nactive against 2 and 3 test organisms respectively (Figure \n\n2). \n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN \n105 \n\nFigure 2. Pie-chart showing primary screening of actinomycetes \nisolates against test organisms. 36 (62%) of isolates were found \nto be effective in inhibiting at least one test organism. 22 (38%) \nof isolates were effective on inhibiting 2 or more test organisms. \nOf these 22 isolates 14 (24%) of them inhibited 2 test organisms \nwhile 8 (14%) effectively inhibited 3 test organisms. \n\nActinomycetes have broad spectrum \nantibacterial activity \nAmong test organisms, Staphylococcus aureus was found \n\nto be the most susceptible with susceptibility toward 12 \n\nactinomycetes isolates followed by Klebsiella pneumoniae, \n\nsusceptible towards 10 isolates of actinomycetes. The \n\nleast susceptible test organism was found to be Salmonella \n\nparatyphi with susceptibility towards 5 actinomycetes \n\nisolates (Figure 3). On secondary screening (Testing of \n\neffect of antibiotic fraction obtained through \n\nfermentation followed by extraction) against test \n\norganisms, Staphylococcus aureus was found to be the \n\nmost effectively inhibited test organism followed by \n\nKlebsiella pneumoniae. Salmonella paratyphi was found to be \n\nthe least inhibited test organism (Table 3).  \n\nTable 1.", "start_char_idx": 15166, "end_char_idx": 17128, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "035117e1-99e2-401b-a817-344a012dc3b2": {"__data__": {"id_": "035117e1-99e2-401b-a817-344a012dc3b2", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "53807cf7-1468-4dc3-bb25-4d8ab28a459a", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "a3942defb5047d9c7832cebe42f4506a50aabe42eb24a7a1d7f516a7d2851c7a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63db8b24-c124-4b5b-9fdf-59090fc23511", "node_type": "1", "metadata": {}, "hash": "ac35334cc3a61323a424c976c656347732dff3a152adcf3e334baa87082e2c76", "class_name": "RelatedNodeInfo"}}, "text": "Table 1. Number of actinomycetes isolated from Soil samples with a screening of antibacterial activity \n\nS. N Soil Sample Place of collection \nNumber of \n\nColonies \n\nNumber of colonies with Inhibitory activity \n\nAgainst 1 test \norganism \n\nAgainst \u22652 test \norganism \n\n1 A Nuwakot  (Damp Land) 5 1 1 \n\n2 B Lagankhel  (Agri Land) 14 5 3 \n3 C Katunje       (Agri Land) 10 3 2 \n4 D Katunje       (Riverbank Land) 8 3 1 \n5 E Budhanilkantha (Agri Land) 13 4 3 \n\n6 F Chapagaun  (Agri Land) 19 5 4 \n\n7 G Chapagaun  (Forest Land) 8 2 2 \n8 H Godawari      (Damp Land) 3 0 1 \n9 I Chovar       (Forest Land) 3 1 0 \n10 J Sundarijal     (Riverbank Land) 5 4 1 \n11 K Sundarijal     (Forest Land) 9 1 2 \n12 L Lubhu        (Damp Land) 4 0 0 \n13 M Sankhamul   (Riverbank Land) 8 2 1 \n14 N Gokarna        (Forest Land) 7 2 0 \n15 O Chandragiri      (Forest land) 5 3 1 \n\nTotal 121 36 22 \n\nTable 2: Primary screening of effective actinomycetes against test organisms \n\nS.\nN. \n\nActinomycetes \nColony \n\nP. aeruginosa E. coli K. pneumoniae S. typhi S. paratyphi B. subtilis S. aureus \n\n1 A3 - + - - - - + \n2 B2 - + - + + - - \n3 B4 - - + - - + + \n4 B9 + - + - - - + \n5 C5 + + - - - - + \n6 C8 - - - + + - - \n7 D6 - - + - - - + \n8 E3 + - - - - + + \n9 E7 + - + - - - - \n\n10 E12 - + - + - - + \n11 F1 - - + + + - - \n12 F2 + - - - - + + \n13 F17 - + + - - + - \n14 F19 + + - - - - - \n15 G1 - + - + + - - \n16 G3 - - - - - + + \n17 H4 + - + - - - - \n18 J2 - - + - - + + \n19 K1 - + - - - + + \n20 K6 + - + - - - + \n21 M3 + + - - - - - \n22 O4 - - + + + - - \n\n36, 62%\n8, 14%\n\n14, 24%\n\nIsolates effective against 1 test organism \nIsolates effective against 2 test organism \nIsolates effective against 3 test organisms\n\n\n\n Nepal J Biotechnol. 2020 Dec; 8(3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN 106 \n\nFigure 3. Bar diagram showing susceptibility of test organisms \ntowards actinomycetes isolates. Staphylococcus aureus was \nfound to be most susceptible organism toward actinomycetes \nwith 12 isolates inhibiting its growth, followed by Klebsiella \npneumoniae (10 isolates). Salmonella paratyphi was least inhibited \norganism with only 5 actinomycetes isolates able to inhibit its \ngrowth. \n\nActinomycetes have inhibitory activity \nagainst drug-resistant strains \nThe antibiotic fractions found to be effective against \n\nStaphylococcus aureus from secondary screening were \n\ntested against Methicillin Susceptible Staphylococcus \n\naureus (MSSA) and Methicillin Resistant Staphylococcus \n\naureus (MRSA) variants. Among 12 isolates, 10 were \n\nfound to have an inhibitory effect against MSSA while \n\nonly 6 were found to have an inhibitory effect against \n\nMRSA strain (Table 4).", "start_char_idx": 17120, "end_char_idx": 19759, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63db8b24-c124-4b5b-9fdf-59090fc23511": {"__data__": {"id_": "63db8b24-c124-4b5b-9fdf-59090fc23511", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "035117e1-99e2-401b-a817-344a012dc3b2", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "3a0fbf59e686109baca8da4a0432346b18e3de03fad4256f187a0c4ea11a822b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c6c42651-57bf-4425-8fa3-cd12bcb06b18", "node_type": "1", "metadata": {}, "hash": "b634526f9bed6c3ca0ed1921fb106c1c5babeaab7cfa0250e9ba152c5685c5c4", "class_name": "RelatedNodeInfo"}}, "text": "Table 4: Screening Results of Effective Antibiotic Producers \nagainst MSSA and MRSA \n\nS.N\n. \n\nActinomycetes \nColony \n\nPrimary \nScreening \n\nSecondary \nScreening \n\n(ZOI in mm) \nMSSA MRSA MSSA MRSA \n\n1 A3 + + 16 10 \n2 B4 + _ 7 - \n3 B9 - _ - - \n4 C5 + + 9 5 \n5 D6 + + 12 8 \n6 E3 - + 6 7 \n7 E12 + + 8 6 \n8 F2 + - 8 - \n9 G3 + - 6 - \n10 J2 + - 13 - \n11 K1 + + 9 7 \n12 K6 + - 5 - \n\nOut of 58 effective isolates in primary screening, pH \n\nrange (7.4-7.8) showed higher number of isolates \n\nfollowed by pH range (7.9-8.4) with 18 and 17 effective \n\nisolates respectively (Table 5). The least number of \n\neffective isolates were found in acidic pH range while \n\nneutral and basic pH range showed larger proportion of \n\neffective isolates. \n\nOn distribution of effective isolates based on moisture of \n\nsoil, moisture range of (11-20) % showed highest number \n\nof effective isolates (17).  Even though the moisture range \n\n(31-40) and (41-50) % showed high number of isolates, it \n\nhas to be noted that the soil samples of river bank has \n\nhigh moisture due to the constant flow of water over \n\nthem. This moisture range was observed only in river  \n\nbank soil and the growth of actinomycetes on such soil is \n\naffected by humus and various other factors as well. \n\nTable 3: Secondary Screening of Antibiotic fractions against test organisms (zone of inhibition in mm) \n\nS.N. Actinomycetes \nColony \n\nP. aeruginosa E. coli K. pneumoniae S. typhi S. paratyphi B. subtilis S. aureus \n\n1 A3 8 - - - - 20 \n\n2 B2 - 12 - 9 7 - - \n\n3 B4 - - 10 - - 7 11 \n\n4 B9 12 - 12 - - - 7 \n\n5 C5 8 15 - - - - 9 \n\n6 C8 - - - 8 6 - - \n\n7 D6 - - 9 - - - 11 \n8 E3 5 - - - - 3 8 \n9 E7 7 - 6 - - - - \n\n10 E12 - 6 - 4 - - 12 \n11 F1 - 11 12 4 - - \n12 F2 3 - - - 11 8 \n13 F17 - 13 5 - - 12 - \n\n14 F19 2 9 - - - - - \n\n15 G1 - 7 - 8 5 - - \n16 G3 - - - - - 8 6 \n17 H4 5 - 4 - - - - \n\n18 J2 - - 6 - - 9 15 \n\n19 K1 - 9 - - - 8 13 \n20 K6 7 - 9 - - - 7 \n21 M3 9 6 - - - - - \n22 O4 - - 8 5 2 - - \n\n9 9\n10\n\n6\n5\n\n7\n\n12\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\nN\no\n\n o\nf \n\na\nct\n\nin\no\n\nm\ny\n\nce\nte\n\n i\nso\n\nla\nte\n\ns\n\nTest organisms\n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN \n107 \n\nDiscussion \nThe soil samples undertaken for the study were of \n\ndifferent environmental sites which include forest land, \n\nagricultural lands, river-bank land, and damp lands.  \n\nOn the isolation of the Actinomycetes, it was observed \n\nthat the highest number of actinomycetes isolates were \n\nisolated from 4 soil samples of agricultural land (56 out \n\nof 121 isolates i.e.  46.28%).", "start_char_idx": 19762, "end_char_idx": 22299, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c6c42651-57bf-4425-8fa3-cd12bcb06b18": {"__data__": {"id_": "c6c42651-57bf-4425-8fa3-cd12bcb06b18", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63db8b24-c124-4b5b-9fdf-59090fc23511", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "6205d7c88a2e897dda5ff90a1f2d545305b86f3f3f1a6190da1d91dcc8212c94", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "06431c08-3ca6-4631-b47d-22796ab24f41", "node_type": "1", "metadata": {}, "hash": "a959fa0dc279dafaf948a244682de45cbefb58a402fdb6ff2e52fd96f7285155", "class_name": "RelatedNodeInfo"}}, "text": "46.28%). Three soil samples from damp \n\nland were found to have 12 isolates of actinomycetes \n\nwhich were lowest among the soil samples classified into \n\ndifferent ecological conditions. The higher number of \n\nisolates from agricultural and forest land can be due to \n\nthe positive impact of the vegetation in the colonization \n\nof the actinomycetes.   \n\nThis finding is in correspondence to the report by \n\nGhorbani et al which states that the abundance of \n\nactinomycetes isolates decreases from irrigated \n\ncultivated land to pastures i.e., damp lands [22]. \n\nKlemmedson also reported that the actinomycetes may \n\nform the number of roots nodulated symbiotic \n\nrelationship with various plants thus increasing the \n\nactinomycetes population in the lands with vegetation \n\n[23]. \n\nOn the distribution of soil samples and actinomycetes \n\nisolates as per the pH value of soil, slightly alkaline pH \n\n(7.4-7.8) or moderately alkaline pH (7.9-8.4) was found \n\nto be most favorable for the proliferation of \n\nactinomycetes as 70 out of 121 isolates were obtained \n\nfrom these pH range (Table 5). Acidic pH has decreased \n\nnumber of actinomycetes and is not favorable for \n\nactinomycetes Agricultural lands was neutral to slightly \n\nalkaline in pH (7.2-8.2) while the pH of the forest lands \n\nwas in acidic range (5.2-6.4) except for soil sample from \n\nforest land of Gokarna (pH value of 7.6) i.e. neutral pH \n\n(Table 5). The results of the study agree with those of \n\nAkond et al [15], Ameerah et al [24], Palanichamy et al \n\nwhich reported that optimum growth of actinomycetes \n\noccurs in neutral to alkaline pH [25]. However, the \n\nfindings were in contrast to Kontro et al which reported \n\n10 species of Streptomyces isolated from Finland were \n\nfound to have maximum growth at pH range of (4.0-5.5) \n\n[26]. This shows that the major factor limiting \n\nactinomycetes development in forest soils is believed to \n\nbe the low soil pH, as the development of most \n\nactinomycetes is favored by a neutral or slightly alkaline \n\npH as reported by Golinska et al [27]. \n\nIn this study, it was found that black colored soil \n\nharbors most of the antibiotic producing actinomycetes \n\nfollowed by black-brown soil in comparison to the red \n\nsoil. This is in contradiction with the findings by Guo et \n\nal who reported that red soil is ideal habitat for \n\nacidophilic actinomycetes and harbors a diverse group \n\nof actinomycetes which are a promising sources of novel \n\nsecondary metabolites [28]. \n\nThe distribution of effective isolates as per the pH value \n\nof soil of origin showed that pH of the soil has a \n\nsignificant effect on the effectiveness ofthe production of \n\nthe antimicrobial compounds by actinomycetes species. \n\nAlso, the effective isolates were found to be from neutral \n\nto alkaline soil. These findings are in accordance with the \n\nAmeerah et al [24], Akond et al where the maximum \n\ngrowth of actinomycetes was reported in neutral to \n\nalkaline pH range [15].  The findings are in conformity \n\nwith those of Singh et al which reported that maximum \n\ngrowth as well as highest antimicrobial activity by S. \n\nsannanensis SU118 was achieved at pH 7 while it does not \n\nexhibit any activity at pH 9 [29]. \n\nOut of 121 Actinomycetes isolates, 58 isolates were found \n\nto have inhibitory activity against at least 1 test organism \n\ni.e.  47.9% of isolates were active against at least 1 test \n\norganism. A similar result was noted in studies of Kumar \n\net al [30] and Patel   et al [12]. In contrast to the findings, \n\nTable 5: Physical characterization of Soil samples \n\nS.N.", "start_char_idx": 22291, "end_char_idx": 25873, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "06431c08-3ca6-4631-b47d-22796ab24f41": {"__data__": {"id_": "06431c08-3ca6-4631-b47d-22796ab24f41", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c6c42651-57bf-4425-8fa3-cd12bcb06b18", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "4e9aca4bbecf700720cfdb8e6c12313856c2ae1f697eb697539a4ec0c6ee0385", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1b20089b-76ee-4480-be1d-c360142c118e", "node_type": "1", "metadata": {}, "hash": "117177e088b7edfe1e521d973df9343aca70614907818ddc24d334992dc5355e", "class_name": "RelatedNodeInfo"}}, "text": "Soil \n\nSample \nPlace/Location \n\nof collection \nColor pH Moisture \n\n1 A \nNuwakot \n\n(Damp Land) \nRed 5.3 9 % \n\n2 B \nLagankhel, \n(Agri Land) \n\nBlack-\nbrown \n\n7.2 32 % \n\n3 C \nKatunje \n\n(Agri Land) \nBrown 8.2 20% \n\n4 D \nKatunje \n\n(River Bank \nLand) \n\nBlack-\nBrown \n\n7.7 52% \n\n5 E \nBudhanilkantha \n\n(Agri Land) \nBlack 8.1 38% \n\n6 F \nChapagaun \n(Agri Land) \n\nDark \nblack \n\n7.5 43% \n\n7 G \nChapagaun \n\n(Forest Land) \nBrown 6.2 12% \n\n8 H \nGodawari \n\n(Damp Land) \nBrown 5.4 15% \n\n9 I \nChovar \n\n(Forest Land) \nBrown 5.2 18% \n\n10 J \nSundarijal \n\n(River Bank \nLand) \n\nBlackish 8.4 47% \n\n11 K \nSundarijal \n\n(Forest Land) \nBrown 6.4 29% \n\n12 L \nLubhu \n\n(Damp Land) \nBrown 5.8 22% \n\n13 M \nSankhamul \n(River Bank \n\nLand) \nBlack 7.8 42% \n\n14 N \nGokarna \n\n(Forest Land) \nBlack-\nBrown \n\n7.6 15% \n\n15 O \nChandragiri \n(Forest Land) \n\nReddish \nBrown \n\n5.7 12% \n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN \n108 \n\nGurung et al reported that 34.18% of 79 isolates were \n\nactive against test organisms [18] and Pandey et al found \n\n33.96% actinomycetes isolated from the Khumbu region \n\nshowed inhibitory activity against at least 1 of 5 test \n\norganisms (Bacillus subtilis, Staphylococcus aureus, Proteus, \n\nSalmonella typhi) [31] . Among 58 active isolates, only 22 \n\n(37.9 %) were active against 2 or more test organisms. \n\nThis is in accordance with the reports of Sharma et al [32] \n\nand Salam and Rana [33]. On the contrary Devadass et al \n\nstated that only 21 out of 150 i.e. 14% of isolates from \n\nWestern Ghats of Tamil Nadu were active against more \n\nthan one test organism [34]. \n\nFrom the study Staphylococcus aureus was found to be \n\nmost susceptible organism towards actinomycetes \n\nisolates. Also, on comparison of the zone size of \n\ninhibition against the gram-positive organism i.e. \n\nStaphylococcus aureus against that of the gram-negative \n\ntest organisms, it can be inferred that the actinomycetes \n\nisolates were more active against gram-positive \n\nbacterium. This may be due to the difference in cell \n\nmorphology of gram-positive and gram-negative cells \n\nand furthermore, the antibiotic compound could have \n\nbeen more functional against gram positive cell \n\ncomponents than that of gram-negative cell components. \n\nA study carried out by Tyagi et al reported that \n\nactinomycetes isolates were highly active against \n\nStaphylococcus aureus, Streptococcus, Bacillus and E. coli \n\nstrains. It also suggests that actinomycetes have more \n\npotent inhibitory activity against gram-positive bacteria \n\n[35]. \n\nThe secondary screening of the antibacterial activity \n\nshowed that out of 22 antibiotic fractions, only 8 were \n\nineffective against gram-positive bacteria while only one \n\nfraction from isolate (G3) was ineffective against gram-\n\nnegative bacteria. 13 fractions were found to be effective \n\nagainst both gram positive and gram-negative bacteria. \n\nThese findings showed that the 9 fractions were found to \n\nhave a narrow spectrum of activity while the 13 fractions \n\nwere found to have a wide spectrum of inhibitory activity \n\nagainst the test organism. However, they cannot be \n\nconfirmed as broad spectrum and narrow spectrum until \n\nthey are tested multiple times with a wider range of the \n\ntest organisms.", "start_char_idx": 25875, "end_char_idx": 29128, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1b20089b-76ee-4480-be1d-c360142c118e": {"__data__": {"id_": "1b20089b-76ee-4480-be1d-c360142c118e", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "06431c08-3ca6-4631-b47d-22796ab24f41", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "f211c08dc81bbfd11424efa7f1c7d62736b57a7ee6675e933c059500060afb77", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1281edbd-dbe4-4b78-b4cd-386f6846628b", "node_type": "1", "metadata": {}, "hash": "8011cce330162b1199acaa7115e7c2f7efd8465159c83815baa4233043c2ee9e", "class_name": "RelatedNodeInfo"}}, "text": "On testing of inhibitory activity of the 12 isolates active \n\nagainst Staphylococcus aureus towards drug resistant \n\nstrains MRSA and MSSA, 11 were found to inhibit MSSA \n\nwhile 6 of them showed inhibitory activity against \n\nMRSA.  These findings showed that drug-resistant \n\nstrains of Staphylococcus aureus can be treated with the \n\nbioactive secondary metabolites from actinomycetes \n\nisolates.  Sharma et al reported 6 strains of actinomycetes \n\nisolates having highly inhibitory activity \n\nagainst MRSA strains (MRSA-97, MRSA-67, and MRSA \n\nP-169) [32]. In a similar study Chaudhary et al reported \n\nthe inhibitory activity of the 31 isolates towards \n\nStaphylococcus aureus among which number of them \n\nbeing inhibitory towards MRSA [5].     \n\nOn the distribution of effective isolates as per moisture \n\ncontent highest number of effective isolates was found to \n\nbe from soil having a moisture content of (11-20) %. The \n\nfindings are inconformity with those of Mavordi et al \n\nwhich stated that Pseudomonas spp. producing the \n\nantibiotics phenazine-1-carboxylic acid were abundant in \n\nthe rhizosphere of native plant species growing in non-\n\nirrigated areas adjacent to the sampled dryland wheat \n\nfields i.e. the lands with less moisture [36].  \n\nConclusion \nSoil harbor large number of actinomycetes isolates which \n\nexhibit inhibitory activity against number of bacterial \n\nstrain. Acidic pH favors the growth of actinomycetes and \n\nits ability to produce antimicrobial compounds. \n\nActinomycetes has been a constant source of \n\nantimicrobial agents and the further studies exploring the \n\nantimicrobial potential of these isolates from various \n\nunexplored areas need to be done.  \n\nAuthor\u2019s contribution \nSB is the principal investigator, carried out all the \n\nlaboratory works, also prepared the manuscript. AS is the \n\ncorresponding author, an academic supervisor of the \n\nresearch and prepared the manuscript.  \n\nCompeting Interests \nThe authors declare no competing or financial interests. \n\nFunding \nThe materials, reagents and chemicals required for the \n\nlaboratory work was funded by Microbiology Research \n\nSociety, Nepal. \n\nAcknowledgments \nWe would like to thank Kathmandu Centre for Genomics \n\nand Research Laboratory as well as St. Xavier\u2019s College \n\nfor providing the research space. We would like to thank \n\nMicrobiology Research Society for providing the \n\nmaterials, reagents and chemicals required to accomplish \n\nthe research work.  We are thankful to Ms. Sunita \n\nShrestha and Ms. Naina Byanjankar, Research Assistants \n\nof Nepal Academy of Science and Technology (NAST) for \n\ntheir help in the preparation of map of sample collection \n\nsites. \n\nEthical Approval and Consent \nNot applicable. \n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN 109 \n\nReferences \n1. Ganesan P, Host R, Antony D, Appadurai DR, Gandhi MR, Paulraj \n\nMG, Ignacimuthu S and Al-Dhabi NA. Isolation and molecular\ncharacterization of actinomycetes with antimicrobial and \nmosquito larvicidal properties. BeniSuef University Journal of\nBasic and Applied Science. 2017;6(2):209-\n17. https://doi.org/10.1016/j.bjbas.2017.04.002\n\n2. Denyer PS, Hodges N, Gorman PS et al. Hugo and Russell\u2019s\nPharmaceutical Microbiology. Eighth Edition. Willey-Blackwell\nPublication. 2011. 169-172p. \n\n3. Sengupta S, Pramanik A, Ghosh A and Bhattacharyya M.\nAntimicrobial activities of actinomycetes isolated from\nunexplored regions of Sundarbans mangrove ecosystem. BMC\nMicrobiology.2015; 15:170.  https://doi.org/10.1186/s12866-015-\n0495-4 \n\n4.", "start_char_idx": 29131, "end_char_idx": 32696, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1281edbd-dbe4-4b78-b4cd-386f6846628b": {"__data__": {"id_": "1281edbd-dbe4-4b78-b4cd-386f6846628b", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1b20089b-76ee-4480-be1d-c360142c118e", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "347229c93a29ea8eefd2aa9c2e59938d27e91ac1641b17270077d83543931cf0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "00e97c3c-493b-42a1-aba7-b02a18da07e8", "node_type": "1", "metadata": {}, "hash": "a8df16bf42419bf06143a3293f4662ee861fbde77ee9fdb2290f2ca5baf288b6", "class_name": "RelatedNodeInfo"}}, "text": "Ganesan P, Host R, Antony D, Appadurai DR, Gandhi MR, Paulraj \nMG, Al-Dhabi NA and Ignacimuthu S.Antimicrobial activity of\nsome actinomycetes from western ghats of Tamil Nadu, India.\nAlexandria Journal of Medicine. 2016; 53:101\u2013110. \nhttps://doi.org/10.1016/j.ajme.2016.03.004\n\n5. Chaudhary HS, Yadav J, Shrivastava AR, Singh S, Singh AK and\nGopalan N. Antibacterial activity of actinomycetes isolated from\ndifferent soil samples of Sheopur (A city of central India).   Journal\nof Advanced Pharmaceutical Technology &\nResearch.2013;4(2):118-23. https://doi.org/ 10.4103/2231-\n4040.111528 \n\n6. Rahman MA, Islam MZ and Islam MA. Antibacterial activities of\nActinomycete isolates collected from Soils of Rajshahi,\nBangladesh. Biotechnology Research International. 2011. , Article\nID 857925. https://doi.org/ 10.4061/2011/857925 \n\n7. Ruan J. [Bergey's Manual of Systematic Bacteriology (second \nedition) Volume 5 and the study of Actinomycetes systematic in\nChina]. Wei Sheng Wu Xue Bao. 2013 :53(6): 521-30 p. Chinese.\nPMID: 24028053\n\n8. Manandhar S and Sharma S. Practical approach to Microbiology. \nRevised Edition. National Book Centre, Graphic plus\nprinters.2013. 273-375 p. \n\n9. Bizuye A, Moges F and Andualem B.  Isolation and screening of\nantibiotic producing actinomycetes from soils in Godhar town,\nNorth West Ethiopia. Asian Pac J of Tropical Medicine.2013; 3(5):\n375-81. https://doi.org/10.1016/S2222-1808(13)60087-0 \n\n10. Remya M and Vijayakumar R. Isolation and characterization of \nmarine antagonistic actinomycetes from west coast of India.\nMedicine and Biology.2008;15:13-9. \n\n11. Waksman SA, Schatz A, and Reynolds DM. Production of\nAntibiotic Substances by Actinomycetes. Annals of the New York\nAcademy of Sciences.2010;1213:112-24. \nhttps://doi.org/10.1111/j.1749-6632.2010.05861.x \n\n12. Patel JD, Parmar M, Patel P, Rohit P, Taviyad R, Ansari P, et al.\nDynamism of Antimicrobial activity of Actinomycetes- A Case\nstudy from Undisturbed Microbial Niche. Advances in\nMicrobiology.2014;4:324-34. \nhttps://doi.org/10.4236/aim.2014.46039\n\n13. Gopinath BV, Vootla PK, Jyothi R and Reddy KS. Antimicrobial \nactivity of Actinomycetessolated from Coal Mine soils of Godavari \nBelt Region, A.P, India. Asian J ExpBiol Sci.2013;4(4):518-23. \n\n14. Awasthi S, Gahlot S, Kumar S and Bhatnagar T. Screening of\ndifferent bioactive soil Streptomyces Spp. for broad spectrum\nantibacterial and antifungal activity.  Research Journal of \nPharmaceutical Biological and Chemical Sciences.2014; 5(3): 1537-\n44. \n\n15. Akond MA, Jahan MN, Sultana N and Rahman F.  Effect of\nTemperature, pH and NaCl on the isolates of Actinomycetes from\nstraw and Compost samples from Savar, Dhaka, Bangladesh.\nAmerican Journal of Microbiology a Antibacterial activity of some\nactinomycetes isolated from farming soils of Turkey.", "start_char_idx": 32697, "end_char_idx": 35501, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "00e97c3c-493b-42a1-aba7-b02a18da07e8": {"__data__": {"id_": "00e97c3c-493b-42a1-aba7-b02a18da07e8", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1281edbd-dbe4-4b78-b4cd-386f6846628b", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "76e69431558a0a4fed67397401336b9e5fc0be9819bbe9691a5c04fa6aef41dd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "123adabb-b9a7-40f9-8dd1-e2aec8055449", "node_type": "1", "metadata": {}, "hash": "0dfab02383c00228089c5f0259015e27f5d95aba1bac0e6762502e3da4d31ce0", "class_name": "RelatedNodeInfo"}}, "text": "African J\nBiotechnol.2004;3(9):441-6.Available online at\nhttp://www.academicjournals.org/AJB\n\n16. GARP-Global Antibiotic Resistance Partnership. Executive\nSummary: Nepal Situation Analysis and Recommendations:\nAntibiotic use and resistance in\nNepal.2015.https://www.nphfoundation.org/ [Accessed on 10\nJune 2018] \n\n17. Gurung TD, Sherpa C, Agrawal VP and Lekhak B.  Isolation and\nCharacterization of antibacterial Actinomycetes from soil samples\nof Kalapatthar, Mount Everest Region. Nepal Journal of Science \nand Technology.2009; 10:173-82. \n\n18. Heng JLS, Shah UKM and Hamzah H.  Isolation, characterization\nand identification of potential actinobacteria with antifungal\nactivities towards chillianthracnose. African Journal of\nBiotechnology.2011;10(32):5979-87. Available online at:\nhttp://www.academicjournals.org/AJB\n\n19. Alharbi SA, Arunachalam C, Murugan AM and Wainwright M. \nAntibacterial activity of actinomycetes isolated from terrestrial soil\nof Saudi Arabia, J of Food. Agriculture & Environment.\n2012;10(2):1093-7. \n\n20. Cheesbrough M.. District Laboratory Practice in Tropical \nCountries. 2000. Low Price Edition. Cambridge University Press.\n\n21. Dhananjeyan V, Selvan N and Dhanapal K. Isolation,\ncharacterization, screening and Antibiotic sensitivity of\nActinomycetes from locally (Near MCAS) collected soil samples. J\nBoil Sci. 2010; 10(6):514-\n9.https://doi.org/10.3923/jbs.2010.514.519\n\n22. Ghorbani NR, Greiner R, Alikhani HA, Hamedi, J and Yakhchali\nB. Distribution of actinomycetes in different soil ecosystems and\neffect of media composition on extracellular phosphatase activity.\nJ.  Soil Sci.  Plant Nutr. 2013;13(1):223-36. \nhttps://doi.org/10.4067/S0718-95162013005000020\n\n23. Klemmedson JO. Ecological Importance of Actinomycete\nNodulated Plants in the Western United States. BOT GAZ. 1979;\n140: 91-6. Available online at: \nhttps://www.jstor.org/stable/2474210\n\n24. Ameerah HA, Suhaidi A, Sharifah A and Syed M.  Identification\nand Optimal growth conditions of Actinomycetes isolated from\nMangrove Environment. Malaysian Journal of Analytical Sciences.\n2015; 19(4): 904 \u2013 10. \n\n25. Palanichamy V, Hundet A, Mitra B and Reddy N.  Optimization of\ncultivation parameters for growth and pigment production by\nStreptomyces spp. isolated from marine sediment and rhizosphere\nsoil.  Int. J. Plant Animal Environ. Sci.2011;1(3):158-70.Available \nonline at: www.ijpaes.com\n\n26. Kontro M, Lignell U and Nevaleinen A.  pH effects on 10\nStreptomyces spp. growth and sporulation depend on nutrients.\nLett. App. Microbiol.2005;41:32-8. \nhttps://doi.org/10.1111/j.1472-765X.2005. 01727.x\n\n27. Golinska P and Dahm H. Occurrence of Actinomycetes in forest\nsoils. Dendrobiology.2011; 66: 3-13. \n\n28. Guo X, Liu N, Li X, Ding Y, Shang F, Gao Y, Ruan J, and Huang Y.\nRed Soils harbor diverse culturable Actinomycetes that are \npromising sources of novel Secondary Metabolites. Appl. Environ.\nMicrobiol.", "start_char_idx": 35502, "end_char_idx": 38412, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "123adabb-b9a7-40f9-8dd1-e2aec8055449": {"__data__": {"id_": "123adabb-b9a7-40f9-8dd1-e2aec8055449", "embedding": null, "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-119", "node_type": "4", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "131f634bc2d815f4dac5f04b67176c91c4c8f81210e66f87579b3220bae7cba2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "00e97c3c-493b-42a1-aba7-b02a18da07e8", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "c74e3bc581a04d816c1c060cd86f7387a031ce406e002b259f08f777bd38c4d4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5ecbd679-80ba-4184-9c98-3c7ae7a028d2", "node_type": "1", "metadata": {}, "hash": "321a5b524c3a9096e5b9d90d2bafe09815a72c16e0194ef9a1a8a55fcc2ff2de", "class_name": "RelatedNodeInfo"}}, "text": "Appl. Environ.\nMicrobiol. 2015; 81(9): 3086-103.\nhttps://doi.org/10.1128/AEM.03859-14 \n\n29. Singh LS, Sharma H, Talukdar NC. Production of potent \nantimicrobial agent by actinomycete, Streptomyces sannanensis\nstrain SU118 isolated from phoomdi in Loktak Lake of Manipur,\nIndia. BMC Microbiology. 2014; 14: 278-90. \nhttps://doi.org/10.1186/s12866-014-0278-3 \n\n30. Kumar S, Palaniswamy C and Raj K. Antimicrobial activity of\nactinomycetes from Tamil Nadu, India. 2014. [Retrieved from\nElsevier on 13/07/2018].\n\n31. Pandey B, Ghimire P and Agrawal VP.  Studies on the antibacterial\nactivity of the Actinomycetes isolated from the Khumbu Region of\nNepal. Journal Biology Science. 2004; 23:44-53. \n\n32. Sharma D, Kaur T, Chadha BS and Manhas RK.  Antimicrobial \nActivity of Actinomycetes against Multidrug Resistant\nStaphylococcus aureus, E.  coli and various other pathogens. \nTropical Journal of Pharmaceutical Research. 2011; 10(6): 801-8.\nhttps://doi.org/10.4314/tjpr. v10i6.14\n\n33. Salam MD and Rana S. Antimicrobial potential of Actinomycetes\nisolated from soil samples of Punjab. India Journal of Microbiology \n& Experimentation. 2014; 1(2): 3-10. \n\n34. Devadass BJ, Paulraj MG, Ignacimuthu S, Theoder PAS and Dhabi \nNAA. Antimicrobial Activity of Soil Actinomycetes isolated from\nWestern Ghats in Tamil Nadu, India. J BacteriolMycol. 2016; 3(2):\n00059. https://doi.org/10.15406/jbmoa.2016.03.00059\n\n35. Tyagi J, Bhatnagar T and Pandey FK. Isolation and\nCharacterization of Actinomycetes from soil and screening their\n\n\n\n Nepal J Biotechnol. 2020 Dec; 8 (3): 102 -110 Budhathoki & Shrestha \n\n\u00a9NJB, BSN 110 \n\nAntibacterial Activities against different microbial isolates.  \nInternational Journal of Life Sciences Research. 2014; 2(4): 101-5. \n\n36. MavrodiDV, Mavrodi OV, Parejko JA, Bonsall RF, Kwak \nYS, Paulitz TC et al. Accumulation of the Antibiotic Phenazine-1-\nCarboxylic Acid in the Rhizosphere of Dryland Cereals. Applied\nand Environmental Microbiology. 2012; 78(3): 804-\n12. https://doi.org/10.1128/AEM.06784-11", "start_char_idx": 38387, "end_char_idx": 40408, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5ecbd679-80ba-4184-9c98-3c7ae7a028d2": {"__data__": {"id_": "5ecbd679-80ba-4184-9c98-3c7ae7a028d2", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "123adabb-b9a7-40f9-8dd1-e2aec8055449", "node_type": "1", "metadata": {"identifier": "njb-119", "author": "Budhathoki, Shailesh; Shrestha, Anima", "title": "Screening of Actinomycetes from Soil for Antibacterial Activity", "date": "2020-12-30", "file": "njb-119.pdf"}, "hash": "3d7ce67c9eff8e477a1f42abd5f944c438076f6a69914dcbdfa2f89fb8279af8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2cfa2ccc-ec20-49ca-9490-82ddcab7f3c5", "node_type": "1", "metadata": {}, "hash": "e10f094b8311ca982f0ef6261f3b147977242abdd6ea26c654f6861ba8257b99", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2020 Dec;  8 (3): 95-101 DOI: https://doi.org/10.3126/njb.v8i3.33663 Research article \n\n\u00a9NJB, BSN 95 \n\nComparative Study of Bioactive Compounds in Different Varieties of \nPears in Nepal \nBarsha Koirala , Angela Shrestha \n\nSt. Xavier\u2019s College, Kathmandu, Nepal \n\nArticle history:- Received: 5 May 2020; Revised: 7 Dec 2020; Accepted: 19 Dec 2020; Published online: 30 Dec 2020 \n\nAbstract \nThis study was conducted to evaluate the physicochemical parameters, perform qualitative tests (for sugars and \nphytochemicals), and quantitative tests (phenolics, antioxidants, anthocyanins, tannins, vitamin C) of six different varieties \nof pears i.e. Bartlette, Chinese pears, Chojuro, Kosui, Pharping local, and Yakumo. The juices extracted from respective pears \nwere used for the analysis. The phenols were determined by the Folin-Ciocalteu method, antioxidants by the DPPH \nscavenging activity, and anthocyanins by a SO\u2082 bleaching technique. The Pharping local pears were found to have the highest \nanthocyanins (85.95\u00b10.1 mg/l), total phenolic content (600\u00b10.01 mg GAE/l), antioxidants (IC50 value 250\u00b10.00 mg of \nphenol/l) and vitamin C content (12.2\u00b10.01 mg/100 ml) and tannins were observed to be highest in Yakumo pears (0.93\u00b10.01 \ng/l). Likewise, the highest clarity i.e. 1.960\u00b10.00 was observed in Bartlette pears and the highest acidity (2.01\u00b10.01%) in \nChojuro pears. Various sugar/carbohydrate tests like Molisch\u2019s test, Benedict\u2019s test, Barfoed test, Bial\u2019s test, Seliwanoff test, \nFehling\u2019s test and Iodine test were performed for the pear varieties. All the pears gave positive results for all the sugar tests \nexcept Iodine test. The positive results for sugar/carbohydrate signifies the presence of various sugars that help for the better \ntaste, texture, and aroma of pear. The pear varieties showed the presence of phytochemicals like flavonoids, terpenoids, \ncatechins, cyclic glycosides, and proteins. The phytochemicals are responsible for fruit preservation and act as anti-\ncarcinogenic components. Among the varieties of pears, Pharping local pears were observed to be most nutritional because \nof high antioxidants, phenols, anthocyanins, and vitamin C.  \n\nKeywords: Pears, Physicochemical parameters, Antioxidants, Phenolic content, Pharping local\n\nCorresponding author, email: varshakoirala@gmail.com \n\nIntroduction \nPear is the second most important deciduous fruit found \n\nin Nepal [1]. It is cultivated in both mid and high hills \n\nranging from 800 to 1200 m above sea level. The pear \n\ncovers 4396.5 hectors of land, a productive area of 3386 \n\nhectors with productivity of 34151 mt which yields 10.1 \n\nmt/hectors [1]. Every year, tons of pears decay during the \n\nharvesting season, because of their low consumption rate \n\n[1,2]. Pears are rich in carbohydrates, vitamin B6, vitamin \n\nA, vitamin C, sugars, iron, calcium, sodium, potassium, \n\nthiamine, water, dietary fibers, phosphorus, etc. Pears are \n\nalso used as a medication to prevent the lungs\u2019 function, \n\nbones deformation, coughs, and chills, ulcers, \n\npulmonary disease, improving immunity, etc. [1,2]. \n\nUsually, there are two types of pear grown in Nepal, i.e. \n\nEuropean and Asian.", "start_char_idx": 48, "end_char_idx": 3240, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2cfa2ccc-ec20-49ca-9490-82ddcab7f3c5": {"__data__": {"id_": "2cfa2ccc-ec20-49ca-9490-82ddcab7f3c5", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5ecbd679-80ba-4184-9c98-3c7ae7a028d2", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "28ac641e69e8b7624cf1281e7a1f0629e1b40ceef311ba3c1163a38915d33797", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a050a96e-fe66-4674-88d6-9f8f071478bb", "node_type": "1", "metadata": {}, "hash": "71731e0ec33de73d44b3e1d731b0dd38c6ca60090292b59f8c60b7a294971ac3", "class_name": "RelatedNodeInfo"}}, "text": "European and Asian. Asian pears, which are also called \n\nApple pears, Salad pears, Nashi, Oriental, Chinese or \n\nJapanese pears, are  a large group of pears that are crispy \n\nand ready to eat as soon as they are harvested. Kosui, \n\nChojuro, Yakumo, Hosui, Pharping local, etc are the \n\nvarieties of Asian pears. European pears are harvested \n\nwhen they are hard and green, and stored at room \n\ntemperature for the ripening process; so, they are \n\nsweeter. The pears like Bartlette, Comice, D\u2019 Anjou are \n\nthe varieties of European pears [3,4]. \n\nPears are rich in sugars like fructose, sucrose, glucose, \n\nsorbitols, etc. Sucrose is the source of high energy and \n\nhelps in cold tolerance capability of fruits; glucose and \n\nfructose act as antioxidants. Pears are also responsible for \n\nmaintaining the quality of fruits and their maturity [2]. \n\nThe pear juice contains 9-15 % of soluble solids [5]. The \n\nvarious analyses (optical and chemical) are performed to \n\nmaintain the quality of juices which are called \n\nphysicochemical analysis. The acidity and pH of the \n\nfruits are responsible for color, brightness, and freshness \n\nand taste of the juices [6,7]. Polyphenols are a group of \n\ncompounds that use phenol as a building block. Some \n\nphenolic compounds found are gallic acid, quercetin, \n\nflavonoids, anthocyanins, (+)-catechin, tannins, \n\nepigallactocatechins, resveratrol, rutin, myricetin [8,9].  \n\nVarious free radicals are generated by oxidative stress \n\nand their accumulation in the cells causes oxidative \n\ndamage and degeneration leading to various \n\ncomplications like premature aging, cataract, heart \n\ndisease, and neurodegenerative disorders [10]. The \n\ncompounds such as phenolics, antioxidants, and vitamin \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0001-8281-8671\nmailto:varshakoirala@gmail.com\nhttps://orcid.org/0000-0001-7355-2162\nmailto:varshakoirala@gmail.com\n\n\nNepal J Biotechnol. 2020 Dec;  8 (3): 95-101 Koirala & Shrestha \n\n\u00a9NJB, BSN 96 \n\nC are responsible for the prevention as well as damage \n\nrepair caused by the free radicals. Fruits are rich in these \n\ncompounds, therefore, their consumption can help with \n\nbone formation, collagen, anti-inflammatory, anti-\n\ntumors/cancerous functions, etc. [11].  \n\nThis research is focused on the comparative analysis of \n\ndifferent types of pear and can help people to understand \n\nthe nutritional values of pears. The data generated from \n\nthis research could be useful to compare different \n\ncharacteristics of pear available in different origins. This \n\nresearch provides information about the phytochemicals, \n\nnutritional values, and sugar concentration in pears \n\ngrown in Nepal. The nutritional profiling of pears could \n\nbe helpful for its product commercialization in the \n\ninternational market.  \n\nMaterials and Methods \nCollection of samples \nSix varieties of fully ripen pears (Bartlette, Chinese, \n\nChojuro, Kosui, Pharping local, and Yakumo) were \n\ncollected from the Warm Temperate Horticulture Centre, \n\nKirtipur, Nepal in August, 2019. \n\nPhysicochemical analysis \n pH, TSS, acidity, clarity, moisture, and ash content were \n\ndetermined. pH was determined using a pH meter. TSS \n\nwas measured using a Brix refractometer. Clarity was \n\ndetermined by taking the absorbance of pear juice at 660 \n\nnm using a spectrophotometer [11,12,13]. \n\nMoisture content \nFor moisture content, the sample was ground and 10 gm \n\nof the sample was kept on the petri dish. The initial \n\nweight of the petri dish was noted.", "start_char_idx": 3221, "end_char_idx": 6878, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a050a96e-fe66-4674-88d6-9f8f071478bb": {"__data__": {"id_": "a050a96e-fe66-4674-88d6-9f8f071478bb", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2cfa2ccc-ec20-49ca-9490-82ddcab7f3c5", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "58bc48560d481bcf9dfe98148bdaf2c1683a07b108a51d62080d43450a588c1d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5826fc9c-d871-4d59-a988-c9fe10b64e7b", "node_type": "1", "metadata": {}, "hash": "c9c608bce3a110d7933614e90c594146de64a965ec83213b74bdd780664c9c29", "class_name": "RelatedNodeInfo"}}, "text": "The initial \n\nweight of the petri dish was noted. The plates were \n\nfurther placed in the oven at 105\u00b0F and the decrease in \n\nweight was noted every hour till it decreases to \u00b15 mg. \n\nBefore weighing, the plate was kept on a desiccator [12]. \n\nThe moisture content was calculated as: \n\nMoisture content = Initial weight - Final weight \u00d7100 % \n\n  Initial weight \n\nAsh content \nSilica crucible was washed and dried in a hot air oven at \n\n150 degrees for 30 minutes. The samples were ground \n\nand 10 gram was weighed. The sample was burned over \n\na low flame furnace and was transferred to the \n\ntemperature-controlled muffle furnace using long tongs. \n\nThe temperature of muffle furnace was kept at 500 \n\ndegrees and was left for 3-4 hrs to cool. The crucible was \n\nleft to cool and was weighed [12]. The ash content was \n\ncalculated as: \n\nTotal Ash  =     weight of ash    \u00d7 100 % \n weight of sample \n\nWhere, the weight of ash = Wt. of  the crucible with ash - \n\nwt. of  the crucible \n\nVitamin C content \n\nThe dye solution (mixture of sodium salt of 2, 6-\n\ndichlorophenol-indophenol dye and sodium \n\nbicarbonate) was standardized by titrating ascorbic acid \n\nwith the dye solution until the appearance of pink color \n\npersists for 10-15 seconds. The dye factor i.e. mg of \n\nascorbic acid per ml of dye was calculated using the \n\nfollowing formula: \n\n    Dye Factor = 0.25\u00d710/Titre \n\nFor titration, 10-20 ml sample was titrated against dye till \n\nthe appearance of pink color persists for 15 seconds. The \n\nreading in the burette was noted. \n\n Ascorbic acid (%)= \n\nTitre\u00d7Dye factor\u00d7Volume made up \u00d7100 \n\n  Weight of sample \u00d7 Volume of the sample taken \n\nMetaphosphoric acid (HPO\u2083) acetic acid solution (3%) \n\nwas prepared by dissolving 15 g metaphosphoric acid in \n\n450 ml water, and 40 ml glacial acetic acid. The standard \n\nfor Ascorbic acid was prepared by dissolving 0.05 gm of \n\nL-ascorbic to 250 ml with the metaphosphoric acetic acid \n\nsolution.  \n\nFor the preparation of dye solution, 0.05 g of the sodium \n\nsalt of 2, 6-dichlorophenol-indophenol dye, and 0.04 g \n\nsodium bicarbonate were dissolved in 200 ml water. It \n\nwas then filtered and stored in a dark-colored bottle at a \n\nrefrigerated condition [13]. \n\nQualitative tests for sugars/carbohydrates in \npear juice \nThe various tests for sugars (Molisch\u2019s test, Benedict\u2019s \n\ntest, Barfoed\u2019s test, Seliwanoff test, Fehling\u2019s test, Bial\u2019s \n\ntest, and Iodine test), protein test and phytochemicals \n\n(Catechins, Flavonoids, Cyclic glycosides, Terpenoids) \n\nwere performed for pear juices. \n\nMolisch\u2019s test \nInitially, 2 ml of the sample was taken, and 2-3 drops of \n\nMolisch\u2019s reagent was added. After some time, purple \n\nring formation was observed which gives a positive test \n\nindicating the presence of all types of sugars like \n\nmonosaccharides, disaccharides, and polysaccharides \n\n[12]. \n\nBenedict\u2019s test \nOne ml of the sample was taken initially, and 2-3 drops \n\nof benedict\u2019s reagents were added and placed in a water \n\nbath and boiled. After some time, the color of the \n\nprecipitate was observed; the presence of red precipitate \n\n\n\nNepal J Biotechnol. 2020 Dec;  8 (3): 95-101 Koirala & Shrestha \n\n\u00a9NJB, BSN 97 \n\ngives a positive test for Benedict\u2019s test. It indicates a \n\npositive test for reducing sugars like glucose [12]. \n\nBarfoed\u2019s test \nInitially, 1 ml of the sample was taken, and 2 ml of \n\nBarfoed\u2019s reagent was added. It was left to boil in the \n\nwater bath and the color change was observed.", "start_char_idx": 6829, "end_char_idx": 10302, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5826fc9c-d871-4d59-a988-c9fe10b64e7b": {"__data__": {"id_": "5826fc9c-d871-4d59-a988-c9fe10b64e7b", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a050a96e-fe66-4674-88d6-9f8f071478bb", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "30100b89d1b870f3f30ed1e94eacf127203f76f4b8860318dad92638cf22f0cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e2e920cf-99e8-4ac7-a7d8-7cf819b71269", "node_type": "1", "metadata": {}, "hash": "c0b90f7604281bc69d55e971d50f3ef5a549a280fa16f58c49ed4b50299dadc1", "class_name": "RelatedNodeInfo"}}, "text": "It was left to boil in the \n\nwater bath and the color change was observed. Brick red \n\ncolor appearance indicates the positive test for reducing \n\nsugars [12]. \n\nSeliwanoff test \nFirst of all, in 1 ml of the test solution, 2 ml of Seliwanoff \n\nreagent was mixed and kept in a water bath for 1 minute. \n\nThe appearance of deep color gives a positive test for \n\nKeto sugars i.e. fructose and sucrose [14]. \n\nFehling\u2019s test \nFirst of all, 1 ml of the sample was taken, and 1 ml of \n\nFehling\u2019s reagent was added. It was left in a boiling water \n\nbath. The precipitation of red color is an indication of a \n\npositive test for sugars like glucose and fructose [14]. \n\nBial\u2019s test \nBial\u2019s reagent was added initially to the test sample. It \n\nwas then kept in boiling water for some time. The \n\nappearance of blue-green color indicates positive for \n\nRibose sugars [12]. \n\nIodine test \nThis test was carried out by taking 1 ml of the sample in \n\nwhich 4-5 drops of iodine were added. Blue color \n\nindicates a positive test for complex sugars like Starch in \n\na sample [12]. \n\nPhytochemicals in pear juice \nFlavonoids \nOne ml test sample was tested with Mg metal and 5-6 \n\ndrops of conc. HCl. The change in color was observed. \n\nRed color denotes flavonoids, Orange stands for \n\nflavones, and Violet indicates  flavonones [14]. \n\nTerpenoids \nIn the mixture of 2 ml chloroform and 3 ml conc. Sulfuric \n\nacid, the sample was added and heated for 2 minutes. A \n\ngrayish-reddish brown color was observed which \n\nindicates a positive test for terpenoids [14]. \n\nCyclic glycosides \nThe sample was mixed with 2 ml of chloroform. Sulfuric \n\nacid was added and shake gently. The brown ring on the \n\ninterface indicates the presence of cyclic glycosides [14]. \n\nProtein test \nThe sample was boiled with 2 ml of  0.25 % w/v of \n\nninhydrin solution. The presence of violet-blue color is \n\nthe positive test for protein [14]. \n\nCatechin test \nThe sample was mixed with the FeCl\u2083 solution. The olive \n\ngreen color gives the test positive for the Catechins [14]. \n\nTotal Phenolic Content (TPC) \nThe aliquots of 1 ml gallic acid of various concentrations \n\nin methanol were added. Then, 5 ml of 10 %  Folin- \n\nCiocalteu reagent and 4 ml of 7% Na\u2082CO\u2083 were mixed up \n\nmaking the final volume 10 ml. The absorbance was \n\nmeasured at 760 nm against a blank (FC reagent+ \n\nNa\u2082CO\u2083) and the graph of the standard was plotted using \n\nthe data of absorbance versus concentration (\u00b5g/ml) [13]. \n\nFor determining total phenolic content in sample, 20 \u00b5l \n\njuice sample was taken and the further procedure was \n\ncarried out as for the calibration solutions. The \n\nabsorbance was taken and the levels of phenolic content \n\nwere determined using the standard graph as Gallic Acid \n\nEquivalents (GAE) [16]. \n\nAntioxidant Content (AOC) \nThe stock solutions of the sample were prepared by \n\ndiluting 5 ml sample(pear juice) and 10 ml of 13.5% \n\nethanol. Diluted samples of pear juice i.e. 50, 100, 150, 200 \n\nand 250 \u03bcl were mixed with DPPH  maintaining final \n\nvolume 3 ml and left for 30 minutes in dark and \n\nabsorbance was measured at 517 nm. The volume of wine \n\nin the diluted solutions needed to decrease the initial \n\nDPPH concentration by 50% together with the amount of \n\nphenol in mg/l was calculated. The results were used to \n\nobtain the IC\n50\n\n values in mg of phenol/l.", "start_char_idx": 10228, "end_char_idx": 13569, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e2e920cf-99e8-4ac7-a7d8-7cf819b71269": {"__data__": {"id_": "e2e920cf-99e8-4ac7-a7d8-7cf819b71269", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5826fc9c-d871-4d59-a988-c9fe10b64e7b", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "080d6895e02979b26b7de441e13a9f4fb59873343b4c01f1589b22d606f87d56", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "25cd12a3-3ead-4c1d-b9ac-23568fbff230", "node_type": "1", "metadata": {}, "hash": "f8d33531bf0c01bf4cc8a567e9772bd3718d45ed9f85b28ab19bad2bebee9efb", "class_name": "RelatedNodeInfo"}}, "text": "The % inhibition\n\nwas calculated as: \n\n  Inhibition (%) =  \n(Ac - As)     x 100\n\nAc\n\nWhere, Ac = absorbance of the control (100 \u03bcl of MeOH\n\ninstead of the sample)    \n\nAs  =  absorbance of the sample\n\nThe percent inhibition was plotted against volumes of \n\nwine using Microsoft Excel and the volume needed to \n\ndecrease DPPH concentration by 50% was calculated \n\nfrom the graph. The volume of sample (diluted) that is \n\nrequired for decrement in the initial DPPH concentration \n\nby 50 % together with the amount of phenol in mg/l was \n\nused for attaining the value of IC\n50\n\n in mg of phenol/l [15].\n\nTotal Tannin Content (TTC) \n200 \u03bcl of pear juice, 300 \u03bcl conc. HCl and 100 \u03bcl of distilled \n\nwater were mixed in two different test tubes. The first test \n\ntube was incubated at 100\u00b0C for 30 min, whereas in the \n\nsecond sample, 50 \u03bcl alcohol was added and the \n\nabsorbance of both samples was taken at 470 nm. The \n\nabsorbance of the two samples was differentiated and \n\nrepresented as \u0394A520 [17].  \n\n\n\nKoirala & Shrestha \n\n\u00a9NJB, BSN 98 \n\n\u0394A520 = 1.1 \u00d7 \u0394A470 and  \n\n\u0394A520 = 1.54 \u00d7 \u0394A470 \n\nThe lowest \u0394A520  value was chosen for the estimation of \n\ntotal tannin content and was represented as g/l  of juice. \n\nIt is calculated as [17]: \n\n  TTC = 15.7 \u00d7 lowest \u0394A520 \n\nTotal Anthocyanin Content (TAC) \n50 \u03bcl pear juice, 50 \u03bcl HCl in ethanol (0.1%), and 100 \u03bcl \n\naqueous HCl (20%) were mixed in two different test \n\ntubes. To the first test tube, 220 \u03bcl of distilled water was \n\nadded and the same amount of sodium bisulfite (26%) \n\nwas added to the second test-tube. Then the Absorbance \n\nwas measured at 520 nm against a blank (50 \u03bcl HCl in \n\nethanol (0.1%), 100 \u03bcl aqueous HCl (20%), and 270 \u03bcl \n\ndistilled water). The difference was calculated and \n\nrepresented as \u0394A520 [17]. The TAC as mg/l of juice was \n\ncalculated as: \n\n TAC = 875 \u00d7 \u0394A520 \n\nData analysis \nThe tests were performed on triplicates (n=3) and the \n\nresults for quantitative tests were reported as mean\u00b1 \n\nstandard deviation (S.D.). The  level of significance \n\nbetween various parameters were determined using one \n\nway ANOVA in Microsoft Excel 2013 and the data \n\npresented were found to be statistically significant (p < \n\n0.05). \n\nResults \n\nPhysicochemical parameters of pears   \nTotal Acidity and pH were observed to be the highest in \n\nChojuro pears i.e. 2.01\u00b10.01 % and 5.23\u00b10.01, respectively. \n\nLikewise, Clarity was observed to be the highest in \n\nBartlette pears i.e. 1.960\u00b10.00. TSS (\u00b0Bx) was observed to \n\nbe highest in Chinese pears i.e. 11\u00b10.00\u00b0Bx. Likewise, \n\nmoisture content were observed to be highest in Chojuro \n\npears i.e. 17.42\u00b10.01 % and ash content in Kosui and \n\nYakumo pears i.e. 1.5\u00b10.04 and 1.5\u00b10.02 % respectively \n\n(Table 1). \n\nTests for sugars/ carbohydrates \nThe qualitative tests were performed for the pear juices. \n\nAll the varieties gave positive tests for all sugars except \n\nthe Iodine test which indicated absence of starch in pears \n\n(Table 2).  \n\nPhytochemicals Screening \n\nAll the pears gave positive tests for the phytochemicals \n\n(Table 3). The highest concentration of  Flavonoids, \n\nCatechins, and Cyclic glycosides were observed in \n\nBartlette and Pharping local pears. Likewise, Terpenoids \n\nwere found to be highest in Pharping local pears only. \n\nNepal J Biotechnol.", "start_char_idx": 13570, "end_char_idx": 16856, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "25cd12a3-3ead-4c1d-b9ac-23568fbff230": {"__data__": {"id_": "25cd12a3-3ead-4c1d-b9ac-23568fbff230", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e2e920cf-99e8-4ac7-a7d8-7cf819b71269", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "2c0e4cac7d814e83a331788668565005a3a2b9b2742d58150ce394ea6ed9f73c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "87fc9b47-38bc-4006-a66b-4ddc70e4d927", "node_type": "1", "metadata": {}, "hash": "05d36c2c895ba135cb5d0afa4f9a394ae0575313abb2395be9179401a1b83c50", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2020 Dec; 8 (3): 9 5 -1 0 1\n\nTable 1: Physicochemical parameters of pears \n\nS.N. Name of  sample Physicochemical parameters \n\nAcidity (%) pH Clarity \u00b0Bx \nMoisture Content \n\n(%) \nAsh content (%) \n\n1. Bartlette 0.134\u00b10.01 4.13\u00b10.01 1.960\u00b10.00 10\u00b10.00 16.68\u00b10.00 0.6\u00b10.01 \n\n2. Chinese 0.67\u00b10.02 4.77\u00b10.01 2.058\u00b10.00 11\u00b10.00 16.97\u00b10.02 0.21\u00b10.00 \n\n3. Chojuro 2.01\u00b10.01 5.23\u00b10.01 2.159\u00b10.00 8\u00b10.00 17.42\u00b10.01 0.25\u00b10.01 \n\n4. Kosui 0.73\u00b10.01 5.06\u00b10.03 2.303\u00b10.01 8\u00b10.00 16.32\u00b10.02 1.5\u00b10.04 \n\n5. Pharping local 0.87\u00b10.01 4.38\u00b10.01 2.454\u00b10.00 7\u00b10.00 15.85\u00b10.00 1.3\u00b10.01 \n\n6. Yakumo 0.67\u00b10.01 5.05\u00b10.00 2.301\u00b10.02 9\u00b10.00 14.84\u00b10.015 1.5\u00b10.02 \n\nAll the values (n=3) were expressed as mean \u00b1 standard deviation and found to be statistically significant (p < 0.05) \n\nTable 2: Various sugar tests in Pear varieties \n\nS.N. Name of sample \nMolisch\u2019s \n\nTest \n\nIodine \n\ntest \n\nBenedict\u2019s \n\ntest \n\nBarfoed \n\ntest \n\nBial\u2019s \n\ntest \n\nSeliwanoff \n\nTest \n\nFehling\u2019s \n\ntest \n\n1. Bartlette +++ - + ++ + ++ + \n\n2. Chinese +++ - + ++ + +++ + \n\n3 Chojuro +++ - ++ +++ + +++ ++ \n\n4. Kosui +++ - ++ +++ + +++ ++ \n\n5. Pharping local +++ - ++ +++ + ++ ++ \n\n6. Yakumo +++ - + ++ + ++ + \n\nNote: (-) denote absence, (+) denote trace,  (++)  denote moderate and  (+++) denote high amount of sugars . The comparison \nwere done on the basis of colour intensities \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 9 5 -1 0 1 Koirala & Shrestha \n\n\u00a9NJB, BSN 99 \n\nDetermination of Tannins, Anthocyanins, \nTotal Phenolic Content(TPC), Antioxidant \nContent(AOC) and Vitamin C in pear juice \nTannins were observed to be highest in Yakumo pears i.e. \n\n0.93\u00b10.01 g/l, and Anthocyanins, Total Phenolic Content, \n\nAntioxidants, and Vitamin C were observed to be highest \n\nin Pharping local pears i.e. 85.95\u00b10.1 mg/l, 600\u00b10.01 mg \n\nGAE/l, IC\n50\n\n value 250\u00b10.00 mg of phenol/l and 12.2\u00b10.01\n\nmg/100 ml respectively (Table 4). \n\n Discussion \nTable 1 focuses on the physicochemical parameters like \n\npH, TSS, clarity, moisture, and ash content. The highest \n\nclarity was found in Bartlette pears i.e. 1.960\u00b10.00, as it \n\nshowed the lowest absorbance (clarity is inversely \n\nproportional to absorbance). On similar research \n\nconducted on two different pears i.e.", "start_char_idx": 16837, "end_char_idx": 19057, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "87fc9b47-38bc-4006-a66b-4ddc70e4d927": {"__data__": {"id_": "87fc9b47-38bc-4006-a66b-4ddc70e4d927", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "25cd12a3-3ead-4c1d-b9ac-23568fbff230", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "2831a553e8c248a05730f3823911fb348c102ea6044a7bf1207dd249834a3fb9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ef601bfc-6a46-4388-b828-9a8f139562aa", "node_type": "1", "metadata": {}, "hash": "4c366cfde6db1829049e078b1ab3fd2c9dd5fc6a5b3d060ad79ffd812616b3fa", "class_name": "RelatedNodeInfo"}}, "text": "On similar research \n\nconducted on two different pears i.e. Shughri and \n\nPhyshun pears, Shugri pears had TSS 13.58\u00b0Bx, 83.1% \n\nmoisture, 3.94% ash, and  13.71\u00b0Bx, and 54.51% moisture, \n\n1.86% ash respectively [18]. Chinese pears were found to \n\nhave highest sucrose (i.e.11\u00b10.00\u00b0Bx) among the 6 \n\nvarieties. Likewise, the moisture was observed to be the \n\nhighest in Chojuro pears i.e. 17.42\u00b10.01% and ash content \n\nin Kosui and Yakumo pears i.e. 1.5\u00b10.04% and 1.5\u00b10.02% \n\nrespectively. Shughri and Physhun pears had higher \n\nmoisture content, Ash content, and TSS than the pears in \n\nthis research. Bartlette pears in earlier research was found \n\nto have the acidity 3.50-4.60% while the Bartlette pears in \n\nthis research had the acidity of 0.134\u00b10.01%. Different \n\nacidity for the same variety of pear could be because of \n\nthe different climatic conditions, storage temperature, \n\nand other environmental parameters [19].   \n\nTable 2 shows the presence of sugars in different pear \n\njuice. All the pears gave positive tests for Molisch\u2019s test, \n\nBenedict\u2019s test, Barfoed test, Bial\u2019s test, Seliwanoff test, \n\nand Fehling\u2019s test but negative for Iodine test. This \n\nindicates the presence of various sugars like glucose, \n\nfructose, and, sucrose and the absence of complex sugars \n\nlike starch in the pears varieties. Sugar acts as a flavor \n\nenhancer, making pear sweet increase the taste, texture, \n\ncolor, and aroma. They also act as food preservatives. All \n\nthe pears gave positive test for Molisch\u2019s test which \n\nindicates the presence of  various sugars in high amount. \n\nPositive test for Benedict\u2019s test and Barfoed test indicate \n\npresence of simple sugars like glucose, fructose, \n\ngalactose, etc. [11]. Those sugars were found high in \n\nChojuro, Kosui and Pharping local pears. Positive Bial\u2019s \n\ntest indicates presence of ribose sugars. The positive \n\nSeliwanoff test indicates presence of sugars like sucrose. \n\nSucrose was found to be high in Chinese, Chojuro and \n\nKosui pears. Positive fehling\u2019s test indicates presence of \n\nreducing sugars like glucose, fructose, lactose, etc. [14]. \n\nThey were present in high amount in Chojuro, Kosui and \n\nPharping pears. Likewise, a study was done for the \n\nanalysis of various sugars in two different pear cultivars \n\n(Pyrus communis) [18,20]. Fructose was found to be in \n\nhighest concentration, followed by other sugars like \n\nglucose, sucrose, fructose, and sorbitol. \n\nTable 3 highlights the presence and absence of different \n\nphytochemicals in pear juice. The pears gave positive \n\ntests for phytochemicals like flavonoids, terpenoids, \n\ncatechins, cyclic glycosides, and proteins. Another \n\nresearch conducted on pear [21] showed a high amount \n\nof catechins and flavonoids present in pears. Likewise, in \n\nTable 3: Qualitative analysis of Phytochemicals \n\nS.N. Name of sample Flavonoids Terpenoids Catechins Cyclic glycosides Proteins \n\n1. Bartlette ++ + ++ ++ + \n\n2. Chinese + + + + + \n\n3 Chojuro + + + + + \n\n4. Kosui + + + + + \n\n5. Pharping local ++ ++ ++ ++ + \n\n6.", "start_char_idx": 18998, "end_char_idx": 22034, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ef601bfc-6a46-4388-b828-9a8f139562aa": {"__data__": {"id_": "ef601bfc-6a46-4388-b828-9a8f139562aa", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "87fc9b47-38bc-4006-a66b-4ddc70e4d927", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "55f0df46cf22857e62aec491270c364401c93793c0da5a69de9c2bf0792e747c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "730adb2f-6875-43d4-bf26-f323d7f2b7ac", "node_type": "1", "metadata": {}, "hash": "55182de02edf80a1c62a765e4462b9fc38f27fb8e146f2f59d56af57ea1349eb", "class_name": "RelatedNodeInfo"}}, "text": "Pharping local ++ ++ ++ ++ + \n\n6. Yakumo + + + + + \n\nNote: (+) denote trace,  (++)  denote moderate and  (+++) denote high amount and (\u2013) indicates the absence of phytochemicals \n\nTable 4: Tannins, Anthocyanins, Total Phenolic Content (TPC), Antioxidant Content (AOC), and Vitamin C in Pears \n\nS.N. Name of pears Tannins \n (g/l) \n\nAnthocyanins \n(mg/l) \n\nTPC (mg \n\nGAE/l) \nAOC \n(IC\n\n50= \nmg of phenol/l)\n\nVit. C \n(mg/100 ml) \n\n1. Bartlette 0.05\u00b10.01 45.55\u00b10.01 501.1\u00b10.05 299.40\u00b10.05 6.57\u00b10.05 \n\n2. Chinese 0.91\u00b10.00 55.65\u00b10.05 273.5\u00b10.2 549.45\u00b10.01 6.94\u00b10.00 \n3. Chojuro 0.06\u00b10.02 30.63\u00b10.00 399.5\u00b10.28 375.93\u00b10.00 5.55\u00b10.02 \n4. Kosui 0.02\u00b10.00 40.85\u00b10.00 304.9\u00b10.01 501.68\u00b10.01 2.3\u00b10.05 \n5. Pharping local 0.04\u00b10.00 85.95\u00b10.1 600\u00b10.01 250\u00b10.00 12.2\u00b10.01 \n6. Yakumo 0.93\u00b10.01 68.75\u00b10.02 301.8\u00b10.05 509.2\u00b10.01 2.2\u00b10.01 \n\nAll Al the values (n=3) were expressed as mean \u00b1 standard deviation and found to be statistically significant (p < 0.05). \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 9 5 -1 0 1 Koirala & Shrestha \n\n\u00a9NJB, BSN 100 \n\nsimilar research [22], various phytochemicals like \n\ncatechins were found. \n\nIn Table 4, the amount of tannins, anthocyanins, total \n\nphenolic content, antioxidants, and vitamin C in pear \n\nvarieties are presented. Tannins were observed to be the \n\nhighest in Yakumo pears i.e. 0.93\u00b10.01 g/l; anthocyanins, \n\ntotal phenolic content, antioxidants, and vitamin C were \n\nobserved to be the highest in Pharping local pears i.e. \n\n85.95\u00b10.1 mg/l, 600\u00b10.01 mg GAE/l, IC\n50\n\n value 250\u00b10.00\n\nmg of phenol/l, and 12.2\u00b10.01 mg/100 ml respectively. \n\nCompared to a previous similar research [23],  where  the \n\nlevel of tannin was observed to be 1.6 g/l, this study, \n\nfound the level of tannin in Yakumo pear equal to \n\n0.93\u00b10.01 g/l [24]. Anthocyanins are responsible for the \n\nred coloration in pear fruits and its development depends \n\non heat and light. The anthocyanin level was found to be \n\n89.5 mg/l in pears which is higher compared to pear in \n\nthis research i.e. 85.95\u00b10.1 mg/l. Given that, the \n\nanthocyanin level is higher in high temperature, those \n\npears might have grown in high temperatures as \n\ncompared to the pears cultivated in Nepal [25]. \n\nVarieties of Oriental pear and Occidental pear had total \n\nphenols 78.5-83.9 mg GAE/l and high antioxidant \n\nactivities. Jules d\u2019Airolles and Abate Fetal pears showed \n\nthe lowest DPPH scavenging capacity; and Cheongbae, \n\nNiitaka, and Hanareum pears were found to have high \n\ntotal phenolic, flavonoid contents, and higher \n\nantioxidants than other varieties [26,27].", "start_char_idx": 22001, "end_char_idx": 24555, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "730adb2f-6875-43d4-bf26-f323d7f2b7ac": {"__data__": {"id_": "730adb2f-6875-43d4-bf26-f323d7f2b7ac", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ef601bfc-6a46-4388-b828-9a8f139562aa", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "8c5335e94e1157fb4b64861b5719381384f42edae3c5cc27370cb4a70ddc5fab", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a454f435-51be-427e-8226-27d1191eda74", "node_type": "1", "metadata": {}, "hash": "f42b42aa8e98a497f6ed08ed8fe947fdfbf33ec66addc5a0094155b6b068ee59", "class_name": "RelatedNodeInfo"}}, "text": "The highest \n\nphenolic content was observed in Pharping local pears \n\ni.e. 600\u00b10.01 mg GAE/l which is higher compared to \n\nOriental and Occidental pears. The amount of the \n\nphenolic compounds present is based on fruit source and \n\nenvironmental factors as well [26]. It also acts as a \n\nprimary antioxidant or free radical terminators and are \n\neffective hydrogen donors [26]. The lower IC\n50\n\n value\n\nindicates greater antioxidant activity because the value \n\nindicates the level of antioxidants essential for the \n\nreduction of free radical i.e. DPPH by 50% of initial \n\nconcentration. The vitamin C content was observed to be \n\n12.2\u00b10.01 mg/100 ml in Pharping local pears while in \n\nsimilar research conducted  [27], it was found in the range \n\n2.2- 6.57 mg/100 ml which is less than that of this \n\nresearch. This could be because of the difference in the \n\nvarious factors like variety, seasonal variation, \n\nenvironment, climate, and the difference in protocols for \n\nthe determination of vitamin C.  \n\nConclusion \nPharping local pears are found to be the most nutritious \n\nwhen compared to the other five varieties. Pears are the \n\nfruits that are rich in Vitamin C, antioxidants, phenolic \n\ncontents, anthocyanins etc. Along with those \n\ncomponents, various sugars, phytochemicals like \n\ncatechins, flavonoids, terpenoids, glycosides, and little \n\nprotein as well. Given such richnesspears in general and \n\nPharping pears in particular are recommended as rich \n\nsources of vitamins, antioxidants, health-promoting \n\nfactors. \n\nAuthor\u2019s Contribution \nBK performed the experiment in the lab under the \n\nsupervision of AS. BK and AS contributed for original \n\ndraft preparation and during revision. BK and AS \n\ncontributed significantly in editing, revising and \n\nrendering the write-up. All authors have read and \n\napproved the final manuscript. \n\nCompeting Interests \nThe authors have no competing interests regarding the \n\npublication. \n\nFunding \nNo funds have been provided for the research by any \n\nfunding agency.   \n\nAcknowledgments \nI would like to acknowledge my supervisor Ms. Angela \n\nShrestha (Lecturer) for her guidance, support and \n\nsupervision during the entire work. I would also like to \n\nexpress deepest gratitude to Mr. Sudhakar Pant (Head of \n\nDepartment), teachers and staffs of Microbiology \n\ndepartment, family and friends for their help and support \n\nduring this research.  \n\nEthical Approval and Consent \nNot Applicable \n\nReferences \n1. Atreya P. Varieties of Pear. Improved cultivation practice of Pear. 1st \nedition.  Central Horticulture Division, Kathmandu 2019. \n2. Hu W, Sun DW, Pu H, Pan T. Recent developments in methods and\ntechniques for rapid monitoring of sugar metabolism in Fruits. \nComprehensive Reviews in Food Science and Food safety. 2016;15:1067-\n79. https://doi.org/10.1111/1541.4337.12225\n3. Beutel JA. Asian pears. Advances in new crops. Timber Press,\n\nhttps://hort.purdue.edu/newcrop/Portland. 1990;304-309. \nproceedings1990/V1-304.html \n4. Watson M. 10 varieties of pears from Anjou to Williams and their\ntextures, tastes and seasons. The spruce eats. \nhttps://www.thespruceeats.com/pear-varieties-2216839 Accessed \nOctober 10 2019. \n5. Gotame TP, Subedi GD, Dhakal M, Khatiwada PP . Postharvest \nhandling of pears.  Pindeshwori Press. 2015. \n6. Giosanu D, V\u00eejan LE, Deliu I. The analyse of physico-chemical\nparameters means to appreciate the typicity of some red wines. Food \nand Environment Safety. 2011;1(10):31\u201335. \n7. Athirah A. Measurement of pH, total soluble solid and titrable\nacidity.", "start_char_idx": 24556, "end_char_idx": 28105, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a454f435-51be-427e-8226-27d1191eda74": {"__data__": {"id_": "a454f435-51be-427e-8226-27d1191eda74", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "730adb2f-6875-43d4-bf26-f323d7f2b7ac", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "866ff7c7c61e4388a67d63c1169d9672609997b93c0048893bb2a719d034d57e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "34646340-52ba-494b-9cfe-53e5337eaeae", "node_type": "1", "metadata": {}, "hash": "64d335f829deca76750833891b1b52f1a7175a8517c292ad2d3a1060991c38d1", "class_name": "RelatedNodeInfo"}}, "text": "Academia.https://www.academia.edu/6955370/measurement_of_ph\n_titratable_acidity Accessed 25 May 2019. \n\nhttps://hort.purdue.edu/newcrop%20/proceedings1990/V1-304.html\nhttps://hort.purdue.edu/newcrop%20/proceedings1990/V1-304.html\nhttps://www.thespruceeats.com/pear-varieties-2216839%20Accessed%20October%2010%202019\nhttps://www.thespruceeats.com/pear-varieties-2216839%20Accessed%20October%2010%202019\nhttps://www.academia.edu/6955370/measurement_of_ph_titratable_acid\nhttps://www.academia.edu/6955370/measurement_of_ph_titratable_acid\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 9 5 -1 0 1 Koirala & Shrestha \n\n\u00a9NJB, BSN 101 \n\n8. Frankel EL, Waterhouse A, Teissedre PL. Principal phenolic \nphytochemicals in selected California wines and their antioxidant \nactivity in inhibiting oxidation of human low-density lipoproteins. J \nAgric Food Chem; 1995;43:890-894. \n9. Lingua MS,  Fabani MP,  Wunderlin DA, Baroni MV. From grape to\nwine: changes in phenolic composition and its influence on antioxidant \nactivity. Food Chem. 2016;208:228\u2013238. \nhttps://doi.org/10.1016/j.foodchem.2016.04.009 \n10. Padma R, Parvathy NG, Renjith V, Kalpana PR. Quantitative \nestimations of tannins, phenols and antioxidant activity of methanolic \nextract of Imperata cylindrica. Int J Res Pharm Sci. 2013;4(11):73-77.  \n11. Danet AF, Pisoschi AM, Kalinowski S. Ascorbic acid determination\nin commercial fruit juice samples by cyclic voltammetry. Journal of \nAutomated Methods. 2008;1:1-8. \nhttps://doi.org/10.1155/2008/937651. \n12. Shrestha B. Practical Biochemistry and Biotechnology.1st edition,\nSNEMP. Kathmandu, Nepal. 2009;123-125. \n13. Patharkar SR, Kawadkar DK, Khapre AP. Development of Orange \n(Citrus reticulate Blanco) Wine from Mixed Culture Fermentation. Int J \nCurr Microbiol App Sci. 2017;6(8): 3375-83. \nhttps://doi.org/10.20546/ijcmas.2017.607.403. \n14. Bhandari L, Bista BB, Bhatta MR, Khanal C, Khanal S, Ranjitkar R,\nBhandari DP. Phytoconstituents, Antioxidant and Bitterness Value of \nSwertia chirayita from Four Different Geographical Region of Nepal. \nJournal of Plant Resources. 2019;17(1):104-11. https://dpr.gov.np/wp-\ncontent/uploads/2019/08/Journal-2019.pdf. \n15. Lugemwa FN, Snyder AL, Shaikh K. (2013). Determination of \nRadical Scavenging Activity and Total Phenols of Wine and Spices: A \nRandomized Study. Antioxidants. 2013;2: 110-121. \nhttps://doi.org/10.3390/antiox2030110 \n16. Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total \nphenols and other oxidation substrates and antioxidants by means of \nFolin-Ciocalteu Reagent. Methods in Enzymology. 1990;299:152-178. \nhttps://doi.org/10.1016/S0076-6879(99)99017-1 \n17. Pandeya A, Rayamajhi S, Pokhrel P, Giri B. Evaluation of secondary \nmetabolites, antioxidant activity, and color parameters of Nepali wines.", "start_char_idx": 28107, "end_char_idx": 30886, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "34646340-52ba-494b-9cfe-53e5337eaeae": {"__data__": {"id_": "34646340-52ba-494b-9cfe-53e5337eaeae", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a454f435-51be-427e-8226-27d1191eda74", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "2a5ee62dd30c187c2e131841db13d0d7c442909286f964596df11820d42e8a06", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "966d0234-7477-4037-94f8-a8f52c8bfce9", "node_type": "1", "metadata": {}, "hash": "8ddfaa2680bafe8d241df3c093346530dcbf6d2fbccd1e8f510c8a538422731f", "class_name": "RelatedNodeInfo"}}, "text": "Food Science and Nutrition. 2018; 6:2252-64. \nhttps://doi.org/10.1002/fsn3.794 \n18. Hussain S. Comparative Study of Two Pear (Pyrus communis L.) \nCultivars in Terms of Nutritional Composition. Food Science and \nQuality Management. 2015. https://www.semanticscholar.org/ \npaper/Comparative-Study-of-Two-Pear-(Pyrus-communis-L.)-\nHussain/cf1fd30f331397758d764b bb7795 28b11712094b \n19. Brewer L, Volz R. The Pear Genome, Genetic and Breeding of Pear. \nSpringer. 2019;63-73.  https://doi.org/10.1007/978-3-030-11048-2 \n20. Gaifang Y, Shaoling Z, Yufen C, Jun L, Jun W, Jiang Y, Huping Z,\nChangcheng X. Characteristics of components and contents of soluble \nsugars in pear fruits from different species. Agricultura Sinica. \n2010;20:4227-29. \n21. Reiland H, Slavin J. Systematic Review of Pears and Health. \nNutrition today. 2015;6:301-305. https://doi.org/10.1097/ \nNT.0000000000000112 \n22. Babincev LM, Guresic DM, Simonovic RM. Spectrophotometric \ncharacterization of Red wine colour from the vineyard region of \nMetohia. Journal of Agricultural Sciences. 2016; 61(3):281-290. \nhttps://doi.org/10.2298/JAS1603281B \n23. Tarko T, Duda-chodak A, Sroka P. Physicochemical and Antioxidant\nProperties of selected polish grape and fruit wines. Acta Scientific \nPolonorum. 2008; 7(3): 35-45. http: // www. food.actapol.net/ issue3/ \nvolume/4_3_2008.pdf \n24. Abaci TZ, Sevindik E, Ayvaz M. Comparative study of bioactive,\ncomponents in pear genotypes from Aradhan/ Turkey. Biotechnology \nand biotechnological equipment. 2015;30: 36-43. \nhttps://doi.org/10.1080/13102818.2015.1095654 \n25. Doka O, Bicanic D. Determination of Total Polyphenolic Content in\nRed Wines by Means of the Combined He-Ne Laser Optothermal \nWindow and Folin-Ciocalteu Colorimetry Assay. Anal Chem. \n2002;74:2157-61. DOI: 10.1021/ac011001s \n26. Yim SH, Nam SH. Physiochemical, nutritional and functional\ncharacterization of 10 different pear cultivars (Pyrus spp.). Journal of \nApplied Botany and Food Quality. 2016;89:73 - 81. DOI: \n10.5073/JABFQ.2016.089.009 \n\n27. Ozturk A, Demirsoy L, Demirsoy H, Asan A, Gul O. Phenolic\nCompounds and Chemical Characteristics of Pears (Pyrus Communis L.) \nInternational Journal of Food Properties. 2015; 18:536\u2013546.", "start_char_idx": 30888, "end_char_idx": 33103, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "966d0234-7477-4037-94f8-a8f52c8bfce9": {"__data__": {"id_": "966d0234-7477-4037-94f8-a8f52c8bfce9", "embedding": null, "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-120", "node_type": "4", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "e55c127e0d3407e52e725003d8e440683b52372f45bb3d6f57599a1e85269b27", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "34646340-52ba-494b-9cfe-53e5337eaeae", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "0348df308dd415664f2ba6050fc16399c3b8c9800bca79f27680c0d880edfb3e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a7ace83d-1d99-4487-b8b7-e48390714491", "node_type": "1", "metadata": {}, "hash": "d9845da383ee9f377162d56bfa19927e579652fdea27b30734610e226002f1e0", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of Food Properties. 2015; 18:536\u2013546. Doi: \nhttps://doi.org/10.1080/10942912.2013.835821\n\nhttps://doi.org/10.1016/j.foodchem.2016.04.009\nhttps://dx.doi.org/10.1155%2F2008%2F937651\nhttps://doi.org/10.20546/ijcmas.2017.607.403\nhttps://dpr.gov.np/wp-content/uploads/2019/08/Journal-2019.pdf\nhttps://dpr.gov.np/wp-content/uploads/2019/08/Journal-2019.pdf\nhttps://dx.doi.org/10.3390%2Fantiox2030110\nhttps://doi.org/10.1016/S0076-6879(99)99017-1\nhttps://dx.doi.org/10.1002%2Ffsn3.794\nhttps://www.semanticscholar.org/%20paper/Comparative-Study-of-Two-Pear-(Pyrus-communis-L.)-Hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b\nhttps://www.semanticscholar.org/%20paper/Comparative-Study-of-Two-Pear-(Pyrus-communis-L.)-Hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b\nhttps://www.semanticscholar.org/%20paper/Comparative-Study-of-Two-Pear-(Pyrus-communis-L.)-Hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b\nhttps://doi.org/10.1007/978-3-030-11048-2\nhttps://dx.doi.org/10.1097%2FNT.0000000000000112\nhttps://dx.doi.org/10.1097%2FNT.0000000000000112\nhttps://dx.doi.org/10.1097%2FNT.0000000000000112\nhttps://doi.org/10.1021/ac011001s\nhttps://doi.org/10.1080/10942912.2013.835821", "start_char_idx": 33044, "end_char_idx": 34249, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a7ace83d-1d99-4487-b8b7-e48390714491": {"__data__": {"id_": "a7ace83d-1d99-4487-b8b7-e48390714491", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "966d0234-7477-4037-94f8-a8f52c8bfce9", "node_type": "1", "metadata": {"identifier": "njb-120", "author": "Koirala, Barsha; Shrestha, Angela", "title": "Comparative Study of Bioactive Compounds in Different Varieties of Pears in Nepal", "date": "2020-12-30", "file": "njb-120.pdf"}, "hash": "39e10d0bddbf7b6695e9c429acdd1a4949c23afbc3033d79714e341891224d2b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "219d6b16-2e75-4a87-88c8-feeafc401230", "node_type": "1", "metadata": {}, "hash": "adf90becd3aef2146eb9807f8e9aafbff93e149016f7c8b2740be2a3ba1348ad", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 79-84 Research article DOI: https://doi.org/10.3126/njb.v9i1.38672 \nSPECIAL ISSUE: ICBSN 2021 \n\n\u00a9NJB, BSN 79 \n\nScreening of Potential Plant Growth Promoting Properties of Bacillus \nSpecies Isolated from Different Regions of Nepal \nEnish Pathak1, Arjun Sanjyal1, Chhatra Raj Regmi1, Saroj Paudel2, Anima Shrestha1\n\n1 Department of Microbiology, Tri-Chandra Multiple Campus, Kathmandu, Nepal \n2 Nepalese Farming Institute, Kathmandu, Nepal \nReceived: 29 Apr 2021; Revised: 28 Jun 2021; Accepted: 28 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract \nThe deleterious effects of intensive use of chemical fertilizers and pesticides in agriculture has led to the \nsubstantial research efforts on finding the alternatives to these agrochemicals. This study was aimed to isolate \nBacillus species from soil of different regions of Nepal and screen for their ability to promote plant growth directly \nor indirectly by testing their ability to produce plant growth hormone indole acetic acid, hydrogen cyanide, \nammonia and protease as well as phosphate solubilization. Thirty nine Bacillus strains were isolated from 25 soil \nsamples of different regions of Kathmandu and Chitwan districts of Nepal. These isolates were tested for plant \ngrowth promoting traits in vitro. Among the total isolates, about 48.7% were indole acetic acid producers, 38.4% \nof the isolates showed the ability to solubilize the phosphate, 71.8% were able to produce ammonia and all the \nisolates had the ability to produce hydrogen cyanide and protease. The isolated strains showed positive results \nto maximum PGPR traits and exhibited a potential to be used as alternatives to chemical fertilizers and pesticides \nand could be used as low-cost bio-based technology to promote plant growth in the agricultural sector. \n\nKeywords: PGPR, Biocontrol Agents, Plant Growth Promotion, Bacillus, Biofertilizers \n\n Corresponding author, email: animashrestha77@gmail.com \n\nIntroduction \nThe application of chemical fertilizers has long been used \n\nin conventional agriculture. While chemical fertilizers \n\nhave aided farmers in increasing crop production there \n\nare also several harmful effects of chemical fertilizers \n\nwhich may include water pollution, chemical burn to \n\ncrops, increased air pollution, acidification of the soil and \n\nmany other direct and indirect effects to the human \n\nhealth ecosystem itself [1]. So, substantial research efforts \n\nare now focused on finding new alternatives to \n\nsupplement the use of chemicals in agriculture. The use \n\nof beneficial rhizobacteria to increase the productivity \n\nand growth of plants could be one of the substitutes to \n\nagrochemicals. \n\nThe strains of bacteria available in the rhizosphere that \n\nstimulate plant growth are termed as Plant Growth \n\nPromoting Rhizobacteria (PGPR) [2].  The mechanism by \n\nwhich PGPR promotes plant growth can be direct or \n\nindirect [3]. PGPR can promote plant growth directly by \n\nfacilitating resource acquisition i.e., fixation of \n\natmospheric nitrogen, solubilization and mineralization \n\nof soil phosphorus, sequestering iron producing \n\nphytohormone and modulating phytohormones level by \n\nproducing phytohormones like cytokinin, gibberellins, \n\nethylene, indole acetic acid etc. Biocontrol bacteria \n\nproduce antibiotics, siderophores and lytic enzymes \n\nincluding chitinases, cellulases, proteases that cause \n\ndeleterious effects to phytopathogens and indirectly \n\npromote plant growth. Competition between pathogens \n\nand plant growth promoting bacteria  can also check the \n\ndisease incidence and severity [3].  \n\nBacillus species are abundant in the rhizosphere, so they \n\ncan be one of the major aspects of bio-based products to \n\nsupplant agrochemicals.", "start_char_idx": 48, "end_char_idx": 3817, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "219d6b16-2e75-4a87-88c8-feeafc401230": {"__data__": {"id_": "219d6b16-2e75-4a87-88c8-feeafc401230", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a7ace83d-1d99-4487-b8b7-e48390714491", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "caa427b34cbf34fee4448ec8d7142a661f00995ff5ac81a3b2844ab1e3eb1f9b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bc397d15-c37e-42e3-8d3d-cf48002a6688", "node_type": "1", "metadata": {}, "hash": "cf2cbbcbc0a64131b3ddf99c37585aa779ece48c7137eaa658b7ebbe225f959a", "class_name": "RelatedNodeInfo"}}, "text": "Bacillus spp are Gram positive \n\ncommon rhizobacteria and widely considered as a major \n\naspect of plant growth promoting rhizobacteria [4]. The \n\nability to replicate rapidly and resistant to adverse \n\nenvironmental conditions provide a unique feature to \n\nBacillus species [5]. Their ability to produce hard, \n\nresistant endospores and antibiotics that limit wide \n\nranges of phytopathogens make Bacillus spp an attractive \n\noption for biocontrol agents [6]. \n\nVarious researches done worldwide identified Bacillus \n\nspp as PGPR [2,4-6]. But studies regarding PGPR Bacillus \n\nspp in Nepal are limited [7,8]. This study was thus aimed \n\nto isolate Bacillus spp from soil samples of different areas \n\nof Kathmandu and Chitwan of Nepal and screen the \n\nisolates for some direct and indirect plant growth \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-9919-476X\nmailto:animashrestha77@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 9 - 8 4      Pathak et al.  \n\n\u00a9NJB, BSN  80 \n\npromoting traits. This includes the test for the production \n\nof indole acetic acid (IAA), solubilization of phosphate \n\nand production of hydrogen cyanide (HCN), ammonia \n\nand protease. \n\nMethodology \nSample collection and processing \nSoil samples (10 g each) were collected from rhizospheric \n\nsoils of different regions namely Khairahani and Fasera \n\nin the Chitwan District and Hanumate Khola, Jagati in \n\nthe Bhaktapur district of Nepal. The soil samples were \n\ncollected from a depth of 5-10 cm in sterile plastic bags \n\nand carried to the laboratory, Nepalese Farming \n\nIndustry, Kathmandu for further processing and \n\nanalysis. The study was conducted from January to \n\nMarch 2019. \n\nOne gram of soil sample was dispensed into 99 mL of \n\nsterile distilled water and homogenized. One mL of \n\nhomogenized soil sample was transferred into 9 mL \n\nsterile distilled water and serial dilution was carried out \n\nup to 10-8 dilution. Serially diluted bacterial cultures (100 \n\n\u00b5L) were spread on nutrient agar media and incubated at \n\n37\u00b0C for 24 h and examined for the appearance of \n\ncolonies. Identification of the isolated colonies was done \n\non the basis of colony characteristics, Gram reaction, \n\nspore staining and catalase test [5]. Screening of isolates \n\nwas done for plant growth promoting properties \u2013 IAA \n\nproduction, phosphate solubilization activity, HCN \n\nproduction, ammonia production and protease enzyme \n\nproduction.  \n\nIAA production \nIAA production was qualitatively estimated [9). All the \n\nisolates were incubated in nutrient broth containing \n\n5\u00b5g/mL L-tryptophan for 48 h at 28\u00b0C. Following \n\nincubation, culture was centrifuged at 5,000 rpm for 20 \n\nmin and 1 mL of supernatant was mixed with 2 mL of \n\nSalkowski reagent and kept in a dark room for 20 min. \n\nAppearance of pink color indicated the IAA production. \n\nPhosphate solubilization activity \nScreening for phosphate solubilization ability of isolate \n\nwas done using Pikovskaya\u2019s agar medium [10]. The \n\nisolates were spot inoculated on Pikovskaya\u2019s agar plates \n\nand incubated at 28\u00b0C for 48 h. Clear zones around the \n\ncolonies indicated the positive test. The diameter of the \n\nhalo zone was measured. \n\nHCN production \n\nQualitative estimation of HCN was done by using the \n\nmethods described by Lorck (as cited by [11]). Each \n\nisolate was streaked on nutrient agar plate supplemented \n\nwith 4% glycine.", "start_char_idx": 3818, "end_char_idx": 7359, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bc397d15-c37e-42e3-8d3d-cf48002a6688": {"__data__": {"id_": "bc397d15-c37e-42e3-8d3d-cf48002a6688", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "219d6b16-2e75-4a87-88c8-feeafc401230", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "64fe10c593530ff0eb02707330155650ff3804a779a95e9ec34ddd55bbb345f6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "49ccfe39-d623-4a70-9872-fdf835f547a6", "node_type": "1", "metadata": {}, "hash": "a39a4ff0d7445103338e846d9a3049935f5e242a31c9a05857d68b549422def7", "class_name": "RelatedNodeInfo"}}, "text": "A Whatmann filter paper soaked in a \n\nsolution of 2% Na2CO3 in 0.5% picric acid was placed \n\nbetween base and lid of petriplate and incubated at 28 \u00b0C \n\nin inverted position for 48 h and observed for color \n\nchange from yellow to brown. \n\nAmmonia production \nAll the bacterial isolates were tested for the production of \n\nammonia [12]. For ammonia production strain was \n\ninoculated into 5 mL peptone medium and incubated for \n\n48 h at 28 \u00b0C. After the bacterial growth, Nessler\u2019s reagent \n\n(0.5 mL) was added to the tube in 2:1 ratio. Development \n\nof brown to yellow color indicated positive test for \n\nammonia production.  \n\nProtease test \nFor protease production test, isolated Bacillus spp were \n\nspot inoculated on skim milk agar plate and kept for \n\nincubation for 24h at 28 \u00b0C.  Appearance of halo zone \n\naround the colonies was considered as positive for \n\nprotease production [10]. The diameter of the halo zone \n\nwas measured. \n\nResults \nThirty nine isolates of Bacillus were obtained from 25 soil \n\nsamples. The isolates were Gram positive rods (Figure 1), \n\nendospore forming, and catalase enzyme producers. The \n\ncolonies on nutrient agar were rough, creamy white, dry \n\nand folded, opaque and irregular edged.  \n\nFigure 1. Gram stained Bacillus species under oil immersion \n\nobjective  \n\nPlant growth promoting properties \nThe plant growth promoting properties of the isolated \n\nwere evaluated based on the ability to produce IAA, \n\nsolubilization of phosphate, ability to produce HCN, \n\nammonia and protease.  \n\nIAA production \nOut of 39 isolates of Bacillus, 19 isolates (48.7%) showed \n\nthe ability to produce IAA. Development of pink color \n\nafter 20 min of addition of 2 drops of Salkowski reagent", "start_char_idx": 7361, "end_char_idx": 9078, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "49ccfe39-d623-4a70-9872-fdf835f547a6": {"__data__": {"id_": "49ccfe39-d623-4a70-9872-fdf835f547a6", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bc397d15-c37e-42e3-8d3d-cf48002a6688", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "efa527e0fecff90c5c4a5203d168dc3ea217c0ba9409b1edf8374bc8ceedcf08", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5c1b54e6-012b-454f-885b-7fde92f78974", "node_type": "1", "metadata": {}, "hash": "123f9f3a6f7431c414dc3ae827a60cbfb84f24ea30be6bbceb0c70c25769f172", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 9 - 8 4      Pathak et al.  \n\n\u00a9NJB, BSN  81 \n\nFigure 2. Appearance of pink color after the addition of \n\nSalkowski reagent in IAA test \n\nin 2mL of the cell free supernatant indicated the positive \n\ntest for IAA (Figure 2). The IAA producing strains were \n\nfurther classified into three groups as +++ (strong), ++  \n\n (moderate) and + (weak) based on the intensity of color \n\nvisible. The strains showing deep pink, pink and light \n\npink were placed in the group +++, ++ and + \n\nrespectively. Four isolates showed deep pink color after \n\n20 min of addition of Salkowski reagent into the cell free \n\nsupernatant liquid whereas 7 isolates showed pink color \n\nand 8 showed light pink color (Table 1).  \n\nPhosphate solubilization  \nThe isolates showing a halo zone around the colonies \n\nafter 48 h of incubation following spot inoculation in \n\nPikovskaya\u2019s agar plate were taken as positive tests for \n\nPhosphate solubilization (Figure 3). Out of 39 isolates, 15 \n\n(38.4%) isolates exhibited the halo zone. The width of the \n\nhalo zone was also measured (Table 1). The width of the \n\nTable 1. Properties of 39 isolates tested for different plant growth promoting traits. \n\nBacillus \nStrain (BS) \n\nIAA \nProduction \n\nP-Solubilization \nWidth of halo zone (mm) \n\nHCN Production \nAmmonia \nProduction \n\nProtease Production Width \nof halo zone (mm) \n\nBS00 - - + + 1 \nBS01 ++ - + ++ 2 \nBS02 - - + + 3 \nBS03 + - + - 1 \nBS04 + - + + 2.5 \nBS05 +++ 1.5 +++ - 1 \nBS08 + 1 ++ - 1.5 \nBS09 + 1 + +++ 1 \nBS10 + 1.5 +++ + 1 \nBS12 - - + +++ 1 \nBS13 ++ 1 +++ ++ 2.5 \nBS14 - - ++ + 1.5 \nBS15 +++ 2 + - 3 \nBS16 + 1.5 + ++ 2.5 \nBS17 - 1 + ++ 3 \nBS18 - - + - 1 \nBS19 - - ++ + 3 \nBS20 - 1 + +++ 1 \nBS21 + - ++ - 1 \nBS22 - - + ++ 2 \nBS23 - - + + 1 \nBS24 - 3 + - 1.5 \nBS25 +++ - +++ + 3 \nBS26 ++ - ++ + 2 \nBS27 - 1.5 + - 2.5 \nBS28 - - + ++ 2 \nBS29 ++ 3 +++ +++ 2 \nBS30 +++ - +++ - 1 \nBS31 - - +++ ++ 2 \nBS32 - - + +++ 1 \nBS33 - - +++ + 1 \nBS34 + 1.5 + - 1.5 \nBS35 - 1 + + 1.5 \nBS36 - - + ++ 1 \nBS37 ++ 0.5 + + 1.5 \nBS38 - - +++ - 1.5 \nBS39 ++ - ++ + 1 \nBS40 ++ - + + 1 \nBS41 - - +++ +++ 1", "start_char_idx": 9084, "end_char_idx": 11185, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5c1b54e6-012b-454f-885b-7fde92f78974": {"__data__": {"id_": "5c1b54e6-012b-454f-885b-7fde92f78974", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "49ccfe39-d623-4a70-9872-fdf835f547a6", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "d11b78c8d6b8e7f2957204b3314d0a09b13d5c1bc146b58f08d3c23c02132bec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b356ab1c-d8c2-4a2f-b6ec-f5b449b271c0", "node_type": "1", "metadata": {}, "hash": "457901370c0d1010eb84f27a57710836f16dbf4c927c972c5cfe9f9cf1d27a14", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 9 - 8 4      Pathak et al.  \n\n\u00a9NJB, BSN  82 \n\nhalo zone was as high as 3mm (BS24 and BS29) to most of \n\nthe Bacillus spp showing the clear zone of 1mm diameter. \n\n \nFigure 3. Appearance of halo zone around colony in phosphate \n\nsolubilization test \n\nHCN production \nAll 39 isolates of Bacillus produced HCN as evidenced by \n\nthe change in color of the Whatmann filter paper from \n\nyellow to brown. In the presence of glycine, the brown \n\ncolor of filter paper was observed giving a clear \n\nindication of HCN production by Bacillus strains (Figure \n\n4). However, different strains produced the different \n\nintensity of brown color as light brown, orange brown to \n\nreddish brown. Based on the distinction in the color of \n\nfilter paper the strains are grouped into +++ (strong), ++ \n\n(moderate) and + (weak) for those producing reddish \n\nbrown, orange brown and light brown respectively \n\n(Table 1). Ten strains changed the color of filter paper to \n\nreddish brown, 6 changed the color to orange brown \n\nwhile 23 strains changed the color to only light brown. \n\n \nFigure 4. Change is color of filter paper to reddish brown \n\nfollowing incubation in HCN production test \n\nAmmonia production \nFor ammonia production, the development of brown to \n\nyellow color after the addition of 0.5 mL of Nessler\u2019s \n\nreagent was observed as a positive test (Figure 5). Among \n\n39 isolates, 28 isolates developed the color of the medium \n\nto brown or yellow following the addition of Nessler\u2019s \n\nreagent. However, different strains produced different \n\nintensity of color as yellow, light brown and deep brown. \n\nBased on the distinction in the color of media the strains \n\nare grouped into +++ (strong), ++ (moderate) and + \n\n(weak) for those producing deep brown, light brown and \n\nyellow respectively (Table 1). Six strains changed the \n\ncolor of media to deep brown, 8 changed the color to light \n\nbrown while 14 strains changed the color to yellow. \n\n \n\n \nFigure 5. Change in color of peptone water in ammonia test \n\nFigure 6. Appearance of halo zone in protease test \n\nProtease production \nThe isolates showing the halo zone around the colonies \n\nafter 28h of incubation following spot inoculation on \n\nskim milk agar were taken as positive tests for protease \n\nproduction (Figure 6). From the 39 isolates taken for \n\nexperiment, all the isolates produced the halo zone. The \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 9 - 8 4      Pathak et al.  \n\n\u00a9NJB, BSN  83 \n\nwidth of the halo zone was also measured (Table 1). The \n\nwidth of the halo zone was as high as 3 mm (5 isolates) to \n\nmost of the Bacillus spp showing the clear zone of 1mm \n\ndiameter. \n\nDiscussion \nCertain strains of Bacillus spp have gained worldwide \n\nattention in recent years due to their abilities in \n\npromoting plant growth. Therefore, in this study, isolates \n\nof Bacillus spp. obtained from soil of different regions of \n\nKathmandu valley and Chitwan district were primarily \n\ntested for the plant growth promoting traits in vitro.  \n\nThe capability to increase plant growth parameters is \n\nhighly related to the IAA level, which was produced by \n\nBacillus spp isolates. IAA, the major auxin in plants, plays \n\na major role in both the shoot and root development [13]. \n\nAmong the 39 isolates, 19 isolates gave pink color on \n\nincubating the supernatant liquid with Salkowski\u2019s \n\nreagent.", "start_char_idx": 11190, "end_char_idx": 14593, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b356ab1c-d8c2-4a2f-b6ec-f5b449b271c0": {"__data__": {"id_": "b356ab1c-d8c2-4a2f-b6ec-f5b449b271c0", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5c1b54e6-012b-454f-885b-7fde92f78974", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "83ab309ebfcda7fa804064900343757b926c9b2d5424fccd5fbbb8831c23bf07", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "74019679-8f9d-4810-b0ca-5ca404ae90ce", "node_type": "1", "metadata": {}, "hash": "f2ab0a1f283de04c03f246180b1aaf90426ec6c2d87a7aac018aebb062c4423c", "class_name": "RelatedNodeInfo"}}, "text": "Salkowski\u2019s reagent when reacted with IAA, \n\ntris-(indole-3-acetato) iron (III) complex is formed which \n\ndisplays pink coloration due to the formation of IAA \n\ncomplex and reduction of Fe3+ [14].. In a similar study, 7 \n\nof 12 test strains of Bacillus  produced IAA [15]. Similar \n\nobservation of IAA production by Bacillus strains has \n\nbeen reported in a study [16]. In their study, 76.3% (n=90) \n\nwere able to produce IAA.  Similar method applied for \n\nIAA production in Pseudomonas exhibited all the isolates \n\nwere able to produce IAA [10]. The availability of \n\ndifferent level of precursors affects the ability of bacteria \n\nto produce IAA.  \n\nDespite phosphorus being abundant in soil it is insoluble \n\nand cannot contribute to the plant growth [3]. So, the \n\nsolubilization and mineralization of insoluble phosphate \n\nin soil by rhizobacteria makes an important property of \n\nplant growth promoting bacteria. Rodriguez and Fraga \n\nstudied that Bacillus and other phosphate solubilizing \n\nbacteria (PSB) like Pseudomonas and Rhizobium were \n\ncapable of converting insoluble phosphate available in \n\nthe soil into soluble form [17] . Phosphate solubilizing \n\nability of isolated strains was tested using Pikovskaya\u2019s \n\nagar medium. Fifteen out of the total isolates produced \n\nhalo zone on Pikovskaya\u2019s agar medium after incubation \n\nfollowing the spot inoculation on the plates as a result of \n\nphosphate solubilization. The halo zone around the \n\ncolony was due to the polysaccharides, organic acids or \n\nphosphatase produced by the phosphate solubilizing \n\nBacillus strains (18). The bigger diameter of halo zones \n\nmay be due to their greater ability to solubilize \n\nphosphate. In a similar study of phosphate solubilization \n\nby Bacillus strains out of 12 isolates that promoted \n\nsoybean seedling significantly, 11 isolates showed \n\nphosphate solubilization (16). Another study \n\ndocumented only 11.5 % of Bacillus spp. isolated from the \n\nsamples obtained from various sites such as vineyard \n\nsoil, fig orchard soil, forest soil, sewage soil, coastal area \n\nsoil, compost of mushroom and paddy field were able to \n\nsolubilize phosphate  (19).  The study indicates that the \n\nphosphate solubilization capacity of Bacillus differs \n\naccording to their site of existence.  \n\nProduction of HCN by rhizobacteria is believed to \n\npromote plant growth by indirect mechanism. Hydrogen \n\ncyanide is supposed to act synergistically with bacterially \n\nencoded antibiotics [3]. Rijavec and Lapanje in their study \n\nconcluded that HCN increases the availability of \n\nphosphate for rhizobacteria and plant hosts, especially in \n\noligotrophic alpine environments and thus indirectly \n\ncontributing to plant growth [20]. Picric acid present in \n\nthe filter paper reacts with free cyanide produced by the \n\nbacteria to produce colored iso purpuric acid, thus the \n\nchange in color of filter paper is visible. The color \n\ndeveloped is directly proportional to free cyanide.  \n\nPlants can only utilize the reduced forms of the nitrogen; \n\nhence, nitrogen first must be fixed and converted to a \n\ncombined form (either ammonia/nitrate) and then \n\ntrapped by the plants [21]. Twenty eight isolates (71.8%) \n\nwere able to produce ammonia. These rates of ammonia \n\nproduction are lower than 95% and 80% demonstrated by \n\nother researchers [11, 22]. \n\nThe enzyme protease,  produced by most of the \n\nmicroorganisms, causes the hydrolysis of the peptide \n\nbonds that link amino acids  in the polypeptide chain [23]. \n\nThe production of protease by beneficial rhizobacteria \n\nhelps to lyse a portion of pathogenic fungi and act as a \n\nbiocontrol agent [24]. Screening of protease producing \n\nability of Bacillus isolates was carried using skim milk \n\nagar medium.", "start_char_idx": 14595, "end_char_idx": 18348, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "74019679-8f9d-4810-b0ca-5ca404ae90ce": {"__data__": {"id_": "74019679-8f9d-4810-b0ca-5ca404ae90ce", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b356ab1c-d8c2-4a2f-b6ec-f5b449b271c0", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "5f27098fda67e04051f09eb1a9ed405dd3397b9e38624bcde96d30a1e9462805", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "30a64c9b-8b6d-480a-b68f-c8f44b5e877d", "node_type": "1", "metadata": {}, "hash": "43fa8383b0a46c2e10b4906a81465040ac584254d23f9f63242b28205d44aba1", "class_name": "RelatedNodeInfo"}}, "text": "The casein present in the Skim milk agar \n\nmedium is hydrolyzed by the proteolytic bacteria, \n\nBacillus which is indicated by the formation of clear zones \n\naround the colonies [25]. All the isolates tested for \n\nprotease enzyme production produced the halo zone \n\naround the colonies on skim milk agar medium following \n\nthe incubation displaying the ability of Bacillus to \n\nproduce protease enzymes. The greater the ability to \n\nproduce protease, the bigger is the diameter of halo \n\nzones. \n\nSix isolates BS09, BS10, BS13, BS16, BS29 and BS37 \n\nshowed positive results for PGPR traits assessed in the \n\nstudy. Among these, BS29 showed maximum IAA \n\nproduction, phosphate solubilization, HCN production, \n\nammonia production and proteolytic activity. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):7 9 - 8 4      Pathak et al.  \n\n\u00a9NJB, BSN  84 \n\nConclusion \nIt is evident from the present studies that the Bacillus \n\nspecies tested were able to demonstrate multiple plant \n\ngrowth promoting traits.  The results concluded that \n\nBacillus strains have a huge potential to be used as \n\nalternatives to chemical fertilizers and pesticides for the \n\npromotion in growth and production of plants. The study \n\non Bacillus strains can be further detailed so that these \n\nstrains can commercially be developed as low-cost bio-\n\nbased products to promote plant growth in the \n\nagricultural sector.  \n\nAuthor\u2019s contribution  \nEP is the principal investigator, carried out laboratory \n\nworks, and also prepared the manuscript. A Sanjyal and \n\nCRR are co-investigators, and carried out laboratory \n\nworks. SP is a laboratory supervisor and helped to carry \n\nout laboratory works. AS is the corresponding author, an \n\nacademic supervisor of the research and prepared the \n\nmanuscript.  \n\nCompeting Interests  \nThe authors declare no competing or financial interests.  \n\nAcknowledgements \nThe authors are thankful to Nepalese Farming Institute, \n\nKathmandu, Nepal for the laboratory support. \n\nFunding \nThe author(s) declared that no grants were involved in \n\nsupporting this work. \n\nEthical Approval and Consent   \nNot applicable. \n\nReferences \n1.  Kumar R, Kumar R, Prakash O. The Impact of Chemical Fertilizers \n\non our Environment and Ecosystem Chapter - 5 The Impact of \nChemical Fertilizers on Our Environment and Ecosystem. \n2019;(February).  \n\n2.  Kloepper JW, Schroth M. Plant growth-promoting rhizobacteria on \nradishes. Vol. 2, In: Proceedings of the 4th international conference \non plant pathogenic bacteria. 1978. p. 879\u201382.  \n\n3.  Glick BR. Plant Growth-Promoting Bacteria\u202f: Mechanisms and \nApplications. 2012;2012.  \n\n4.  Fan B, Blom J, Klenk HP, Borriss R. Bacillus amyloliquefaciens, \nBacillus velezensis, and Bacillus siamensis Form an \u201cOperational \nGroup B. amyloliquefaciens\u201d within the B. subtilis species complex. \nFront Microbiol. 2017;8(JAN):1\u201315.  \n\n5.  Shafi J, Tian H, Ji M. Bacillus species as versatile weapons for plant \npathogens: a review. Biotechnol Biotechnol Equip [Internet]. \n2017;31(3):446\u201359. Available from: \nhttps://doi.org/10.1080/13102818.2017.1286950 \n\n6.  Cavaglieri L, Orlando J, Etcheverry M. In vitro influence of bacterial \nmixtures on Fusarium verticillioides growth and fumonisin B1 \nproduction: Effect of seeds treatment on maize root colonization. \nLett Appl Microbiol. 2005;41(5):390\u20136.  \n\n7.  Shrivastava UP. Isolation and initial characterization of \ndiazotrophic plant growth promoting rhizobacteria ( PGPR ) from \n\nrice rhizosphere of Parsa and Bara district of Nepal.", "start_char_idx": 18349, "end_char_idx": 21865, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "30a64c9b-8b6d-480a-b68f-c8f44b5e877d": {"__data__": {"id_": "30a64c9b-8b6d-480a-b68f-c8f44b5e877d", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "74019679-8f9d-4810-b0ca-5ca404ae90ce", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "279628b83b2ec51eaab1730cf34805ad4b97f180860a77cca66bf1e0d8f8ebfa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c30ae690-faf0-40c4-87b6-db867bcea69a", "node_type": "1", "metadata": {}, "hash": "4af326b9e1ebd36115a38f3d0a4cb6242dad5186ba278bed8f2e6f1ba7897e5a", "class_name": "RelatedNodeInfo"}}, "text": "Int J Pharm Life \nSci. 2013;4(3):2481\u20138.  \n\n8.  Pathak R, Shrestha A, Lamichhane J, Gauchan DP. PGPR in \nbiocontrol: mechanisms and roles in disease suppression. Int J \nAgron Agri R [Internet]. 2017;11(1):69\u201380. Available from: \nhttp://www.innspub.net/wp-content/uploads/2017/08/IJAAR-\nVol-11-No-1-p-69-80.pdf \n\n9.  Zakry, F.A.A, Halimi, M.S. Abdul Rahim, K. B.\u202f; Osumanu, H. A.\u202f; \nWong, S. K.\u202f; Franklin, R. K.\u202f; Stephen, L. C. T.\u202f; Make J. Isolation and \nplant growth-promoting properties of Rhizobacterial Diazotrophs \nfrom pepper vine (Piper nigrum L). Malaysian Appl Biol. \n2010;39(2):41\u20135.  \n\n10.  Suresh A, Pallavi P, Srinivas P, Kumar VP, Chandra SJ, Reddy SR. \nPlant growth promoting activities of fluorescent pseudomonads \nassociated with some crop plants. African J Microbiol Res. \n2010;4(14):1491\u20134.  \n\n11.  Agbodjato NA, Noumavo PA, Baba-Moussa F, Salami HA, Sina H, \nS\u00e8zan A, et al. Characterization of potential plant growth promoting \nrhizobacteria isolated from Maize (Zea mays L.) in central and \nNorthern Benin (West Africa). Appl Environ Soil Sci. 2015;2015.  \n\n12.  Cappuccino JG, Sherman N. Microbioloy: A laboratory manual. 5th \ned. Benjamin/Cummings Science Publishing; 1998.  \n\n13.  Prusty R, Grisafi P, Fink GR. The plant hormone indoleacetic acid \ninduces invasive growth in Saccharomyces cerevisiae. Proc Natl \nAcad Sci U S A. 2004;101(12):4153\u20137.  \n\n14.  Kamnev AA, Shchelochkov AG, Perfiliev YD, Tarantilis PA, \nPolissiou MG. Spectroscopic investigation of indole-3-acetic acid \ninteraction with iron(III). J Mol Struct. 2001;563\u2013564:565\u201372. \nhttps://doi.org/10.1016/S0022-2860(00)00911-X \n\n15. Akinrinlola RJ, Yuen GY, Drijber RA, Adesemoye AO. Evaluation \nof Bacillus Strains for Plant Growth Promotion and Predictability of \nEfficacy by In Vitro Physiological Traits. Int J Microbiol. 2018;2018. \nhttps://doi.org/10.1155/2018/5686874 \n\n16.  Wahyudi, Aris Tri Astuti, Rina Puji Widyawati A, Meryandini A, \nNawangsih AA. Characterization of Bacillus sp. strains isolated \nfrom rhizosphere of soybean plants for their use as potential plant \ngrowth for promoting Rhizobacteria. Int J Curr Microbiol Appl Sci. \n2013;2(10):406\u201317.  \n\n17.  Rodr\u00edguez H, Fraga R. Phosphate solubilizing bacteria and their role \nin plant growth promotion. Biotechnol Adv. 1999;17(4\u20135):319\u201339.  \n\n18.  Paul D, Sinha SN. Isolation and characterization of phosphate \nsolubilizing bacterium Pseudomonas aeruginosa KUPSB12 with \nantibacterial potential from river Ganga, India. Ann Agrar Sci. \n2017;15(1):130\u20136.  \n\n19.", "start_char_idx": 21866, "end_char_idx": 24387, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c30ae690-faf0-40c4-87b6-db867bcea69a": {"__data__": {"id_": "c30ae690-faf0-40c4-87b6-db867bcea69a", "embedding": null, "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-129", "node_type": "4", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "9abb6cc8a3ddaab32af7bbf0e54d94e11e5726b6795ba0bc743d6167dd9ce3cf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "30a64c9b-8b6d-480a-b68f-c8f44b5e877d", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "3e6d06501dc72b11ba8ccb4045ac62261e5aa0f08676d31f901830e4daf98297", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1ed3dd89-18ee-4ab9-a4a6-b9d62ee5ac64", "node_type": "1", "metadata": {}, "hash": "6e7285148cb1a6e9621ab4704fa2560911435b99406de64f1df133a1a78e2657", "class_name": "RelatedNodeInfo"}}, "text": "2017;15(1):130\u20136.  \n\n19.  S\u00f6zer Bahadir P, Liaqat F, Eltem R. Plant growth promoting \nproperties of phosphate solubilizing Bacillus species isolated from \nthe Aegean Region of Turkey. Turk J Botany. 2018;42(2):183\u201396.  \n\n20.  Rijavec T, Lapanje A. Hydrogen cyanide in the rhizosphere: Not \nsuppressing plant pathogens, but rather regulating availability of \nphosphate. Front Microbiol. 2016;7(NOV):1\u201314.  \n\n21.  Smil V. Nitrogen cycle and world food production. World Agric. \n2011;2(Smil):9\u201313.  \n\n22.  Joseph B, Patra RR, Lawrence R. Characterization of plant growth \npromoting rhizobacteria associated with chickpea ( Cicer arietinum \nL .). Plant Prod. 2007;1(September).  \n\n23.  Singh P, Rani A, Chaudhary N. Isolation and Characterization of \nProtease producing Bacillus strain FS-1. Agric Eng Int CIGR J. \n2007;6(04):633\u20139.  \n\n24.  Kundan R, Pant G, Jadon N, Agrawal PK. Plant Growth Promoting \nRhizobacteria: Mechanism and Current Prospective. J Fertil Pestic. \n2015;06(02).  \n\n25. Yasmin F, Othman R, Saad MS, Sijam K. Screening for beneficial \nproperties of rhizobacteria isolated from sweetpotato rhizosphere. \nBiotechnology. 2007;6(1):49\u201352. \nhttp://dx.doi.org/10.3923/biotech.2007.49.52", "start_char_idx": 24363, "end_char_idx": 25560, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1ed3dd89-18ee-4ab9-a4a6-b9d62ee5ac64": {"__data__": {"id_": "1ed3dd89-18ee-4ab9-a4a6-b9d62ee5ac64", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c30ae690-faf0-40c4-87b6-db867bcea69a", "node_type": "1", "metadata": {"identifier": "njb-129", "author": "Pathak, Enish; Sanjyal, Arjun; Regmi, Chhatra Raj; Paudel, Saroj; Shrestha, Anima", "title": "Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal", "date": "2021-07-31", "file": "njb-129.pdf"}, "hash": "71f93ee857ee6043f26d084309aad19fddba69cc6362bf4d327bd0657b44f961", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bfede939-bc82-4fbe-a772-b2235cfda71c", "node_type": "1", "metadata": {}, "hash": "49b039529b13639191bc93f5cfba517b3ac471133ae1725bc2ba7ee236617242", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 6 3 - 7 4  Review article DOI: https://doi.org/10.3126/njb.v9i1.38669  \n\n\u00a9NJB, BSN 63 \n\nPlant and Plant Associated Microflora: Potential Bioremediation \nOption of Indoor Air Pollutants \nY.H.K.I.S. Gunasinghe1 , I.V.N.Rathnayake1 , M.P.Deeyamulla2\n1Department of Microbiology, Faculty of Science, University of Kelaniya, Kelaniya Sri Lanka \n2Department of Chemistry, Faculty of Science, University of Kelaniya, Kelaniya Sri Lanka \n\nReceived: 17 Oct 2020; Revised: 10 Jul 2021; Accepted: 18 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract \nIndoor air pollution is a significant problem today because the release of various contaminants into the indoor air has \ncreated a major health threat for humans occupying indoors. Volatile Organic Compounds (VOCs) are pollutants released \ninto the environment and persist in the atmosphere due to its low boiling point values. Various types of indoor activities, \nsources, and exposure to outdoor environments enhance indoor VOCs. This poor indoor air quality leads to adverse \nnegative impacts on the people in the indoor environment. Many physical and chemical methods have been developed to \nremove or decompose these compounds from  indoors. However, those methods are interrupted by many environmental \nand other factors in the indoor atmosphere, thus limit the applications. Therefore, there is a global need to develop an \neffective, promising, economical, and environmentally friendly alternatives to the problem. The use of the plant and \nassociated microflora significantly impact reducing the environmental VOC gases, inorganic gases, particulate matter, and \nother pollutants contained in the air. Placing potted plants in indoor environments not only helps to remove indoor air \npollutants but also to boost the mood, productivity, concentration, and creativity of the occupants and reduces stress, \nfatigue, sore throat, and cold.  Plants normally uptake air pollutants through the roots and leaves, then metabolize, \nsequestrate, and excrete them. Plant-associated microorganisms help to degrade, detoxify, or sequestrate the pollutants, the \nair remediation, and promote plant growth. Further studies on the plant varieties and microorganisms help develop eco-\nfriendly and environmentally friendly indoor air purifying sources. \n\nKeywords: Plants, Microorganisms, VOC, Air pollution, Biological remediation \n\n Corresponding author, email: kaviisugunasinghe@gmail.com \n\nIntroduction \nPeople spend the bulk of their lifetime indoors, either in \n\nresidential or public areas. Number of pollutants in the \n\nindoor air are higher than the outdoor air; hence poor \n\nair quality in these indoor environments will lead to \n\nseveral health issues.  Today, it has become one of the \n\nbiggest environmental threats [1]. Therefore, most \n\nstudies have been disclosed the connection between \n\nindoor air pollution and associated adverse health \n\neffects [2,3]. Continuous exposure of individuals to poor \n\nindoor air quality can lead to \"sick building syndrome\" \n\n(SBS);  health problems such as headache, fatigue, eye \n\nand skin irritation, or respiratory illnesses, etc. [4]. In \n\n2012, World Health Organization (WHO) reported that \n\nindoor air pollution by households cooking over coal, \n\nwood, and biomass stoves caused about 4.3 million \n\ndeaths worldwide [5].  \n\nIndoor air contaminants are generated through several \n\nsources such as occupational activities, household \n\nproducts, chemical reactions indoors, pets, materials, \n\nunderground garages, and outside air sources [6,7]. \n\nParticles, biological agents, radon, asbestos, and gaseous \n\ncontaminants such as CO, CO2, NOx, SOx, aldehydes, \n\nand Volatile Organic Compounds (VOC) are released as \n\nmain indoor air contaminants from the sources as \n\nmentioned above [8]. Removing the pollutant \n\ngenerating sources from indoors, increasing the \n\nventilation rates, improving air distribution and \n\ncleaning the indoor air, etc. are the primary air purifying \n\nprinciples at indoors.", "start_char_idx": 48, "end_char_idx": 4080, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bfede939-bc82-4fbe-a772-b2235cfda71c": {"__data__": {"id_": "bfede939-bc82-4fbe-a772-b2235cfda71c", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1ed3dd89-18ee-4ab9-a4a6-b9d62ee5ac64", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "3feffd0a94e2194e0b36f0f36a84740b14775e8634363ef11611490db894ee29", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "47706aed-2bf8-4809-9d80-0ca9034927b7", "node_type": "1", "metadata": {}, "hash": "2b0667f5ee1f7bfb34b4fc9597aef9d064bf7867dcbab3dc5708190e0b8cd102", "class_name": "RelatedNodeInfo"}}, "text": "are the primary air purifying \n\nprinciples at indoors. Increasing the ventilation rate is \n\nthe easiest way to reduce indoor air pollutants. \n\nHowever, it is usually affected by outdoor weather and \n\nexternal pollution condition [9]. Other current strategies \n\nused to remove indoor air pollutants are filtration, \n\nelectrostatic precipitator with ionization, adsorption, \n\nozonization, photolysis, photocatalysis etc. [8]. Among \n\nthe above mentioned treatment strategies, some are very \n\nmuch expensive and complex methods. However, \n\nbiological purification is a simple, low cost, and \n\nenvironmental friendly technique. Therefore has been \n\ninvestigated in many studies [10,11]. This review covers \n\nthe potential use of plant and plant associated \n\nmicroflora for indoor air pollutant removal and \n\ndegradation. \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-1293-5562\nhttps://orcid.org/0000-0002-1293-5562\nhttps://orcid.org/0000-0003-3476-7018\nhttps://orcid.org/0000-0002-3085-4280\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4  Gunasinghe et al. \n\n\u00a9NJB, BSN 64 \n\nIndoor Air Quality \nAn average person needs 30 lb of air per day to live. \n\nHowever, he needs only 1.360 kg (3 lb) and 0.680 kg (1.5 \n\nlb) of water and food per day [12]. It indicates why air \n\nbecomes the foremost necessary thing for the survival of \n\nhumans and other living beings.  According to the U.S. \n\nNational Institute for Occupational Safety and Health \n\n(NIOSH) reports in 2007, the average total VOCs \n\nconcentration in air samples could reach 2.90 mg \n\nm\u22123[13].  Inadequate building ventilation is the leading \n\ncause of the high level of pollutant content indoors [14], \n\nand high pollutant content also causes severe public \n\nhealth threats [1]. Humans spend most of their time \n\nindoors, thus more researches are focused on indoor air \n\nquality and related studies.  \n\nAmbient air is often contaminated with high amounts of \n\nindoor air pollutants like particulate matter (PM), VOCs \n\nlike benzene, toluene, ethylbenzene, xylene, \n\npolyaromatic hydrocarbons (PAHs), formaldehyde, and \n\ninorganic pollutants as sulfur dioxide (SO2), nitrogen \n\noxides (NOx), carbon monoxide (CO), carbon dioxide \n\n(CO2) and Ozone (O3). Although many of those \n\ncompounds are outdoor air pollutants, can also be \n\nfound indoors in higher amounts than outdoors [15]. \n\nBenzene is a ubiquitous trace element in indoor air [16], \n\nand its indoor concentration is higher than outdoors. A \n\nsafe level for benzene exposure cannot be \n\nrecommended. PAHs presence in the atmosphere is \n\ntypically attached to air particles and present as \n\ncomplex mixtures. Therefore, the composition of PAH \n\nmay vary from site to site. However, WHO (2000) \n\nreported that 8.7\u00d710-5 ng/m3 of PAHs have a risk for \n\nlung cancers.  Exposure of 0.01 mg/m-3 Naphthalene is \n\ndescribed as a safe level. Still, long term inhalation can \n\ncause respiratory tract lesions leading to inflammation \n\nand malignancy of animals. Formaldehyde exposure of \n\n0.36 mg/m-3 for 04 hours causes sensory irritations of \n\nthe eyes in humans [17]. Furniture, carpets, construction \n\nmaterials, sprays, cleaning, restoration activities, and \n\nsurrounded industries are the foremost sources of the \n\nvarious volatile organic compounds, aliphatic and \n\naromatic hydrocarbons, alcohols, and aldehydes, and \n\nchlorinated compounds [6,7,18,19].", "start_char_idx": 4026, "end_char_idx": 7555, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "47706aed-2bf8-4809-9d80-0ca9034927b7": {"__data__": {"id_": "47706aed-2bf8-4809-9d80-0ca9034927b7", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bfede939-bc82-4fbe-a772-b2235cfda71c", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "5bef57fba21cd1b3d907eb8420eac014aa249a48f4c1a8615eb66820e6597c6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "853d7eae-dfa0-42d7-9c76-77045abef90b", "node_type": "1", "metadata": {}, "hash": "ff69fe3753b9a1ce4a09d04548753c80c598d255353de72cebe1f02db6d9f0be", "class_name": "RelatedNodeInfo"}}, "text": "Inorganic gaseous \n\npollutants, SO2, NOx, CO, and CO2 are generated \n\nthrough the combustion of fossil fuels, gas fired \n\nappliances (stoves and ovens), kerosene heaters, tobacco \n\nsmoking [7,20,21], and outdoor sources exposure [22]. \n\nPotential health hazards  \nThe presence of toxic volatiles and other pollutants in \n\nindoor air can cause various illnesses in humans. The \n\nEuropean Environmental Agency has shown that indoor \n\nair quality is one of the priority considerations in \n\nchildren\u2019s health [23]. Prevalence of SBS is higher in \n\nbuildings with air conditioners than in natural \n\nventilation systems [24]. Typically this has been \n\nreported in offices, schools, aged care homes, and \n\napartments like building-associated environments [2]. \n\nSBS is often associated with various symptoms such as \n\nheadache and nausea, nasal congestion (runny nose, \n\nstuffy nose, shortness of breath, wheezing,  sneezing, \n\nsinus, chest tightness, and chest congestion), throat \n\nproblems (dry throat, sore throat, hoarseness), eye \n\nproblems (dry eye, itching, tearing, blurry vision, \n\nburning eyes, sore eyes, and problems with contact \n\nlenses), fatigue (sleepiness, or drowsiness and unusual \n\ntiredness,), chill and fever, muscle pain (aching muscles \n\nor joints, pain or stiffness in the lower back, pain or \n\nstiffness in the upper back, and pain or numbness in \n\nshoulder/neck), and even neurological symptoms \n\n(feeling depressed, difficulty remembering or \n\nconcentrating, and tension or nervousness), dry skin, \n\nand dizziness as well [25].  \n\nApart from these illnesses, sometimes poor indoor air \n\nconditions also cause adverse health effects like \n\nrespiratory tract illnesses, lung cancers, and heart \n\ndiseases [26]. Potential harmful effects of benzene, \n\ntoluene, xylene, and formaldehyde exposure were \n\nsummarized below (Table 01). Prevalence of illnesses \n\ndue to indoor air contaminants depends on factors like \n\nindividual sensitivity to the contaminant, concentration \n\nof the contaminant,  current physical health state of the \n\nindividual, and also  duration of exposure to the \n\ncontaminant [27]. According to the International Agency \n\nfor Research on Cancer (IARC), benzene is a  toxic \n\nchemical proven as a carcinogen [28]. Benzene can cause \n\nmost hematological diseases, such as acute and chronic \n\nlymphocytic leukemia,  acute and myeloid leukemia, \n\nnon-Hodgkin\u2019s lymphoma, multiple myeloma, and \n\naplastic anemia even at the low dose of exposure [29\u2013\n\n31]. The safe level for benzene exposure is still \n\nunknown, but the European Union recommended in \n\n2000 that the benzene concentration in the ambient air \n\nshould not exceed 5 \u00b5g m-3 [32]. Impure indoor air with \n\nparticulate matter (PM\u226410 \u00b5m) is often correlated with \n\ncardiovascular or respiratory disorders, and recently it \n\nis revealed that exposure to PM during the period of \n\npregnancy or early life may cause autism spectrum \n\ndisorder (ASM) [33,34]. These potential health hazards \n\nassociated with poor indoor air quality highlight the \n\nneed to review indoor air pollution and purification \n\nmethods more seriously. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4  Gunasinghe et al. \n\n\u00a9NJB, BSN 65 \n\nHow to avoid indoor air pollutants? \nMany strategies can be used for the reduction of indoor \n\nair pollutants. Those are supported by several efforts, \n\nsuch as removing the pollutant source from indoors, \n\nenhancing the ventilation rate, improving indoor air \n\ndistribution, and cleaning [37]. Many industries have \n\ntaken steps to scale down the usage of possible sources \n\nof indoor air pollutants during their product \n\nmanufacturing cycle.  \n\nCurrent strategies applied to remove or reduce indoor \n\nair pollutants are filtration, electronic precipitator with \n\nionization, adsorption, ozonation, photolysis, and \n\nphotocatalysis [8,38]. Manipulation of filtration is \n\nsuitable for particle removal in indoor air [39].", "start_char_idx": 7556, "end_char_idx": 11489, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "853d7eae-dfa0-42d7-9c76-77045abef90b": {"__data__": {"id_": "853d7eae-dfa0-42d7-9c76-77045abef90b", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "47706aed-2bf8-4809-9d80-0ca9034927b7", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "e2a6b8816c9fe0d670e7ebcede2ccc490d934b873148e13daffc898ef9716d91", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "44e9d201-69a5-47e8-91d8-0f570e2dc445", "node_type": "1", "metadata": {}, "hash": "ba641861dba3dc0d5b67995bbc9c559d5c722ff99e6df088f1e11ebfda1a6037", "class_name": "RelatedNodeInfo"}}, "text": "Manipulation of filtration is \n\nsuitable for particle removal in indoor air [39]. However \n\nmicrobial colonization on the filters will hinder the \n\nfiltration. During electrostatic precipitation, by \n\ngenerating an electrical field, charged particles of air can \n\nbe trapped. However, there is a risk of generating \n\nhazardous charged particles. Removing air pollutants \n\nusing adsorption might be a highly specific technique, \n\nwhich is used as a post-treatment. The problem \n\nassociated with oxidizing the pollutant may be the \n\ngeneration of unhealthy toxic products. Researchers are \n\nstill proposing strategies to address this case with non-\n\nadverse impacts. Membrane separation, enzymatic \n\noxidation, botanical purification, biofilters, and \n\nbiotrickling filters are number of those strategies. Out of \n\nthose plants and plant associated microflora, lowering \n\nthe toxicity of contaminants in indoor environments is \n\nbecoming a popular alternative as an economical air \n\nrestoration technology [38]. \n\nIndoor pollutant removal capability of plants \nPlants remove VOC, through aerial plant parts and \n\nplant associated microflora. Growing media and plant \n\nroots are also capable of removing VOC in the air. \n\nRecent studies showed that plants are one of the best air \n\npollutant absorbing and metabolizing agents [40]. Plant \n\nvolatile organic matter removal or degradation rate and \n\nefficiency rely upon the plant species, light, \n\ntemperature, growing media, and VOC (concentration, \n\nidentity, and VOC mixture effects). Stomata, cuticle, and \n\nadsorption to the plant wax layer are the critical VOC \n\nremoval sites of the aerial plant parts. After entering \n\ninto the leaf, the compound often undergoes \n\ndegradation, storage, excretion, and translocation to \n\nalternative plant elements. Microorganisms present in \n\nthe plant pot soil and plant root also can remove VOC \n\nfrom indoor air [41]. These plant pollutant removal and \n\ndegradation strategies have been confirmed using \n\nseveral plant species using radiolabeling [42,43]. Several \n\nstudies on plants with 14C labeled aromatic \n\nhydrocarbons revealed that aromatic rings of those \n\nhydrocarbons were cleaved during their metabolic \n\ntransformations and utilization of aromatic \n\nhydrocarbons under sterile conditions [44].  \n\nPlants can sink air pollutants through their large surface \n\narea of foliage and canopies because it provides a \n\nsurface for the pollutant substances. Also, plant leaves \n\ncan sorb several gaseous substances as nutrients or as \n\nmicronutrients [45]. The plant uses processes like \n\ncomplexation, precipitation, and oxidation-reduction to \n\ndetoxify or utilize those substances as nutrients. These \n\nplant and atmospheric interactions result in the \n\nreduction of these harmful particulate substances and \n\nVOC\u2019s [46]. VOCs removal and degradation capability \n\nof many indoor and outdoor plant species have been \n\nrecorded in the literature.  As reported in the literature, \n\nTable 1 Potential health hazards - Benzene, Toluene, Xylene, and formaldehyde exposure. \n\nVOCs \nLimit of Exposure (\u00b5g m-3) \n\nPotential health hazards \nRef \n\nShort term Long term \n\nToluene 15,000 (8h) \n2,300 \n(one day \naverage) \n\nShort-term exposure \u2013 Eye, nose, and throat irritation, dizziness, \nheadaches, and feelings of intoxication. \nLong term exposure \u2013Neurological effects including reduced \nscores in tests of short-term memory, attention, and \nconcentration \n\n[35] \n\nBenzene \n\nNo safe level \nof exposure \nrecommended\n. \n\nNo safe level of \nexposure \nrecommended \n\nCarcinogenic  chemical (Group1) to humans- Cause adult acute \nmyeloid leukaemia. Positive associations have been observed \nfor non-Hodgkin lymphoma, chronic lymphoid leukaemia, \nmultiple myeloma, chronic myeloid leukaemia, acute myeloid \nleukaemia in children Lung cancer \n\n[36] \n\nXylene \n- 100 (1year) \n\nIrritation to the lungs, throat, and nose.", "start_char_idx": 11408, "end_char_idx": 15303, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "44e9d201-69a5-47e8-91d8-0f570e2dc445": {"__data__": {"id_": "44e9d201-69a5-47e8-91d8-0f570e2dc445", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "853d7eae-dfa0-42d7-9c76-77045abef90b", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "469953d5ce5d427ec513211854132c2fb5f928c06ef5120d5ff1809bde5bd71d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2c4280ea-6833-44a9-acae-8a9897ed3e57", "node_type": "1", "metadata": {}, "hash": "973456806cc30348fa2e730a75ef53b19d6260b03ee262d4fbe52f3bd412c1b4", "class_name": "RelatedNodeInfo"}}, "text": "Severe inhalation \nexposure can cause dizziness, headache, confusion, liver and \nkidney damage, heart problems, and coma \n\n[35] \n\nFormaldehyde 100 (30 min) 10 (1year) \n\nSensory irritation of  eyes, nose, and throatexposure-dependent \ndiscomfort, lachrymation, sneezing, coughing, nausea, and \ndyspnoea. Human carcinogenic chemical. Long-term exposure \nlinked to nasal cancer. \n\n[36] \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4  Gunasinghe et al. \n\n\u00a9NJB, BSN 66 \n\nplant species and their potential in removing or \n\ndetoxifying toluene (Table 2), benzene (Table 3), xylene \n\n(Table 4), and formaldehyde (Table 5) removal or \n\ndegradation are summarized below.  \n\nHowever, there could be some deleterious effects like \n\nimpairment of plant physiological activity and plant \n\ninjuries due to these chemicals. Chronic exposure to  \n\nhigher concentrations of air pollutant substances can \n\naffect plant photosynthesis, vitality, and productivity. \n\nThis stress makes the plant more susceptible to diseases \n\nand insect infections [47] \n\nTable 2 Plant species and their potential for Toluene removal. \n\nPlant species Results Ref \n\nZamioculcas zamiifolia \nToluene uptake per unit area of Z. zamiifolia plant leaf at 72 h of exposure \n0.93\u00b10.02 mmol m\u22122 \n\n[48] \n\nHemigraphis alternate, Hedera helix, Hoya \ncarnosa, Asparagus densifloru \nTradescantia pallida, Fittonia argyroneura \n\nRemoval efficiency of toluene and total VOC by twenty-eight selected \nornamental plants varied substantially among the species tested, \nRange of pollutant removal,  Toluene - 1.54 - 9.63 \u00b5g m\u20133 m\u20132 h\u20131      \n Total VOC - 5.55 -44.04 \u00b5g m\u20133 m\u20132 h\u20131. \n\n[6] \n\nO. microdasys, D. dermensis \n Time taken for the complete removal of 2 ppm toluene from an airtight \nchamber was 55 h and 120 h, respectively for O. microdasys and D. \ndermensis plants. \n\n[49] \n\nDieffenbachia maculate, Spathiphyllum wallisii \nAsparagus densiflorus \n\nToluene removal rate constant ranged from 3.4 to 5.7 L h\u22121m\u22122 leaf area \nwhen exposed to 20.0 mg m\u22123 of toluene \n\n[50] \n\nHedera helix ,Spathiphyllum wallisii  \nSyngonium podophyllum, Cissus rhombifolia \n\nToluene (initial 1 \u03bcL L\u20131 ) removal efficiencies of \nH. helix -220.2 \u00b1 31.8 ng m\u20133 h\u20131 cm\u20132 \nS. podophyllum, - 161.6 \u00b1 19.2 ng m\u20133 h\u20131 cm\u20132 \n S. wallisii - 203.7 \u00b1 24.3 ng m\u20133 h\u20131 cm\u20132 \nLowest efficiency - C. rhobifolia. - 85.7 ng m\u20133 h\u20131 cm\u20132 \n\n[51] \n\nHerbs \nAloysia triphylla, Brittonz Melissa officinalis \nMentha piperita , Mentha piperita  \nMentha suaveolens ,Mentha suaveolens  \nPelargonium graveolens, \nPlectranthus tomentosus  \nRosmarinus officinalis ,Salvia elegans  \nHerbaceous foliage plants  \nBegonia maculata ,Davallia mariesii \nFarfugium japonicum, Fittonia verschaffeltii  \nHedera helix  Philodendron spp.", "start_char_idx": 15304, "end_char_idx": 18037, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2c4280ea-6833-44a9-acae-8a9897ed3e57": {"__data__": {"id_": "2c4280ea-6833-44a9-acae-8a9897ed3e57", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "44e9d201-69a5-47e8-91d8-0f570e2dc445", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "c5badbe7f02da1440dccdedd9dcb83572df4990493a5f47307505a7047065687", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8605673e-c564-4cc5-bd48-f0973edafdc4", "node_type": "1", "metadata": {}, "hash": "e6c94a08762b6794a7f4e849856e5d84d1674130d753f4d4ce2c8ceebcf02926", "class_name": "RelatedNodeInfo"}}, "text": "Soleirolia soleirolii  \nWoody foliage plants \nArdisia crenata , Ardisia japonica \nArdisia pusilla, Cinnamomum camphora  \nSchefflera elegantissima, Eurya emarginata , \nIlex cornuta, Ligustrum japonicum, \nPinus densiflora, Pittosporum tobira, \nRhododendron fauriei \n\nEfficiency of toluene removal ranged from 378 to 16.6 \u00b5g m\u20133 h\u20131 m\u20132 [52] \n\nFatsia japonica, Draceana fragrans \nVolatile toluene and xylene removal efficiencies were increased as the \nplant\u2019s root zone volume increased. \n\n[53] \n\nSchefflera actinophylla, Ficus benghalensis \n\nToluene and total xylene (m, p, o) removal efficiency of leaf area over a 24h \nperiod in  \nS. actinophylla,  - 13.3 \u03bcg m\u22123 m\u22122   \nF. benghalensis  -  7.0 \u03bcg m\u22123 m\u22122 \n\n[54] \n\nPhoenix roebelenii \nPurification capability (Pa) increased with an increase in room temperature \nfrom 21 to 26\u00b0C , reaching a range of 15\u201335 (V/h) \nInitial toluene 1.5 ppm, Pa for toluene was 6.5 (V/h) \n\n[55] \n\nAzalea indica Time taken to remove 339 mg m-3 of Toluene 76 h [56] \n\n Epipremnum aureum, Spathiphyllum Removal rate for TVOC was 74%, and  68%respectively [57] \n\nEpipremnum  aureum, Davallia fejeensis \nEpipremnum  aureum plant had a positive impact on mixed VOC(decane, \ntoluene, 2 ethylhexanol, benzene, octane, xylene, \u03b1- pinene)  filtration than \nDavallia fejeensis \n\n[58] \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  67 \n\nDiversity of plant associated microflora \n\nMicrobial reservoirs like soil, rhizosphere, phyllosphere, \n\nanthosphere (external environment of flower), \n\nspermosphere (the exterior of germinating spores), and \n\ncarposphere (external area of the fruit) indicate plant \n\nmicrobial relationships [77]. Diverse groups of bacterial \n\ntaxa namely proteobacteria, acidobacteria, \n\nactinobacteria, bacteroidetes, Verrucomicrobia, \n\nPlanctomycetes, Cloroflexi, Firmicutes, and \n\nGemmatimonatedes are present as root endophytes \n\n[78,79]. Among those, a representative amount of taxa \n\nhave been derived from the soil environments [80]. \n\nPlant root microbiota is mostly transferred horizontally. \n\nHowever, bacteria can sometimes be transferred via \n\nseeds by relocating microorganisms to proliferating \n\nplants [81,82]. The narrow layer of soil on plant roots \n\nhas high microbial diversity, it\u2019s one of the most \n\ncomplex ecosystems and is called as a rhizosphere [83]. \n\nRoot exudate containing organic acids, phenolic \n\ncompounds, plant growth regulators, sugars, sterols, \n\nvitamins, amino acids, fatty acids, and nucleotides \n\nensures good microbial growth around roots [84,85]. \n\nPlant root endophytes enter into tissues through passive \n\nmechanisms (root cracks or emerging points of lateral \n\nroots) or active mechanisms [86].  \n\nAerial plant tissues are different in ecology from \n\nbelowground parts; however, it\u2019s a good source for \n\nphyllosphere and endosphere bacteria. Normally \n\nendophytes spread systemically to the leaves, fruits, and \n\nstems via the xylem. In addition, endophytes enter plant \n\ntissues through aerial plant parts; as fruits and flowers. \n\nPhyllosphereic bacterial community is highly dependent  \n\nTable 3 Plant species and their potential for Benzene removal.", "start_char_idx": 18039, "end_char_idx": 21221, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8605673e-c564-4cc5-bd48-f0973edafdc4": {"__data__": {"id_": "8605673e-c564-4cc5-bd48-f0973edafdc4", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2c4280ea-6833-44a9-acae-8a9897ed3e57", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "9aaa152bdadd0c86e6dbc98fc4c5f6a97a28779fd13950b96f3280e48e9a4c03", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5254c04f-be91-43d5-9177-6794970e60e7", "node_type": "1", "metadata": {}, "hash": "e9be7df63598ac94a4061788e7eef66a2313982e08192c7b92821c01530244f5", "class_name": "RelatedNodeInfo"}}, "text": "Plant species Results Ref \nHowea forsteriana, Spathiphyllum floribundu, \nDracaena deremensis , Spathiphyllum sensation, \nDracaena marginata, Epipremnum aureum , \nScheflera actinophylla  \n\nFrom seven potted plant species, benzene removal was \nranged from 12-28 ppm day-1. \n\n[59] \n\nDracaena deremensis, Spathiphyllum wallisii \n\nBenzene removal per leaf area of  \nDracaena deremensis  - 606 \u00b1 155 mg m\u22123 d\u22121 m\u22122  \nSpathiphyllum wallisii 686 \u00b173 mg m-3 d-1 m-2;   \nHowea forsteriana 537\u00b1 69 mg m-3 d-1 m-2.  \n\n[60] \n\nZamioculcas zamiifolia \nBenzene uptake per unit area of Z. zamiifolia leaf was \n0.96\u00b1 0.01 mmol m\u22122 \n\n[48] \n\nCrassula portulacea, Hydrangea macrophylla, \nCymbidium, Ficus microcarpa var. fuyuensis, \nDendranthema morifolium, Citrus medica var. sarcodactylis, \nDieffenbachia amoena, Spathiphyllum, Nephrolepis exaltata,  \nDracaena deremensis  \n\nRemoval of benzene was in the range of  22.1- 561.3 \u00b5g \nm-2 min-1 \n\n[61] \n\nSuperior removal efficiency \nHemigraphis alternate, Hedera helix \nTradescantia pallida, Asparagus densifloru \nHoya carnosa \nIntermediate removal efficiency \nFicus benjamina, Polyscias fruticose, \nFittonia argyroneura, Sansevieria trifasciata \nGuzmania spp., Anthurium andreanum, \nSchefflera elegantissima \n\nBenzene removal efficiency of  \nHemigraphis alternata -5.54 \u00b5g m\u20133 m\u20132 h\u20131 \n\nTradescantia pallida- 3.86 \u00b5g m\u20133 m\u20132 h\u20131 \nHedera helix - 3.63 \u00b5g m\u20133 m\u20132 h\u20131 \nFittonia argyroneura -2.74 \u00b5g m\u20133 m\u20132 h\u20131 \nAsparagus densiflorus,- 2.65 \u00b5g m\u20133 m\u20132 h\u20131 \nHoya carnosa -  2.21 \u00b5g m\u20133 m\u20132 h\u20131 \n\n[6] \n\nDracaena deremensis  \nOpuntia microdasy \n\nRemoval rates of  2 ppm of benzene from the test \nchambers by  \nO. microdasys -3.2 mg/ m3 d1 \nD. dermensis  - 1.46 mg/ m3d1 \n\n[49] \n\nHedera helix, Spathiphyllum wallisii  \nSyngonium podophyllum, Cissus rhombifolia  \n\nHighest removal efficiency -S. wallisii.", "start_char_idx": 21224, "end_char_idx": 23049, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5254c04f-be91-43d5-9177-6794970e60e7": {"__data__": {"id_": "5254c04f-be91-43d5-9177-6794970e60e7", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8605673e-c564-4cc5-bd48-f0973edafdc4", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1949e8ecdf5d8199f4db1a740ef4a94a36098b99e102189739645e12a7311d2b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b9dd623d-3c78-49da-8f5d-f0f4e80e637a", "node_type": "1", "metadata": {}, "hash": "afa2577ee4a8512ca20ef54ed24091d96d8f25aef14f15a38504cf7c5fb33cff", "class_name": "RelatedNodeInfo"}}, "text": "Medium level removal efficiency - S. podophyllum and \nH. helix lowest removal efficiency - C. rhombifolia  \n\n[51] \n\nChamaedorea seifrizii, Scindapsus aureus \nSansevieria trifasciata, Philodendron domesticum \nIxoraebarbata craib, Monster acuminate \nEpipremnum aureum, Dracaena sanderiana \n\nhighest benzene uptake  \nD. sanderiana - 10.00 \u00b11.04 mmol of benzene at 72 h \nCrude wax 46 % and stomata 54 % \n \n\n[62] \n\nSyngonium podophyllum \nBenzene removal - 25 ppmv from the test chambers \nwithin 7 days \n\n[63] \n\nEpipremnum  aureum, Davallia fejeensis \n\nEpipremnum  aureum plant had a positive impact on \nmixed VOC (decane, toluene, 2 ethylhexanol, benzene, \noctane, xylene, \u03b1- pinene)  filtration than Davallia \nfejeensis \n\n[58] \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  68 \n\nTable 4. Plant species and their potential for Xylene removal. \n\nPlant species Results Ref \n\nAlternanthera bettzickiana,Drimiopsis botryoides, \nAloe vera, Chlorophytum comosum,  \nAglaonema commutatum, Cordyline fruticose, \nPhilodendron martianum, Sansevieria hyacinthoides, \nAglaonema rotundum, Fittonia albivenis, \nMuehlenbeckia platyclada, Tradescantia spathacea, \nGuzmania lingulata, Zamioculcas zamiifolia, \nCyperus alternifolius \n\nbest xylene removing plant - Zamioculcas zamiifolia  \n88% xylene removal within 72 hours.  \nxylene uptake was 0.81 \u00b10.01 mmol m\u22122 leaf area \n as  \n \n\n[64] \n\nZamioculcas zamiifolia \nAt 72 h of xylene exposure, Z. zamiifolia leaf uptake about \n0.86\u00b10.07 mmol m\u22122 per unit area. \n\n[48] \n\nD. deremensis \nO. microdasys  \n\nTime taken for complete removal of 2 ppm xylene from the \nairtight chamber of O. microdasys and D. dermensis plants \nwere respectively 47 hours and 98 hours.   \n\n[49] \n\nxora coccinea, Muraya paniculat, Ficus benjamina, \nEuphorbia milii, Adenium obesum, Millingtonia hortensis, \nDalbergia cochinchinensis, Pterocarpus indicus, \nPhyllanthus acidus, Cassia fistula, B. buttiana, Gardenia \njasminoides, Ehretia microphyllaLam \n\nUptake of xylene by B. buttiana plant parts \n   stems 53.1\u00b11.9% \n   epicuticular waxes 32.3\u00b10.9% \n   plant stomata - 14.6\u00b10.0% \n\n[65] \n\nFatsia japonica \nDraceana fragrans \n\nVolatile toluene and xylene removal efficiencies were \nincreased as the plant\u2019s root zone volume increased.", "start_char_idx": 23052, "end_char_idx": 25330, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b9dd623d-3c78-49da-8f5d-f0f4e80e637a": {"__data__": {"id_": "b9dd623d-3c78-49da-8f5d-f0f4e80e637a", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5254c04f-be91-43d5-9177-6794970e60e7", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "0b5694caa1d43f5d472dcfafc88b3007dda10a83f8c7d04d9a08a4d74231a1d0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "11b8c2a9-1ac2-4687-ab9c-503bcc711495", "node_type": "1", "metadata": {}, "hash": "308088150bc668c26217b29e826607f36975f3dbed1e06b75db99ecc0af128ae", "class_name": "RelatedNodeInfo"}}, "text": "[53] \n\nSchefflera actinophylla \nFicus benghalensis \n\nToluene and total xylene (m, p, o) removal efficiency leaf \narea over a 24-h period was  in  \n    S. actinophylla- 13.3 \u03bcg m\u22123 m\u22122 and 7.0 \u03bcg m\u22123 m\u22122 \n   F. benghalensis -  13.0 \u03bcg m\u22123 m\u22122  and 7.3 \u03bcg m\u22123 m\u22122 \n \n\n[54] \n\nPhoenix roebelenii \nPurification capability (Pa) increased with an increase in \nroom temperature from 21 to 26 \u00b0C, reaching a range of 15\u2013\n35 (V/h) \n\n[55] \n\nEpipremnum aureum \nSpathiphyllum \n\nRemoval rate for  \nTVOC -74%  \nOdor - 68%. \n\n[57] \n\nEpipremnum  aureum \nDavallia fejeensis \n\nEpipremnum  aureum plant had a positive impact on mixed \nVOC (decane, toluene, 2 ethylhexanol, benzene, octane, \nxylene, \u03b1- pinene)  filtration than Davallia fejeensis \n \n\n[58] \n\nTable 5. Plant species and their potential for Formaldehyde removal. \n\nPlant species Results Ref \n\nOsmunda japonica, Selaginella tamariscina, \nDavallia mariesii, Polypodium formosanum, \nPsidium guajava, Lavandula spp.,Pteris dispar, \nPteris multifidi, Pelargonium spp \n\nFormaldehyde removal 86 plant species were analyzed and \nOsmunda japonica showed the best 6.64 \u00b5g m\u20133 \nformaldehyde/cm2 of leaf area over 5 h \n\n[66] \n\nHedera helix, Chrysanthemum morifolium \nDieffenbachia compacta Epipremnum aureum \n\n90% removal by -Hedera Helix, Chrysanthemum morifolium, \nDieffenbachia compacta, Epipremnum aurenum (from the initial \namount of 1.63 ppm within 24 hours).  \n\n[67] \n\nFatsia japonica  \nFicus benjamina \n \n\nTime interval required to reduce 50% of benzene from the \ninitial concentration (2 \u00b5L L-1)  \n     F. japonica - 96 min  \n     F. benjamina.- 123 min \n\n[68] \n\nTillandsia velutina \nThe plant decreased Formaldehyde concentration by 22.51 % \nin 12 h \n\n[69] \n\nPhoenix roebelenii \nPurification capability (Pa) increased with an increase in \nroom temperature from 21 to 26 \u2103, reaching a range of 15\u2013\n35 (V/h) \n\n[55] \n\nSchefflera arboricola Nephrolepis exaltata These plants reported a high air purification ability [70] \n\nFatsia japonica \nReducing rate, \n225 \u03bcg m\u22123 the first 2 h  \naround 80 \u03bcg\u00b7m\u22123 for the final 3 h. \n\n[71] \n\nEpipremnum aureum Removal rate for  [57] \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  69 \n\nSpathiphyllum  TVOC - 74% \n Odor - 68%. \n\nNicotiana tabacum Transgenic plants increase formaldehyde removal by 20 % [72] \n\nChlorphytum comosum \nAloe vera \nEpipremnum aureum \n\nFormaldehyde removal efficiencies; spider plant-soil system \nat the light intensities of 90%, 92%, and 95% were \nrespectively 80 \u00b5molm\u22122s\u22121, 160 \u00b5mol m\u22122 s\u22121, and 240 \u00b5mol \nm\u22122 s\u22121 in the daytime.", "start_char_idx": 25333, "end_char_idx": 27908, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "11b8c2a9-1ac2-4687-ab9c-503bcc711495": {"__data__": {"id_": "11b8c2a9-1ac2-4687-ab9c-503bcc711495", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b9dd623d-3c78-49da-8f5d-f0f4e80e637a", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "fe22b5f2f05c21a0adba7608b2fa4a3527ed31ff5e1ce5ae1834232130e9c4e0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "40a3bc18-0810-407a-908a-923fba0c77e7", "node_type": "1", "metadata": {}, "hash": "5947d2bfa9857b8a9e821db9b8203f44f83a216cbf941ff6df09649117480330", "class_name": "RelatedNodeInfo"}}, "text": "[73] \n\nAglaonema commutatum, Spathiphyllum floribundum, \nCommutatum, Agave potatorum, Dracaena fragrans, \nD. reflexa, Cordyline fruticose, Gasteria gracilis, \nD. angustifolia , D. sanderiana, D. deremensis, \nSansevieria trifasciata, A.commutatum ,  \nAlocasia macrorrhiza, S. trifasciata, Aloe nobilis, \nScindapsus aureus, D. amoena, A.commutatum, \nScindapsus pictus, Philodendron sodiroi, \nSyngonium podophyllum , Asparagus setaceus, \nAloe aristata, Chlorophytum comosum, \nPhilodendron martianum , Zamioculcas zamiifolia, \nPhilodendron selloum  \n\nScindapsus aureus, Asparagus setaceus, S. trifasciata, C. \ncomosum, A. commutatum, A. commutatum , A. commutatum, S. \npictus, G. gracilis, and P. sodiroi reported a high formaldehyde \npurification capabilities with less damages. \n\n[74] \n\nChamaedorea elegans \nInitial formaldehyde concentration - 14.6 mg m-3 \n\nMaximum formaldehyde elimination capacity of 1.47 \nmg/m2h  \n\n[75] \n\nHedera helix \nHedera helix reported a  70% reduction of the required time to \nreach 0.5 ppm of gaseous HCHO when compared with \nnatural dissipation \n\n[76] \n\non environmental factors such as temperature, \n\nhumidity, and air pollutants [87,88]. Plant associated \n\nmicroflora plays a crucial role in VOC degradation by \n\nincreasing the bioavailability of VOCs to plants via the \n\nproduction of biosurfactants and the formation of \n\nbiofilms [89]. These microbial associations with plants \n\nincrease the ability of microorganisms to metabolize \n\nlarge numbers and varieties of organic compounds, \n\ntogether with improving plant strength of VOC \n\nremediation. Therefore, many studies have focused on \n\nthe ability of microbial air remediation and its potential \n\napplications. \n\nRole of microflora during air pollutant \nremoval and degradation \nPlant associated microbial flora helps the growth and \n\ndevelopment of the plant by enhancing the availability  \n\n of nutrients through the production of siderophores, \n\norganic acids, and plant growth promoters (Indole \n\nAcetic Acid (IAA)). It helps the plant\u2019s survival in biotic \n\nand abiotic stress conditions. As an example, during \n\nstressful conditions, ethylene is produced from 1-\n\naminocyclopropane-1-carboxylate (ACC). Bacteria can \n\nproduce 1-amino cyclopropane-1-carboxylatedeaminase \n\nand degrades ACC into ammonia and \u03b1-ketobutyrate \n\nand lowers the amount of ACC inside the plant \n\nresulting in the reduction of ethylene production and \n\nstress [10,90,91]. They not only support \n\nphytoremediation; through the detoxification, \n\ndegradation, and sequestration of the contaminants, but \n\nalso promote plant growth [92]. Phyllosphere bacteria \n\nfacilitate the absorption of pollutants into the plants. \n\nEndophytes and phyllosphere bacteria can degrade \n\nabsorbed pollutants by detoxification, transformation, or \n\nsequestration [93]. In soil pollution, root endophytes can \n\ndecrease phytotoxicity by enhancing the pollutant \n\naccumulation inside the plant [94]. Biological nitrogen \n\nfixation of Rhizobium bacteria incorporate carbon and \n\nnitrogen into the soil. These plant root nodule associated \n\nbacterial flora provide nutrients to plants. Natural \n\nbehaviors of bacteria improve the nutrient availability to \n\nthe plant and the environmental tolerance [95] through \n\nremediation of organic and metal contaminants by \n\nabsorbing, accumulating, detoxifying, and degrading \n\nthose pollutants [94]. Plant associated microflora \n\ndetoxifies the PM, which the host plant absorbs. PM \n\nactivates Reactive Oxygen Species (ROS) that adversely \n\naffect bacteria, but bacteria have mechanisms to detoxify \n\nROS toxicity [96,97].", "start_char_idx": 27911, "end_char_idx": 31516, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "40a3bc18-0810-407a-908a-923fba0c77e7": {"__data__": {"id_": "40a3bc18-0810-407a-908a-923fba0c77e7", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "11b8c2a9-1ac2-4687-ab9c-503bcc711495", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "197c96c7575e7e9fcdf4dc6aa793cc6bd0dc9d96f5e089c328c336adb54e4b01", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7834997f-8ccf-4b92-9d7b-11dfcc7f3882", "node_type": "1", "metadata": {}, "hash": "992ad4f788797d8422ce080dbe1a5e88024e68c9363a3ba6dfa2b83b1d46579a", "class_name": "RelatedNodeInfo"}}, "text": "Microorganisms have degradation \n\npathways to degrade and reduce the phytotoxicity of \n\npollutants. Therefore it reduces the evapotranspiration \n\nof volatile pollutants [93].  \n\nIn some cases, plants produce biogenic volatile organic \n\ncompounds. Thus VOC degrading microorganisms \n\nshould present in the phyllosphere. However, a limited \n\nnumber of studies are available about phyllosphere \n\nmicroflora since they are transient flora that occupies the \n\nphyllosphere temporarily, and the diversity changes \n\ndepending on various factors. Therefore, the study of \n\nthis transient flora is somewhat difficult. Many root \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  70 \n\nassociated VOC degrading microflora are used to treat \n\ngroundwater and soil and air remediation [10,92,98,99]. \n\nAir-remediation through soil is somewhat different; \n\nthere are trapped air and moisture inside the soil \n\nparticles. Once soil contains low moisture conditions air \n\nparticles with pollutants penetrates through the soil so \n\nthat the soil microflora can degrade those pollutants. \n\nAfter the water is supplied to the soil, cleaned air is \n\nreleased into the atmosphere. This is how soil and \n\nrhizosphere microflora contribute to removing indoor \n\nair pollution [59]. Microbial pollutant degrading \n\ncapabilities are enhanced when they are associated with \n\nplants [100].  Air pollution due to inorganic pollutants \n\n(NOx, SOx, and O3, etc.) also remediated through the \n\nmicroorganisms. It is a well understood fact that \n\nchemoorganotrophic bacteria (nitrogen producers, \n\nsulfur depositors, photosynthetic bacteria) use these \n\ninorganic compounds to generate energy. Ozone is a \n\ntoxic compound to bacteria, and it is used as a \n\nbactericidal agent. Therefore the use of bacteria in \n\ndetoxifying ozone is difficult [96,97]. \n\nMetabolic activities of bacteria in \nbioremediation of air pollutants  \nSeveral aromatic compounds have become significant \n\nair pollutants. Their persistence and widespread \n\noccurrence throughout the environment are facilitated \n\nby the thermodynamic stability of the benzene ring \n\n[101]. Microorganisms adapted to use these pollutants \n\nas their carbon sources through their catabolic pathways \n\n[102]. During aerobic respiration of microbes, oxygen is \n\nthe final electron acceptor, and it provides energy yield \n\nto the cell. In addition, oxygen helps to activate the \n\nsubstrates via oxygenation reactions [103]. Most of the \n\nPseudomonas sp. are aerobic therefore, many studies \n\nhave been conducted on its ability to degrade many \n\nenvironmental contaminants aerobically [104].   \n\nBacterial biodegradation of VOC relies on the type of \n\ndegrading enzymes and the microorganisms [105]. In \n\nthe aerobic catabolic funnel, most of the peripheral \n\npathways involve oxygenation reactions which are \n\ncarried out by monooxygenases and hydroxylating \n\ndioxygenases and generate dihydroxy aromatic \n\ncompounds such as catechol, homogentisate,  \n\nprotocatechuate, gentisate, homoprotocatechuate, \n\nhydroquinone, and hydroxyquinol. These intermediate \n\ncompounds are the substrates for ring cleavage \n\nenzymes. These enzymes use oxygen to open the \n\naromatic ring between the two hydroxyl groups like \n\northo cleavage, catalyzed by intradiol dioxygenases or \n\nproximal to at least one of the two hydroxyl groups \n\n(catalyzed by extradiol dioxygenases, and meta \n\ncleavage) [102]. \n\nAccording to Murray (1972) and Williams (1974), \n\nPseudomonas putida mt-2 strain utilizing toluene also \n\ngrown on the substrates like 1,2,4-trimethylbenzenem-\n\nethyltoluene, m-xylene, and p-xylene and oxidize all \n\nthese substrates to corresponding benzylalcohols, \n\nbenzaldehydes [106,107]. Subsequently, the above \n\nproducts were mineralized by meta-cleavage pathways.", "start_char_idx": 31517, "end_char_idx": 35334, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7834997f-8ccf-4b92-9d7b-11dfcc7f3882": {"__data__": {"id_": "7834997f-8ccf-4b92-9d7b-11dfcc7f3882", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "40a3bc18-0810-407a-908a-923fba0c77e7", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "082fe10714749a6e443264169c2d130017f86b21693fd9d65c7c63bc0306449c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "27a9f60c-b0d4-4ae4-9900-4700b38427c4", "node_type": "1", "metadata": {}, "hash": "78111c7e36405663f935e86f4abe775a8848b2da78e2ed11679417e35f373fc1", "class_name": "RelatedNodeInfo"}}, "text": "Subsequently, the above \n\nproducts were mineralized by meta-cleavage pathways. \n\nP.mendocina KR1, Ralstonia picketti PKO1, and \n\nBurkholderia vietnamiensis G4  reported degradation of \n\nbenzene as well as toluene using toluene-4-\n\nmonooxygenase  (TmoA), toluene 3-monooxygenase \n\n(TbuA1), and toluene 2-monoocygenase (TomA), \n\nrespectively [108\u2013110]. Nitrosomonas europea produced \n\namminomonooxygenase enzyme, which activates by \n\nammonia and oxidize BTEX compounds [111]. \n\nBacterial mobile genetic elements like plasmids and \n\ntransposons contain genes responsible for these \n\ncatabolic activities. Once bacteria are exposed to the \n\ncontaminated environment, they facilitate the horizontal \n\ngene transformation and rapid adaptation to utilize the \n\npollutants [104]. Bacterial natural adaptations and \n\npollutant remediation is a slow and time-consuming \n\nprocess. However, their utilization for in-situ \n\nbioremediation of polluted sites, and biotransformation \n\nof toxic compounds into non-toxic compounds such as \n\nfine chemicals and other value added products, \n\ndevelopment of in-situ high sensitive biomonitoring \n\ndevices such as biosensors are the techniques that can be \n\nused to enhance the remediation process [112\u2013114]. \n\nConclusion  \nSeveral methods have been proposed to reduce the \n\nindoor air pollution caused by various chemicals \n\nreleased into the air due to anthropogenic activities \n\noccurring indoors. Although chemical and physical \n\nmethods are available, most of them have issues in \n\nefficiency, short-life span, high cost, need for recovery \n\nsystems, high maintenance demand, and secondary \n\npollutants generated during VOC removal. Use of \n\nplants and their associated microflora provides a \n\nsolution to these issues as an economical and \n\nenvironmentally friendly alternative. This review \n\nprovides an overview of the use of ornamental plants \n\nand their associated microflora in removing the air \n\npollutants indoors. According to the literature \n\nZamioculcas zamiifolia, Spathiphyllum wallisii, Sansevieria \n\ntrifasciata, Hedera helix, and Ficus benjamina plants can be \n\nsuggested as the effective plants for benzene, toluene, \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  71 \n\nxylene, and formaldehyde removal.  Microbial \n\nassociations with plants benefit in VOC remediation \n\nbecause it increases the microbial capability in \n\nmetabolizing large numbers and varieties of organic \n\ncompounds. Also microflora influence the plant \n\nstrength during VOC remediation. More laboratory and \n\nfield studies are needed to increase the efficiency in \n\nusing plants for indoor air purification as well as to \n\nunderstand their mechanisms of air purification.  \n\nAcknowledgment \nThis research was supported by the Accelerating Higher \n\nEducation Expansion and Development (AHEAD) \n\nDevelopment Oriented Research (DOR) grant of the \n\nMinistry of Higher Education, Sri Lanka funded by the \n\nWorld Bank \n\nAuthor\u2019s Contribution  \nThe authors confirm contribution to the paper as \n\nfollows: study conception and design: \n\nY.H.K.I.S.Gunasinghe, I.V.N.Rathnayake, and \n\nM.P.Deeyamulla; draft manuscript preparation: \n\nY.H.K.I.S.Gunasinghe; Review, and editing the final \n\ndraft: Y.H.K.I.S.Gunasinghe, I.V.N.Rathnayake; All \n\nauthors read and approved the final manuscript. \n\nCompeting Interest  \nThe authors declare that they have no competing \n\ninterest, which includes personal, financial, or any other \n\nkind of relationship with people or organizations that \n\ncould inappropriately affect this review. \n\nEthics approval \nNot applicable. \n\nReferences \n1. Kirchner S, Derbez M, Duboudin C, Elias P, Gregoire A, Lucas J-\n\nP, et al. Indoor air quality in French dwellings. Indoor Air 2008 \n[Internet]. 2008. p. Paper-ID.", "start_char_idx": 35256, "end_char_idx": 39047, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "27a9f60c-b0d4-4ae4-9900-4700b38427c4": {"__data__": {"id_": "27a9f60c-b0d4-4ae4-9900-4700b38427c4", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7834997f-8ccf-4b92-9d7b-11dfcc7f3882", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "0c0754cdb89d0bdf30cc9798b4c0492117252a9c467856c3e954dc98a1439209", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2aa6f11e-e5c8-45ef-b0bc-9566f4e35e34", "node_type": "1", "metadata": {}, "hash": "9e0f50f97002e00d5ede9b67fff29c9a637b86d6329fc1a2170da38fc71f7216", "class_name": "RelatedNodeInfo"}}, "text": "Indoor Air 2008 \n[Internet]. 2008. p. Paper-ID. Available from: https://hal.archives-\nouvertes.fr/hal-00688556 \n\n2. Lahtinen M, Huuhtanen P, Reijula K. Sick Building Syndrome \nand Psychosocial Factors - a Literature Review. Indoor Air \n[Internet]. 1998;8:71\u201380. Available from: \nhttp://doi.wiley.com/10.1111/j.1600-0668.1998.tb00012.x \n\n3. Newby DE, Mannucci PM, Tell GS, Baccarelli AA, Brook RD, \nDonaldson K, et al. Expert position paper on air pollution and \ncardiovascular disease. Eur Heart J [Internet].2015;36:83\u2013\n93.Available from: https://academic.oup.com/eurheartj/article-\nlookup/doi/10.1093/eurheartj/ehu458 \n\n4. Burge PS. Sick building syndrome. Occup Environ Med. BMJ \nPublishing Group Ltd; 2004;61:185\u201390.  \n\n5. WHO IAQ. Household air pollution and health [Internet]. 2018. \nAvailable from: https://www.who.int/en/news-room/fact-\nsheets/detail/household-air-pollution-and-health \n\n6. Yang DS, Pennisi S V., Son K-C, Kays SJ. Screening Indoor Plants \nfor Volatile Organic Pollutant Removal Efficiency. HortScience \n[Internet]. 2009;44:1377\u201381. Available \nfrom:https://journals.ashs.org/view/journals/hortsci/44/5/article-\np1377.xml \n\n7. Yua BF, Hu ZB, Liu M, Yang HL, Kongb QX, Liu YH. Review of \nresearch on air-conditioning systems and indoor air quality \ncontrol for human health. Elsevier [Internet]. Elsevier Ltd and \nIIR; 2008; Available from:https://journals.ashs.org/view/ \njournals/hortsci/44/5/article-p1377.xml \n\n8. Guieysse B, Hort C, Platel V, Munoz R, Ondarts M, Revah S. \nBiological treatment of indoor air for VOC removal: Potential and \nchallenges. Biotechnol Adv [Internet]. 2008;26:398\u2013410. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/S0734975008000426 \n\n9. Casset A, Braun J-J. Relation entre allerg\u00e8nes de l\u2019environnement \nint\u00e9rieur, sensibilisation et sympt\u00f4mes de rhinite et asthme \nallergiques. Rev Mal Respir [Internet]. 2010;27:913\u201320. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/S0761842510003669 \n\n10. Weyens N, van der Lelie D, Taghavi S, Vangronsveld J. \nPhytoremediation: plant\u2013endophyte partnerships take the \nchallenge. Curr Opin Biotechnol [Internet]. Elsevier; 2009;20:248\u2013\n54. Available from: https://linkinghub.elsevier.com/retrieve \n/pii/S0958166909000238 \n\n11. Khaksar G, Treesubsuntorn C, Thiravetyan P. Endophytic \nBacillus cereus ERBP\u2014Clitoria ternatea interactions: Potentials \nfor the enhancement of gaseous formaldehyde removal. Environ \nExp Bot [Internet]. Elsevier B.V.; 2016;126:10\u201320. Available from: \nhttp://dx.doi.org/10.1016/j.envexpbot.2016.02.009 \n\n12. Kumar SR, Arumugam T, Anandakumar CR, Balakrishnan S, \nRajavel DS. PROPERTIES Use of Plant Species in Controlling \nEnvironmental Pollution- A Review. Acad Environ Life Sci \n[Internet]. 2013;2:52\u201363. Available from: www.bepls.com \n\n13.", "start_char_idx": 39000, "end_char_idx": 41791, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2aa6f11e-e5c8-45ef-b0bc-9566f4e35e34": {"__data__": {"id_": "2aa6f11e-e5c8-45ef-b0bc-9566f4e35e34", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "27a9f60c-b0d4-4ae4-9900-4700b38427c4", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "655011c682acdf845463d969510f638dcaa7bd5b0dd1f3ecfcf8098095330922", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "464924bc-4685-4141-97a4-991c2db93582", "node_type": "1", "metadata": {}, "hash": "04680c1cf996874d48a24eb2ef01a20c05f792feedd3cbf045cf9a5140f0bd07", "class_name": "RelatedNodeInfo"}}, "text": "Available from: www.bepls.com \n\n13. Volatile Organic Compounds\u2019 Impact on Indoor Air Quality | \nIndoor Air Quality (IAQ) | US EPA [Internet]. 2019. Available \nfrom: https://www.epa.gov/indoor-air-quality-iaq/volatile-organic-\ncompounds-impact-indoor-air-quality \n\n14. Spengler J, Sexton K. Indoor air pollution: a public health \nperspective. Science (80- ) [Internet]. 1983;221:9\u201317. Available \nfrom: https://www.sciencemag.org/lookup/doi/10.1126/science.6857273 \n\n15. Myers I, Maynard RL. Polluted air\u2014outdoors and indoors. \nOccup Med (Chic Ill) [Internet]. Oxford University Press; \n2005;55:432\u20138. Available from: http://academic.oup.com/occmed/ \narticle/55/6/432/1415876/Polluted-airoutdoors-and-indoors \n\n16. Bono R, Traversi D, Maestri L, Schilir\u00f2 T, Ghittori S, Baiocchi C, et \nal. Urban air and tobacco smoke in benzene exposure in a cohort \nof traffic policemen. Chem Biol Interact [Internet]. 2005;153\u2013\n154:239\u201342. Available from: https://linkinghub.elsevier.com/ \nretrieve/pii/S0009279705000918 \n\n17. WHO Regional Office. Air quality guidelines for Europe. Environ \nSci Pollut Res [Internet]. 1996;3:23\u201323. Available from: \nhttp://link.springer.com/10.1007/BF02986808 \n\n18. Ao CH, Lee SC. Removal of indoor air ppb level volatile organic \ncompounds (VOCs) and NOx by heterogeneous photocatalysis. \nProceeding Better Air Qual Asian Pacific Rim Cities [Internet]. \n2002;16\u20138. Available from: https://scholar.google.com/scholar \n?hl=en&as_sdt=0%2C5&q=Ao+CH%2C+Lee+SC.+Removal+of+indo\nor+air+ppb+level+volatile+organic+compounds+%28VOCs%29+and+\nNOx+by+heterogeneous+photocatalysis.+Proceeding+Better+Air+Qua\nl+Asian+Pacific+Rim+Cities.+2002%3B16\u20138.&btnG= \n\n19. Kim S-S, Kang D-H, Choi D-H, Yeo M-S, Kim K-W. Comparison \nof strategies to improve indoor air quality at the pre-occupancy \nstage in new apartment buildings. Build Environ [Internet]. \nElsevier; 2008;43:320\u20138. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S0360132306003453 \n\n20. MESICK HH. 1. Foliage Plants for Indoor Removal of the Primary \nCombustion Gases Carbon Monoxide and Nitrogen Dioxide. J \nMississippi Acad Sci. 1985;30.  \n\n21. Smith KR. Indoor air pollution in developing countries: \nrecommendations for research. Indoor Air. Wiley Online Library; \n2002;12:198\u2013207.  \n\n22. Huang Y, Ho S, Lu Y, Niu R, Xu L, Cao J, et al. Removal of Indoor \nVolatile Organic Compounds via Photocatalytic Oxidation: A \nShort Review and Prospect. Molecules [Internet]. 2016;21:56. \nAvailable from: http://www.mdpi.com/1420-3049/21/1/56 \n\n23. 23. Bose-O\u2019Reilly S, McCarty KM, Steckling N, Lettmeier B. \nMercury Exposure and Children\u2019s Health. Curr Probl Pediatr \nAdolesc Health Care [Internet]. 2010;40:186\u2013215. Available from: \n\n\n\nNepal J Biotechnol.", "start_char_idx": 41756, "end_char_idx": 44483, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "464924bc-4685-4141-97a4-991c2db93582": {"__data__": {"id_": "464924bc-4685-4141-97a4-991c2db93582", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2aa6f11e-e5c8-45ef-b0bc-9566f4e35e34", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "05875fdd22851aea99477ba7c6be0b1cd95811c89f1075e2f8e452e04d8202da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c11b5970-75f4-447b-b03a-087bbda18cae", "node_type": "1", "metadata": {}, "hash": "c2bb38b75a33cdc84f32a8f3e184c79cbb28aaf5e6baaf1b044dc5dd7e0ccbce", "class_name": "RelatedNodeInfo"}}, "text": "Available from: \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  72 \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S153854421000093\n3 \n\n24. Sepp\u00e4nen O, Fisk WJ. Association of ventilation system type with \nSBS symptoms in office workers. Indoor Air [Internet]. \n2002;12:98\u2013112. Available from: http://doi.wiley.com/10.1034/j.1600-\n0668.2002.01111.x \n\n25. Lahtinen M, Huuhtanen P, Reijula K. Sick Building Syndrome \nand Psychosocial Factors - a Literature Review. Indoor Air \n[Internet]. Wiley Online Library; 1998;8:71\u201380. Available from: \nhttp://doi.wiley.com/10.1111/j.1600-0668.1998.tb00012.x \n\n26. WHO. Exposure to air pollution: a major public health concern. \nWHO Doc Prod Serv Geneva [Internet]. 2010; Available from: \nhttps://www.who.int/ipcs/features/air_pollution.pdf \n\n27. Bernstein JA, Alexis N, Bacchus H, Bernstein IL, Fritz P, Horner \nE, et al. The health effects of nonindustrial indoor air pollution. J \nAllergy Clin Immunol [Internet]. Elsevier; 2008;121:585\u201391. \nAvailable from: https://linkinghub.elsevier.com/retrieve \n/pii/S0091674907022099 \n\n28. Cogliano VJ, Baan R, Straif K, Grosse Y, Lauby-Secretan B, El \nGhissassi F, et al. Preventable Exposures Associated With Human \nCancers. JNCI J Natl Cancer Inst [Internet]. 2011;103:1827\u201339. \nAvailable from: https://academic.oup.com/jnci/article-\nlookup/doi/10.1093/jnci/djr483 \n\n29. Bussel JB, Berkowitz RL, McFarland JG, Lynch L, Chitkara U. The \nNew England Journal of Medicine Downloaded from nejm.org at \nPENN STATE UNIVERSITY on November 25, 2015. For personal \nuse only. No other uses without permission. From the NEJM \nArchive. Copyright \u00a9 2010 Massachusetts Medical Society. All \nrights reserved. N Engl J Med. 1988;319:1374\u20138.  \n\n30. Collins JJ. Lymphohaematopoeitic cancer mortality among \nworkers with benzene exposure. Occup Environ Med [Internet]. \n2003;60:676\u20139. Available from: https://oem.bmj.com/lookup/doi/ \n10.1136/oem.60.9.676 \n\n31. VAN RAALTE HGS. Leukemia and Benzene. Ann Intern Med \n[Internet]. 1983;99:885. Available from: \nhttp://annals.org/article.aspx?doi=10.7326/0003-4819-99-6-885 \n\n32. Directive C. 69/EC of 16 November 2000 relating to limit values \nfor benzene and carbon monoxide in ambient air. Off J Eur \nCommunities, No L. 2000;313:12\u20133.  \n\n33. Becerra TA, Wilhelm M, Olsen J, Cockburn M, Ritz B. Ambient \nAir Pollution and Autism in Los Angeles County, California. \nEnviron Health Perspect [Internet]. National Institute of \nEnvironmental Health Sciences; 2013;121:380\u20136. Available from: \nhttps://ehp.niehs.nih.gov/doi/10.1289/ehp.1205827 \n\n34. Raz R, Roberts AL, Lyall K, Hart JE, Just AC, Laden F, et al.", "start_char_idx": 44444, "end_char_idx": 47102, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c11b5970-75f4-447b-b03a-087bbda18cae": {"__data__": {"id_": "c11b5970-75f4-447b-b03a-087bbda18cae", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "464924bc-4685-4141-97a4-991c2db93582", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "010d2a95c25dd38ea808e22a7670fb1eec708466155b9fe220a722279acff1de", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "631fac86-dad1-4cd3-962b-a49f0e7d54ab", "node_type": "1", "metadata": {}, "hash": "deaadb92fe91691abb15f1389aef654f6fcf6c5e277703df4cfe3e411c4382e8", "class_name": "RelatedNodeInfo"}}, "text": "Autism Spectrum Disorder and Particulate Matter Air Pollution \nbefore, during, and after Pregnancy: A Nested Case\u2013Control \nAnalysis within the Nurses\u2019 Health Study II Cohort. Environ \nHealth Perspect [Internet]. NLM-Export; 2015;123:264\u201370. \nAvailable from: https://ehp.niehs.nih.gov/doi/10.1289/ehp.1408133 \n\n35. Health Canada [Internet]. 2018. Available from: \nhttps://www.canada.ca/en/health-canada.html \n\n36. WHO guidelines for air quality. Indian Pediatr. 2010;35:812\u20135.  \n37. EPA US. Environmental Protection Agency (2002). Mercur Hum \n\nExpo http//www epa gov/hg/exposure htm [Internet]. 2008; \nAvailable from: https://www.epa.gov/ \n\n38. Luengas A, Barona A, Hort C, Gallastegui G, Platel V, Elias A. A \nreview of indoor air treatment technologies. Rev Environ Sci \nBio/Technology [Internet]. 2015;14:499\u2013522. Available from: \nhttp://link.springer.com/10.1007/s11157-015-9363-9 \n\n39. Liu G, Xiao M, Zhang X, Gal C, Chen X, Liu L, et al. A review of \nair filtration technologies for sustainable and healthy building \nventilation. Sustain Cities Soc [Internet]. 2017;32:375\u201396. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS221067071630734X \n\n40. Brilli F, Fares S, Ghirardo A, de Visser P, Calatayud V, Mu\u00f1oz A, \net al. Plants for Sustainable Improvement of Indoor Air Quality. \nTrends Plant Sci [Internet]. Elsevier Ltd; 2018;23:507\u201312. \nAvailable from: http://dx.doi.org/10.1016/j.tplants.2018.03.004 \n\n41. Dela Cruz M, Christensen JH, Thomsen JD, M\u00fcller R. Can \nornamental potted plants remove volatile organic compounds \nfrom indoor air? \u2014 a review. Environ Sci Pollut Res [Internet]. \n\n2014;21:13909\u201328. Available from: http://link.springer.com/10.1007/ \ns11356-014-3240-x \n\n42. SCHMITZ H, HILGERS U, WEIDNER M. Assimilation and \nmetabolism of formaldehyde by leaves appear unlikely to be of \nvalue for indoor air purification. New Phytol [Internet]. \n2000;147:307\u201315. Available from: http://doi.wiley.com/10.1046/ \nj.1469-8137.2000.00701.x \n\n43. Zhang W, Tang L, Sun H, Han S, Wang X, Zhou S, et al. C1 \nmetabolism plays an important role during formaldehyde \nmetabolism and detoxification in petunia under liquid HCHO \nstress. Plant Physiol Biochem [Internet]. Elsevier; 2014;83:327\u201336. \nAvailable from: https://linkinghub.elsevier.com/retrieve/ \npii/S0981942814002642 \n\n44. Durmishidze S V. Cleavage of the aromatic ring of some \nexogenous compounds in plants. Metsniereba, Tbilisi. 1975.  \n\n45. Hill AC. Vegetation: A Sink for Atmospheric Pollutants. J Air \nPollut Control Assoc [Internet]. 1971;21:341\u20136. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/00022470.1971.10469535 \n\n46. Hosker RP, Lindberg SE. Review: Atmospheric deposition and \nplant assimilation of gases and particles. Atmos Environ \n[Internet]. 1982;16:889\u2013910.", "start_char_idx": 47104, "end_char_idx": 49878, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "631fac86-dad1-4cd3-962b-a49f0e7d54ab": {"__data__": {"id_": "631fac86-dad1-4cd3-962b-a49f0e7d54ab", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c11b5970-75f4-447b-b03a-087bbda18cae", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "3455aa24caabb2851c3c8602d346a7269892c1f4e2b72657cb1e93f4ac8d4ae5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "83c016a7-55b2-4d8c-902f-647985cc7532", "node_type": "1", "metadata": {}, "hash": "05b13ae123b1cbc397f937acdfdb24aaee05c7d901218e56ecc58fbf98543e7f", "class_name": "RelatedNodeInfo"}}, "text": "Atmos Environ \n[Internet]. 1982;16:889\u2013910. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/0004698182901755 \n\n47. Florentina I, Io B. The Effects of Air Pollutants on Vegetation and \nthe Role of Vegetation in Reducing Atmospheric Pollution. \nImpact Air Pollut Heal Econ Environ Agric Sources [Internet]. \nInTech; 2011. Available from: http://www.intechopen.com/books/the-\nimpact-of-air-pollution-on-health-economy-environment-and-\nagricultural-sources/the-effects-of-air-pollutants-on-vegetation-and-the-\nrole-of-vegetation-in-reducing-atmospheric-pollu \n\n48. Sriprapat W, Thiravetyan P. Phytoremediation of BTEX from \nIndoor Air by Zamioculcas zamiifolia. Water, Air, Soil Pollut \n[Internet]. 2013;224:1482. Available from: \nhttp://link.springer.com/10.1007/s11270-013-1482-8 \n\n49. Mosaddegh MH, Jafarian A, Ghasemi A, Mosaddegh A. \nPhytoremediation of benzene, toluene, ethylbenzene and xylene \ncontaminated air by D. deremensis and O. microdasys plants. J \nEnviron Heal Sci Eng [Internet]. 2014;12:39. Available from: \nhttp://link.springer.com/10.1186/2052-336X-12-39 \n\n50. H\u00f6rmann V, Brenske K-R, Ulrichs C. Assessment of filtration \nefficiency and physiological responses of selected plant species to \nindoor air pollutants (toluene and 2-ethylhexanol) under \nchamber conditions. Environ Sci Pollut Res [Internet]. \nEnvironmental Science and Pollution Research; 2018;25:447\u201358. \nAvailable from: http://link.springer.com/10.1007/s11356-017-0453-9 \n\n51. Hwa Yoo M, Kwon YJ, Son K-C, Kays SJ. Efficacy of Indoor \nPlants for the Removal of Single and Mixed Volatile Organic \nPollutants and Physiological Effects of the Volatiles on the Plants. \nJ Am Soc Hortic Sci [Internet]. 2006;131:452\u20138. Available from: \nhttps://journals.ashs.org/view/journals/jashs/131/4/article-p452.xml \n\n52. Kim KJ, Yoo EH, Jeong M Il, Song JS, Lee SY, Kays SJ. Changes in \nthe Phytoremediation Potential of Indoor Plants with Exposure to \nToluene. HortScience [Internet]. 2011;46:1646\u20139. Available from: \nhttps://journals.ashs.org/view/journals/hortsci/46/12/article-p1646.xml \n\n53. Kim KJ, Jung HH, Seo HW, Lee JA, Kays SJ. Volatile Toluene and \nXylene Removal Efficiency of Foliage Plants as Affected by Top \nto Root Zone Size. HortScience [Internet]. 2014;49:230\u20134. \nAvailable from: https://journals.ashs.org/view/journals/hortsci/ \n49/2/article-p230.xml \n\n54. Kim KJ, Kim HJ, Khalekuzzaman M, Yoo EH, Jung HH, Jang HS. \nRemoval ratio of gaseous toluene and xylene transported from air \nto root zone via the stem by indoor plants. Environ Sci Pollut Res \n[Internet]. 2016;23:6149\u201358. Available from: \nhttp://link.springer.com/10.1007/s11356-016-6065-y \n\n55. Baosheng K, Shibata SI, Sawada A, Oyabu T, Kimura H. Air \nPurification Capability of Potted Phoenix Roebelenii and Its \nInstallation Effect in Indoor Space. Sensors Mater [Internet]. \n2009;21:445.", "start_char_idx": 49835, "end_char_idx": 52686, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "83c016a7-55b2-4d8c-902f-647985cc7532": {"__data__": {"id_": "83c016a7-55b2-4d8c-902f-647985cc7532", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "631fac86-dad1-4cd3-962b-a49f0e7d54ab", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "51f7868cd4db624f0747470792f4a38d408367099a203fa6f012e83a65c05824", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "249664e0-900c-446e-aea6-87d4669f0512", "node_type": "1", "metadata": {}, "hash": "3c61188e7556c3bfcb6a133929f56e893856037575ef9fd85d45f6d04e6e54e5", "class_name": "RelatedNodeInfo"}}, "text": "Sensors Mater [Internet]. \n2009;21:445. Available from: \nhttp://myukk.org/SM2017/article.php?ss=590 \n\n56. De Kempeneer L, Sercu B, Vanbrabant W, Van Langenhove H, \nVerstraete W. Bioaugmentation of the phyllosphere for the \nremoval of toluene from indoor air. Appl Microbiol Biotechnol \n[Internet]. 2004;64:284\u20138. Available from: \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  73 \n\nhttp://link.springer.com/10.1007/s00253-003-1415-3 \n\n57. TakashiOYABU\", AyakoSAWADA\uff61, HiroyukiKURODA\" T \nandTakayukiYOSHIOKA\". Purification Capabilities o fGolden \nPothos and Peace Lily fbr Indoor Air Pollutants and lts \nApplication to a Relaxation Space. J Agric Meteorol. 2005;6:145\u2013\n114.  \n\n58. Mikkonen A, Li T, Vesala M, Saarenheimo J, Ahonen V, \nK\u00e4renlampi S, et al. Biofiltration of airborne VOCs with green \nwall systems-Microbial and chemical dynamics. Indoor Air \n[Internet]. 2018;28:697\u2013707. Available from: \nhttp://doi.wiley.com/10.1111/ina.12473 \n\n59.  Orwell RL, Wood RL, Tarran J, Torpy F, Burchett MD. Removal \nof Benzene by the Indoor Plant/Substrate Microcosm and \nImplications for Air Quality. Water, Air, Soil Pollut [Internet]. \n2004;157:193\u2013207. Available from: http://link.springer.com/10.1023/ \nB:WATE.0000038896.55713.5b \n\n60. Wood RA, Orwell RL, Tarran J, Torpy F, Burchett M. Potted-\nplant/growth media interactions and capacities for removal of \nvolatiles from indoor air. J Hortic Sci Biotechnol [Internet]. \n2002;77:120\u20139. Available from: http://www.tandfonline.com/doi/full/ \n10.1080/14620316.2002.11511467 \n\n61. Liu Y-J, Mu Y-J, Zhu Y-G, Ding H, Crystal Arens N. Which \nornamental plant species effectively remove benzene from indoor \nair? Atmos Environ [Internet]. 2007;41:650\u20134. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S1352231006008077 \n\n62. Treesubsuntorn C, Thiravetyan P. Removal of benzene from \nindoor air by Dracaena sanderiana: Effect of wax and stomata. \nAtmos Environ [Internet]. Elsevier Ltd; 2012;57:317\u201321. Available \nfrom: http://dx.doi.org/10.1016/j.atmosenv.2012.04.016 \n\n63. Irga PJ, Torpy FR, Burchett MD. Can hydroculture be used to \nenhance the performance of indoor plants for the removal of air \npollutants? Atmos Environ [Internet]. Elsevier Ltd; 2013;77:267\u2013\n71. Available from: http://dx.doi.org/10.1016/j.atmosenv.2013.04.078 \n\n64. Sriprapat W, Boraphech P, Thiravetyan P. Factors affecting \nxylene-contaminated air removal by the ornamental plant \nZamioculcas zamiifolia. Environ Sci Pollut Res [Internet]. \n2014;21:2603\u201310. Available from: http://link.springer.com/10.1007 \n/s11356-013-2175-y \n\n65.", "start_char_idx": 52647, "end_char_idx": 55256, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "249664e0-900c-446e-aea6-87d4669f0512": {"__data__": {"id_": "249664e0-900c-446e-aea6-87d4669f0512", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "83c016a7-55b2-4d8c-902f-647985cc7532", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "bfab951409d80b499ce7db10085ab9458f5c3905872315c3eb82d50444943f66", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4c8cd93f-155f-44c0-a202-56d2df8b9633", "node_type": "1", "metadata": {}, "hash": "e6e822f840ce46830e9a970bb91397cba931e8a3a2f1a2148abb8813dba1c700", "class_name": "RelatedNodeInfo"}}, "text": "Sangthong S, Suksabye P, Thiravetyan P. Air-borne xylene \ndegradation by Bougainvillea buttiana and the role of epiphytic \nbacteria in the degradation. Ecotoxicol Environ Saf [Internet]. \nElsevier; 2016;126:273\u201380. Available from: \nhttp://dx.doi.org/10.1016/j.ecoenv.2015.12.017 \n\n66. Kim KJ, Jeong M Il, Lee DW, Song JS, Kim HD, Yoo EH, et al. \nVariation in Formaldehyde Removal Efficiency among Indoor \nPlant Species. HortScience [Internet]. 2010;45:1489\u201395. Available \nfrom: https://journals.ashs.org/view/journals/hortsci/45/10/article-\np1489.xml \n\n67. Aydogan A, Montoya LD. Formaldehyde removal by common \nindoor plant species and various growing media. Atmos Environ \n[Internet]. Elsevier Ltd; 2011;45:2675\u201382. Available from: \nhttp://dx.doi.org/10.1016/j.atmosenv.2011.02.062 \n\n68. Kim KJ, Kil MJ, Song JS, Yoo EH, Son K-C, Kays SJ. Efficiency of \nVolatile Formaldehyde Removal by Indoor Plants: Contribution \nof Aerial Plant Parts versus the Root Zone. J Am Soc Hortic Sci \n[Internet]. 2008;133:521\u20136. Available from: https://journals.ashs.org \n/view/ journals/jashs/133/4/article-p521.xml \n\n69. Li P, Pemberton R, Zheng G. Foliar trichome-aided formaldehyde \nuptake in the epiphytic Tillandsia velutina and its response to \nformaldehyde pollution. Chemosphere [Internet]. Elsevier Ltd; \n2015;119:662\u20137. Available from: http://dx.doi.org/10.1016/ \nj.chemosphere.2014.07.079 \n\n70. Hasegawa Y, Asada S, Katsube T, Ikeguchi T. Analysis of \nbioelectrical potential when plant purifies air pollution. IEICE \nTrans Electron [Internet]. 2004;E87-C:2093\u20138. Available from: \nhttps://www.researchgate.net/profile/Yuki_Hasegawa7/publication/237\n415885_Analysis_of_Bioelectrical_Potential_When_Plant_Purifies_Air\n_Pollution/links/58f63e050f7e9b67a34b83b2/Analysis-of-Bioelectrical-\nPotential-When-Plant-Purifies-Air-Pollution.pdf \n\n71. Lim Y-W, Kim H-H, Yang J-Y, Kim K-J, Lee J-Y, Shin D-C. \nImprovement of Indoor Air Quality by Houseplants in New-built \nApartment Buildings. J Japanese Soc Hortic Sci [Internet]. \n\n2009;78:456\u201362. Available from: http://joi.jlc.jst.go.jp/JST.JSTAGE \n/jjshs1/78.456?from=CrossRef \n\n72. Sawada A, Oyabu T, Chen L, Hirai N, Izui K. Purification \nCapability of Tobacco Transformed with Enzymes from a \nMethylotrophic BACTERIUM for Formaldehyde. Int J \nPhytoremediation [Internet]. 2007;9:487\u201396. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/15226510701709630 \n\n73. Xu Z, Wang L, Hou H. Formaldehyde removal by potted plant\u2013\nsoil systems. J Hazard Mater [Internet]. Elsevier B.V.; \n2011;192:314\u20138. Available from: http://dx.doi.org/10.1016 \n/j.jhazmat.2011.05.020 \n\n74. Zhou J, Qin F, Su J, Liao J wu, Xu H lian.", "start_char_idx": 55257, "end_char_idx": 57912, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4c8cd93f-155f-44c0-a202-56d2df8b9633": {"__data__": {"id_": "4c8cd93f-155f-44c0-a202-56d2df8b9633", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "249664e0-900c-446e-aea6-87d4669f0512", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "9e6d165ffd7ef2dcbe90426df84d60bcce790f8e60812c9019edc3852bc263c1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "98172b00-77fa-4796-b29d-ee3c43ca234f", "node_type": "1", "metadata": {}, "hash": "780e4c3d09bd5a052902fc3e5a2654a35719e92af7fbe40d6f5104a59bb04b4c", "class_name": "RelatedNodeInfo"}}, "text": "Purification of \nformaldehyde-polluted air by indoor plants of Araceae, \nAgavaceae and Liliaceae. J Food, Agric Environ [Internet]. \n2011;9:1012\u20138. Available from: https://www.researchgate.net \n/profile/Hui-lian_Xu/publication/231184098_Purification _of_ \nformaldehyde-polluted_air_by_indoor_plants_of_Araceae_ \nAgavaceae_and_Liliaceae/links/0fcfd5065a101d697c000000/Purificatio\nn-of-formaldehyde-polluted-air-by-indoor-plants- \n\n75. Teiri H, Pourzamani H, Hajizadeh Y. Phytoremediation of VOCs \nfrom indoor air by ornamental potted plants: A pilot study using \na palm species under the controlled environment. Chemosphere \n[Internet]. Elsevier B.V.; 2018;197:375\u201381. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653518300869 \n\n76. Lin M-W, Chen L-Y, Chuah Y-K. Investigation of A Potted Plant \n(Hedera helix) with Photo-Regulation to Remove Volatile \nFormaldehyde for Improving Indoor Air Quality. Aerosol Air \nQual Res [Internet]. 2017;17:2543\u201354. Available from: \nhttps://aaqr.org/articles/aaqr-17-04-oa-0145 \n\n77. Fierer N. Embracing the unknown: disentangling the \ncomplexities of the soil microbiome. Nat Rev Microbiol [Internet]. \nNature Publishing Group; 2017;15:579\u201390. Available from: \nhttp://dx.doi.org/10.1038/nrmicro.2017.87 \n\n78. Burns KN, Kluepfel DA, Strauss SL, Bokulich NA, Cantu D, \nSteenwerth KL. Vineyard soil bacterial diversity and composition \nrevealed by 16S rRNA genes: Differentiation by geographic \nfeatures. Soil Biol Biochem [Internet]. Elsevier Ltd; 2015;91:232\u2013\n47. Available from: http://dx.doi.org/10.1016/j.soilbio.2015.09.002 \n\n79. Faist H, Keller A, Hentschel U, Deeken R. Grapevine (Vitis \nvinifera) Crown Galls Host Distinct Microbiota. Drake HL, editor. \nAppl Environ Microbiol [Internet]. 2016;82:5542\u201352. Available \nfrom: http://aem.asm.org/lookup/doi/10.1128/AEM.01131-16 \n\n80. Hardoim PR, van Overbeek LS, Berg G, Pirttil\u00e4 AM, Compant S, \nCampisano A, et al. The Hidden World within Plants: Ecological \nand Evolutionary Considerations for Defining Functioning of \nMicrobial Endophytes. Microbiol Mol Biol Rev [Internet]. \n2015;79:293\u2013320. Available from: https://mmbr.asm.org/content \n/79/3/293 \n\n81. Hardoim PR, Hardoim CCP, van Overbeek LS, van Elsas JD. \nDynamics of Seed-Borne Rice Endophytes on Early Plant Growth \nStages. Baker SE, editor. PLoS One [Internet]. 2012;7:e30438. \nAvailable from: https://dx.plos.org/10.1371/journal.pone.0030438 \n\n82. Liu Y, Zuo S, Xu L, Zou Y, Song W. Study on diversity of \nendophytic bacterial communities in seeds of hybrid maize and \ntheir parental lines. Arch Microbiol [Internet]. 2012;194:1001\u201312. \nAvailable from: http://link.springer.com/10.1007/s00203-012-0836-8 \n\n83.", "start_char_idx": 57913, "end_char_idx": 60594, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "98172b00-77fa-4796-b29d-ee3c43ca234f": {"__data__": {"id_": "98172b00-77fa-4796-b29d-ee3c43ca234f", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4c8cd93f-155f-44c0-a202-56d2df8b9633", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "fb7ca33140593435106073218322a64325fa747026beb6b4b76a00ae324c8a4a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "11ef3d27-76d2-4bb1-a5eb-a1c77136084d", "node_type": "1", "metadata": {}, "hash": "0946204b9b8df2ce7d06134e7b0074350a313b5fcb74c1c3d5e14df75cb1869c", "class_name": "RelatedNodeInfo"}}, "text": "HILTNER L t. Uber nevere Erfahrungen und Probleme auf dem \nGebiet der Boden Bakteriologie und unter besonderer \nBeurchsichtigung der Grundungung und Broche. Arbeit Deut \nLandw Ges Berlin. 1904;98:59\u201378.  \n\n84. Hartmann A, Rothballer M, Schmid M. Lorenz Hiltner, a pioneer \nin rhizosphere microbial ecology and soil bacteriology research. \nPlant Soil. 2008;312:7\u201314.  \n\n85. Mendes R, Garbeva P, Raaijmakers JM. The rhizosphere \nmicrobiome: significance of plant beneficial, plant pathogenic, \nand human pathogenic microorganisms. FEMS Microbiol Rev \n[Internet]. 2013;37:634\u201363. Available from: \nhttps://academic.oup.com/femsre/article-lookup/doi/10.1111/1574-\n6976.12028 \n\n86. Compant S, Reiter B, Sessitsch A, Nowak J, Cle\u0301ment C, Ait Barka \nE. Endophytic Colonization of Vitis vinifera L. by Plant Growth-\nPromoting Bacterium Burkholderia sp. Strain PsJN. Appl Environ \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1):6 3 - 7 4     Gunasinghe et al.  \n\n\u00a9NJB, BSN  74 \n\nMicrobiol [Internet]. 2005;71:1685\u201393. Available from: \nhttps://aem.asm.org/content/71/4/1685 \n\n87. Compant S, Cl\u00e9ment C, Sessitsch A. Plant growth-promoting \nbacteria in the rhizo- and endosphere of plants: Their role, \ncolonization, mechanisms involved and prospects for utilization. \nSoil Biol Biochem [Internet]. 2010;42:669\u201378. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S0038071709004398 \n\n88. Compant S, Mitter B, Colli-Mull JG, Gangl H, Sessitsch A. \nEndophytes of Grapevine Flowers, Berries, and Seeds: \nIdentification of Cultivable Bacteria, Comparison with Other \nPlant Parts, and Visualization of Niches of Colonization. Microb \nEcol [Internet]. 2011;62:188\u201397. Available from: \nhttp://link.springer.com/10.1007/s00248-011-9883-y \n\n89. Willem M DV. Mining the microbes \u2013 the human microbiome as \nmodel. Microb Biotechnol [Internet]. 2009;2:128\u201356. Available \nfrom: http://dx.doi.org/10.1111/j.1751-7915.2009.00090.x%5CnAll \nPapers/Other/Microb Biotechnol 2009 Rosenberg E.pd... - Microb \nBiotechnol 2009 Rosenberg E.pdf \n\n90. Hontzeas N, Hontzeas CE, Glick BR. Reaction mechanisms of the \nbacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase. \nBiotechnol Adv [Internet]. 2006;24:420\u20136. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S0734975006000231 \n\n91. Bulgarelli D, Schlaeppi K, Spaepen S, van Themaat EVL, Schulze-\nLefert P. Structure and Functions of the Bacterial Microbiota of \nPlants. Annu Rev Plant Biol [Internet]. 2013;64:807\u201338. Available \nfrom: http://www.annualreviews.org/doi/10.1146/annurev-arplant-\n050312-120106 \n\n92. Weyens N, van der Lelie D, Taghavi S, Newman L, Vangronsveld \nJ. Exploiting plant\u2013microbe partnerships to improve biomass \nproduction and remediation.", "start_char_idx": 60595, "end_char_idx": 63305, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "11ef3d27-76d2-4bb1-a5eb-a1c77136084d": {"__data__": {"id_": "11ef3d27-76d2-4bb1-a5eb-a1c77136084d", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "98172b00-77fa-4796-b29d-ee3c43ca234f", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "67ec47873cb618fc1ad6ffce97d2383ef60298de8a135e2756a04c493ddeaa56", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8ab2835c-1b06-435e-ada0-488e9b35217c", "node_type": "1", "metadata": {}, "hash": "0d393db6fdeef64851307409a38285cc487643595111db9985d87f7ea72a782e", "class_name": "RelatedNodeInfo"}}, "text": "Trends Biotechnol [Internet]. \nElsevier; 2009;27:591\u20138. Available from: \nhttps://linkinghub.elsevier.com/ retrieve/pii/S016777990900136X \n\n93. Weyens N, Thijs S, Popek R, Witters N, Przybysz A, Espenshade \nJ, et al. The Role of Plant\u2013Microbe Interactions and Their \nExploitation for Phytoremediation of Air Pollutants. Int J Mol Sci \n[Internet]. 2015;16:25576\u2013604. Available from: \nhttp://www.mdpi.com/ 1422-0067/16/10/25576 \n\n94. Rajkumar M, Sandhya S, Prasad MNV, Freitas H. Perspectives of \nplant-associated microbes in heavy metal phytoremediation. \nBiotechnol Adv [Internet]. Elsevier B.V.; 2012;30:1562\u201374. \nAvailable from: http://dx.doi.org/10.1016/j.biotechadv.2012.04.011 \n\n95. Hafeez FY, Yasmin S, Ariani D, Ur-Rahman M, Zafar Y, Malik \nKA. Plant growth-promoting bacteria as biofertilizer. Agron \nSustain Dev [Internet]. 2006;26:143\u201350. Available from: \nhttp://www.edpsciences.org/10.1051/agro:2006007 \n\n96. Van Sluys MA, Monteiro-Vitorello CB, Camargo LEA, Menck \nCFM, da Silva ACR, Ferro JA, et al. COMPARATIVE GENOMIC \nANALYSIS OF PLANT-ASSOCIATED BACTERIA. Annu Rev \nPhytopathol [Internet]. 2002;40:169\u201389. Available from: \nhttp://www.annualreviews.org/doi/10.1146/annurev.phyto.40.030402.0\n90559 \n\n97. Wu D, Sun MZ, Zhang C, Xin Y. Antioxidant properties of \nLactobacillus and its protecting effects to oxidative stress caco-2 \ncells. J Anim Plant Sci [Internet]. 2014;24:1766\u201371. Available from: \nhttp://thejaps.org.pk/docs/v-24-6/28.pdf \n\n98. Arslan M, Imran A, Khan QM, Afzal M. Plant\u2013bacteria \npartnerships for the remediation of persistent organic pollutants. \nEnviron Sci Pollut Res [Internet]. 2017;24:4322\u201336. Available \nfrom: http://link.springer.com/10.1007/s11356-015-4935-3 \n\n99. Glick BR. Using soil bacteria to facilitate phytoremediation. \nBiotechnol Adv [Internet]. Elsevier Inc.; 2010;28:367\u201374. Available \nfrom: http://dx.doi.org/10.1016/j.biotechadv.2010.02.001 \n\n100. Xu Z, Wu M, He Y. Toluene Biofiltration Enhanced by Ryegrass. \nBull Environ Contam Toxicol [Internet]. 2013;90:646\u20139. Available \nfrom: http://link.springer.com/10.1007/s00128-013-0973-z \n\n101. DAGLEY S. Biochemistry of aromatic hydrocarbon degradation \nin Pseudomonas. Bact. Academic Press; 1986;10:527\u201356.  \n\n102. Harayama S, Timmis KN. Aerobic biodegradation of aromatic \nhydrocarbons. Met Ions Biol Syst Vol 28 Degrad Environ Pollut \nby Microorg Their Met. CRC Press; 1992;28:99.  \n\n103. D\u00edaz E. Bacterial degradation of aromatic pollutants: A paradigm \nof metabolic versatility. Int Microbiol [Internet]. 2004;7:173\u201380. \nAvailable from: http://hdl.handle.net/10261/2134 \n\n104. Wackett LP.", "start_char_idx": 63306, "end_char_idx": 65902, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8ab2835c-1b06-435e-ada0-488e9b35217c": {"__data__": {"id_": "8ab2835c-1b06-435e-ada0-488e9b35217c", "embedding": null, "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-132", "node_type": "4", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "1299539ea333958e99cb0a4d10257eb9e6010315073978c53fb045087bb8a16e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "11ef3d27-76d2-4bb1-a5eb-a1c77136084d", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "073a3965405d62b70ec696e0de57520d017c2d654d866f0eded735cebc3247f4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f651c6d5-a718-4d07-b8b2-37493d7a4326", "node_type": "1", "metadata": {}, "hash": "7d095bacde296b268e59b53fbb06b2db23b6929ce41dd65012d6a7803645c7f5", "class_name": "RelatedNodeInfo"}}, "text": "Wackett LP. Pseudomonas putida\u2014a versatile biocatalyst. Nat \nBiotechnol [Internet]. 2003;21:136\u20138. Available from: \nhttp://www.nature.com/articles/nbt0203-136 \n\n105. Yoshikawa M, Zhang M, Toyota K. Biodegradation of Volatile \nOrganic Compounds and Their Effects on Biodegradability under \nCo-Existing Conditions. Microbes Environ [Internet]. \n2017;32:188\u2013200. Available from: https://www.jstage.jst.go.jp/ \narticle/jsme2/32/3/32_ME16188/_article \n\n106. Murray K, Duggleby CJ, Sala-trepat JM, Williams PA. Pathway \nby. 1972;310:301\u201310.  \n\n107. Williams PA, Murray K. Metabolism of benzoate and the \nmethylbenzoates by Pseudomonas putida (arvilla) mt 2: evidence \nfor the existence of a TOL plasmid. J Bacteriol [Internet]. \n1974;120:416\u201323. Available from: https://jb.asm.org/content \n/120/1/416.short \n\n108. Byrne AM, Kukor JJ, Olsen RH. Sequence analysis of the gene \ncluster encoding toluene-3-monooxygenase from Pseudomonas \npickettii PKO1. Gene [Internet]. 1995;154:65\u201370. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/037811199400844I \n\n109. Fishman A, Tao Y, Wood TK. Toluene 3-Monooxygenase of \nRalstonia pickettii PKO1 Is a para-Hydroxylating Enzyme. J \nBacteriol [Internet]. 2004;186:3117\u201323. Available from: \nhttps://jb.asm.org/content/186/10/3117 \n\n110. Shields MS, Montgomery SO, Chapman PJ, Cuskey SM, Pritchard \nPH. Novel pathway of toluene catabolism in the \ntrichloroethylene-degrading bacterium G4. Appl Environ \nMicrobiol [Internet]. 1989;55:1624\u20139. Available from: \nhttps://aem.asm.org/content/55/6/1624.short \n\n111. Keener WK, Arp DJ. Transformations of aromatic compounds by \nNitrosomonas europaea. Appl Environ Microbiol [Internet]. \n1994;60:1914\u201320. Available from: https://aem.asm.org/content \n/60/6/1914.short \n\n112. Marchant R. From the test tube to the table. EMBO Rep [Internet]. \n2001;2:354\u20137. Available from: \nhttps://onlinelibrary.wiley.com/doi/abs/10.1093/embo-reports/kve099 \n\n113. Timmis KN, Pieper DH. Bacteria designed for bioremediation. \nTrends Biotechnol [Internet]. Elsevier; 1999;17:201\u20134. Available \nfrom: http://dx.doi.org/10.1016/S0167-7799(98)01295-5 \n\n114. Netter KJ. Xenobiotic conjugation chemistry. Toxicology \n[Internet]. 1987;44:122\u20133. Available from: \nhttps://linkinghub.elsevier.com/ retrieve/pii/0300483X87900527", "start_char_idx": 65891, "end_char_idx": 68173, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f651c6d5-a718-4d07-b8b2-37493d7a4326": {"__data__": {"id_": "f651c6d5-a718-4d07-b8b2-37493d7a4326", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8ab2835c-1b06-435e-ada0-488e9b35217c", "node_type": "1", "metadata": {"identifier": "njb-132", "author": "Gunasinghe, Y.H.K.I.S.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Plant and Plant Associated Microflora: Potential Bioremediation Option of Indoor Air Pollutants", "date": "2021-07-31", "file": "njb-132.pdf"}, "hash": "e57cb25d00f5a0a4c9ac0337377086c4ff44bbd0635338074f0dec7eb50ecea8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "880209cc-c7fc-426c-b7cc-e02a3ccb8b41", "node_type": "1", "metadata": {}, "hash": "4ac3804ea8fe5b0069033eb28f2676b44899f6ddd1a57edefa4e619ca4df2438", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 50-62 Research article DOI: https://doi.org/10.3126/njb.v9i1.38667  \n\n\u00a9NJB, BSN 50 \n\nEvaluation of Phytochemical, Antioxidant and Antibacterial Activities \nof Selected Medicinal Plants \nShrimita Shrestha1 , Sudip Bhandari1 , Babita Aryal2 , Bishnu P Marasini1 , Santosh Khanal1 , Pramod \n\nPoudel3 , Binod Rayamajhee4 , Bikash Adhikari2 , Bibek Raj Bhattarai2  and Niranjan Parajuli2\n\n1Department of Biotechnology, National College, Naya Bazar, Kathmandu, Nepal \n2Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n3Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n4Department of Infectious Diseases and Immunology, Kathmandu Research Institute for Biological Sciences (KRIBS), \nLalitpur, Nepal \nReceived: 08 Mar  2021; Revised: 10 Jul 2021; Accepted: 15 Jul 2021; Published online: 31 Jul 2021 \n\nAbstract \nMedicinal plants are important reservoirs of bioactive compounds that need to be explored systematically. \nBecause of their chemical diversity, natural products provide limitless possibilities for new drug discovery. \nThis study aimed to investigate the biochemical properties of crude extracts from fifteen Nepalese medicinal \nplants. The total phenolic contents (TPC), total flavonoid contents (TFC), and antioxidant activity were \nevaluated through a colorimetric approach while the antibacterial activities were studied through the \nmeasurement of the zone of inhibition (ZoI) by agar well diffusion method along with minimum inhibitory \nconcentrations (MIC) by broth dilution method. The methanolic extracts of Acacia catechu and Eupoterium \nadenophorum showed the highest TPC (55.21 \u00b1 11.09 mg GAE/gm) and TFC (10.23 \u00b1 1.07 mg QE/gm) among \nthe studied plant extracts. Acacia catechu showed effective antioxidant properties with an IC50 value of 1.3 \n\u03bcg/mL, followed by extracts of Myrica esculenta, Syzygium cumini, and Mangifera indica. Morus australis \nexhibited antibacterial activity against Klebsiella pneumoniae (ZoI: 25mm, MIC: 0.012 mg/mL), Staphylococcus \naureus ATCC 25923 (ZoI: 22 mm, MIC: 0.012 mg/mL), Pseudomonas aeruginosa (ZoI; 20 mm, MIC: 0.05 mg/mL), \nand methicillin-resistant Staphylococcus aureus (MRSA) (ZoI: 19 mm, MIC: 0.19 mg/mL). Morus australis extract \nshowed a broad-spectrum antibacterial activity, followed by Eclipta prostrata, and Hypericum cordifolium. Future \nstudy is recommended to explore secondary metabolites of those medicinal plants to uncover further clinical \nefficacy. \n\nKeywords: Antibacterial activity; Medicinal plants; Secondary metabolites; Minimum inhibitory concentration \n\n Corresponding author, email: niranjan.parajuli@cdc.tu.edu.np \n\nIntroduction \nThe separation and identification of physiologically \n\nactive chemicals and molecules from medicinal plants \n\nhas resulted in innovative treatments and \n\npharmaceutical advances. Secondary metabolites \n\nextracted from medicinal plants have played a \n\nsignificant role in upholding human health against \n\nvarious infectious diseases since ancient times. Plant \n\nextracts or their active phytoconstituents have been used \n\nas folk medicine by 80 % of the world's population in \n\nconventional therapies [1].", "start_char_idx": 48, "end_char_idx": 3283, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "880209cc-c7fc-426c-b7cc-e02a3ccb8b41": {"__data__": {"id_": "880209cc-c7fc-426c-b7cc-e02a3ccb8b41", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f651c6d5-a718-4d07-b8b2-37493d7a4326", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "93127260b3e170a3685586c5328949a8ab99fad4478bd2104fc52d8108a9947c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6b10f7e5-cfbd-4cc2-89f1-3398452e8762", "node_type": "1", "metadata": {}, "hash": "5234f9fe4b2cd717b9588a2c152835c62cd28f38f4fe47f4205707fd1138fcbd", "class_name": "RelatedNodeInfo"}}, "text": "It is believed that over 50% of \n\nall modern clinical drugs are of natural product origin \n\n[2].  \n\nMultidrug resistance (MDR) is characterized as an \n\nacquired non-susceptibility to at least one antimicrobial \n\nagent from three or more categories [3]. Mobile genetic \n\nelements such as interferons, plasmids, and transposons \n\nare the most common carriers of antibiotic resistance \n\namong bacteria [4]. The rapid emergence of resistance to \n\nnewly introduced antimicrobial agents, suggests that \n\neven a new antimicrobial agent would not be a complete \n\nsolution to the problem [5]. MDR pathogens have raised \n\na significant problem in public health by undermining \n\nthe existing antibiotic-based treatment era, resulting in \n\nan increased mortality rate in patients [6]. MDR \n\npathogens worsen the disease severity and put the value \n\nof antibiotics at risk, affecting the global economy [7]. It \n\nis anticipated that if the race of antimicrobial resistance  \n\n(AMR) keep rising, it would take the lives of nearly ten \n\nmillion peoples annually by 2050 [8]. Thus, a new \n\nantibacterial agent is urgently needed to treat MDR-\n\ninduced infections caused by pathogens such as \n\nEnterobacteriaceae, Staphylococcus aureus, extended-\n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0003-2896-9799\nhttps://orcid.org/0000-0002-7899-2885\nhttps://orcid.org/0000-0001-9731-8485\nhttps://orcid.org/0000-0001-6153-5234\nhttps://orcid.org/0000-0002-0186-6119\nhttps://orcid.org/0000-0001-5304-7812\nhttps://orcid.org/0000-0003-3007-8901\nhttps://orcid.org/0000-0002-5532-6644\nhttps://orcid.org/0000-0003-0259-5004\nhttps://orcid.org/0000-0002-9233-6489\nmailto:niranjan.parajuli@cdc.tu.edu.np\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  51 \n\nspectrum \u03b2-lactamase (ESBL) producing bacteria, \n\namong others [9]. \n\nTable 1: Description of medicinal plants used in this study. \n\nMedicinal plants \nVoucher \nspecimen \n\nLocal \nname \n\nParts \nused \n\nEclipta prostrata NCDB203 Bhringaraj \nWhole \nplants \n\nShorea robusta NCDB212 Saal Leaves \n\nSmallanthus sonchifolius NCDB214 Yacon Leaves \n\nHypericum cordifolium NCDB201 Arelu Leaves \n\nMangifera indica NCDB211 Mango Leaves \n\nMorus australis NCDB210 Kimbu Barks \n\nPsidium guajava NCDB206 Guava Leaves \n\nChrysanthemum indicium NCDB205 Godawari Leaves \n\nMyrica esculenta NCDB208 Kafal Leaves \n\nUrtica ardens NCDB213 Sisnoo Buds \n\nPterocarpus marsupium NCDB204 Bijayasal Barks \n\nEupoterium adenophoium NCDB202 Banmara Leaves \n\nZingiber officinale NCDB200 Aaduwa Leaves \n\nAcacia catechu NCDB209 Khair Barks \n\nSyzygium cumini NCDB207 Jamun Leaves \n\n Acinetobacter baumannii, Pseudomonas aeruginosa,  \n\nMedicinal plants produce secondary metabolites that \n\ncan tackle MDR pathogens. Furthermore, medicinal \n\nplants have immunomodulatory and antioxidant \n\nactivity, which result in antibacterial properties.", "start_char_idx": 3284, "end_char_idx": 6305, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6b10f7e5-cfbd-4cc2-89f1-3398452e8762": {"__data__": {"id_": "6b10f7e5-cfbd-4cc2-89f1-3398452e8762", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "880209cc-c7fc-426c-b7cc-e02a3ccb8b41", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "7ef0485c54327de1b591a2f746351c80b8fcaaabec81fbb7c17be3b250ac1321", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bd141d92-6624-44f5-92bd-1649e32f0a66", "node_type": "1", "metadata": {}, "hash": "b044a90f41497fa78032b84a173a53c57e5e389cece3799aca59aed6707f6ea8", "class_name": "RelatedNodeInfo"}}, "text": "They \n\nhave a wide range of immunomodulatory effects \n\nstimulating both non-specific and specific immunity \n\n[10]. Antimicrobial and antioxidant activity is found in \n\nphytochemicals such as vitamins (A, C, E, and K), \n\ntannins, carotenoids, polyphenols, flavonoids, alkaloids, \n\nsaponins, pigments, enzymes, terpenoids, and minerals \n\n[11]. Nonetheless, analgesic, antibacterial, deodorizing, \n\nfebrifuge, fungicidal, antiseptic, astringent, \n\ngalactagogue, diuretic, antidepressant, insecticidal, \n\nantipyretic, and sedative properties have been recorded \n\nfor volatile oils from plants (Blanco et al., 2009; Bekoe et \n\nal., 2018; Iscan et al. 2002). \n\nHowever, microorganisms have continuously evolved \n\nwith a wide range of metabolic mechanisms to \n\novercome drug effects [6]. Plant-derived drugs are a \n\nsuperior choice over synthetic drugs because of fewer \n\nside effects and adverse effects (Bindu Jacob & \n\nNarendhirakannan R.T., 2019; Verma et al., 2018). Nepal \n\nis rich in biodiversity and geographical condition with \n\ndiverse flora, and numerous species are believed to \n\npossess curative properties. However, most of these \n\nclaims lack scientific validation. The plants selected for \n\nthis study are being used routinely by the indigenous \n\npeople as remedies against various human diseases \n\nsince ancient times. Therefore, the selected plants may \n\ncontain certain important bioactive compounds that \n\ncould have some medicinal and antimicrobial properties \n\nand some therapeutic value based on phytochemical \n\nconstituents and their secondary metabolites. Hence, the \n\nantibacterial activity of plant extracts reported here \n\nwould be beneficial to identify some potent secondary \n\nmetabolites as future drug candidates for the \n\ntherapeutic measures of MDR-strains-induced infections \n\nin Nepal and beyond. \n\nMaterials and Methods  \nBacterial isolates \nEight MDR bacterial strains: Acinetobacter spp. (628), \n\nCitrobacter freundii (377), methicillin-resistant \n\nStaphylococcus aureus (MRSA) (338), Klebsiella \n\npneumoniae (386), Pseudomonas aeruginosa (484), \n\nEscherichia coli (2A), Morganella morganii (4331), and \n\nXanthomonas spp. (767) were collected from the \n\nNational Public Health Laboratory (NPHL), \n\nKathmandu, and transferred aseptically to the \n\nlaboratory of the Department of Biotechnology, National \n\nCollege for further study. All isolates were obtained \n\nfrom clinical specimens. Besides, ATCC strains such as \n\nE. coli 25922, S. aureus 25923, Salmonella Typhimurium \n\n14028, and K. pneumoniae 700603 were also collected \n\nfrom the NPHL stored at -20\u00b0C for further studies. \n\nCollection of plant materials \nDifferent parts (leaves, bark, fruit, roots, and stem) were \n\ncollected based on the ethnomedicinal and traditional \n\nmedicinal practices from different geographical regions \n\nof Nepal as depicted in Table 1 (Collection period: \n\nJanuary to June 2017). The plant samples were identified \n\nby National Herbarium and Plant Laboratories, \n\nGodawari, Lalitpur, Nepal, and herbarium collections \n\nwere deposited in the Department of Botany, National \n\nCollege, Khusibu, Kathmandu. \n\nPreparation of plant extracts \nThe plant parts (mentioned in Table 1) were dried in the \n\nshade at room temperature, pulverized into the \n\npowders with the help of a grinding mill, and then \n\nsoaked in methanol for 24 hours. Then, they were \n\nfiltered, and the process was repeated three times with \n\nfresh methanol. To obtain plant extracts, the filtrates \n\nwere concentrated in a rotary evaporator at 50 \u00b0C.", "start_char_idx": 6306, "end_char_idx": 9837, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bd141d92-6624-44f5-92bd-1649e32f0a66": {"__data__": {"id_": "bd141d92-6624-44f5-92bd-1649e32f0a66", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6b10f7e5-cfbd-4cc2-89f1-3398452e8762", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "4eb1125b92f960349a7bcbdd73c9dd58b6d2ae8fcefde89f10c064953ebf6063", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "837070a3-9bbc-44c7-a9f7-514c7fd7af15", "node_type": "1", "metadata": {}, "hash": "4d0b75b1e2d73687f125be391e6c0857479839eeb61fc87bdc41b4f15aeb5104", "class_name": "RelatedNodeInfo"}}, "text": "Determination of TPC and TFC  \nUsing Folin-Ciocalteu reagent and a 96-well plate-based \n\ncolorimetric process, The TPC was calculated \n\n(Ainsworth & Gillespie, 2007; Bhandari et al., 2021). \n\nInitially, 20 \u00b5L of plant extract was mixed with 100 \u00b5L of \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  52 \n\nFolin-Ciocalteu's reagent (1:10 v/v) and 80 \u00b5L of sodium \n\ncarbonate (7.5%, w/v) in each well-containing standard \n\nand sample before incubation. Then, the sample was \n\nincubated at room temperature, and absorbance was \n\nmeasured at 765 nm[15]. By comparing TPC to standard \n\ngallic acid, milligrams of gallic acid equivalents per \n\ngram of extract (mg GAE/gm) were determined. \n\nLikewise, for TFC, 20 \u00b5L of plant extract was mixed with \n\n60 \u00b5L of methanol, 5 \u00b5L of potassium acetate (1 M), 5 \u00b5L \n\nof 10% aluminum chloride, and 110 \u00b5L of distilled \n\nwater, then incubated at room temperature for 30 \n\nminutes, and the absorbance was measured at 415 \n\nnm[17]. Likewise, TFC was expressed as milligrams of \n\nquercetin equivalents per gram of extract (mg QE/gm \n\nextract) by comparing to standard quercetin [17]. \n\nDetermination of antioxidant activity \nThe antioxidant property was determined by \n\ndiscoloration assay based on the scavenging of 2, 2- \n\ndiphenyl-1-picrylhydrazyl (DPPH) free radical (0.1 mM \n\n) (Brand-Williams et al., 1995; Aryal et al., 2021) at 517 \n\nnm using a multi-plate reader (Epoch 2, BioTek, \n\nInstruments, Inc., USA), maintaining 1 mg/mL of \n\nquercetin as a control. Crude extracts were allowed to \n\nreact with DPPH free radicals for 30 minutes at room \n\ntemperature. The scavenging of DPPH radical was \n\ncalculated by using the following expression: (where \n\noptical density (OD) is the absorbance). \n\n% Scavenging = 100 \u2212\n(OD of extract)\n\n(OD of control)\n\u00d7 100 \n\nAntibacterial activity \nUsing sterile cotton swabs moistened with the bacterial \n\nsuspension, an inoculum suspension containing 1.5 x108 \n\nCFU/mL of bacteria was spread on firm Muller-Hinton \n\nAgar (MHA) plates (Balouiri et al., 2016; Marasini et al., \n\n2015; Valgas et al., 2007). Using a sterile cork borer, \n\nwells were punched in plates (6 mm diameter) and \n\nmicropipettes were used to fill the wells with a \n\nfunctioning suspension (50\u00b5L) of plant extracts (50 \n\nmg/mL), as well as neomycin (20 \u00b5g /mL), amikacin (30 \n\nmcg), and nitrofurantoin (30 mcg) as positive controls \n\nand 50 % DMSO as negative controls [23]. The MHA \n\nplates were incubated for 24 hours at 37\u00b0C and finally, \n\nthe ZoI was determined after overnight incubation. \n\nDetermination of MIC \nThe broth dilution method was followed to determine \n\nMIC values of plant extracts as recommended by the \n\nClinical and Laboratory Standards Institute [24].", "start_char_idx": 9840, "end_char_idx": 12602, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "837070a3-9bbc-44c7-a9f7-514c7fd7af15": {"__data__": {"id_": "837070a3-9bbc-44c7-a9f7-514c7fd7af15", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bd141d92-6624-44f5-92bd-1649e32f0a66", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "d96eedfc5ab27bb37aea6d154cfc14a785ebbbbe73d469dab0673f185506311e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a8198b02-f977-4523-9422-3757fd4bb356", "node_type": "1", "metadata": {}, "hash": "9d0088abfe8377c1b78c0afc7fc4a2686c9b5e23e45636874166c43a3aa71653", "class_name": "RelatedNodeInfo"}}, "text": "Extracts of E. adenophorum, M. australis, E. prostrata, A. \n\ncatechu, Z. officinale, P. marsupium, S. robusta, M. indica, S. \n\nsonchifolius, M. esculenta, U. ardens, H. cordifolium, S. \n\ncumini, P. guajava, and C. indicium showed significant \n\nantibacterial activity with larger ZoI, so they were \n\nselected for the determination of MIC value. The plant \n\nextracts were two-fold diluted to get a series of \n\nconcentrations ranging from 25 mg/mL to 0.012 mg/mL \n\nin freshly prepared sterile nutrient broth. Then 20 \u00b5L of \n\nbacterial culture adjusted to 0.5 McFarland Standard \n\nwas inoculated in each dilution tube and incubated at \n\n37\u02daC for 24 hours. The set-up included bacterial growth \n\ncontrols containing test tubes with media inoculated \n\nwith 20 \u00b5L of bacterial inoculum only and negative \n\ncontrols with media and plant extract without bacterial \n\ninoculum. The MIC value was measured by choosing \n\nthe lowest concentration of plant extract that inhibited \n\nthe organism's growth in the test tubes, as determined \n\nby unaided observation. The bacterial growth in the \n\ntubes containing the plant extracts was compared to the \n\ncontrol sample without the plant extracts to establish the \n\ngrowth endpoints. Each assay was carried out in \n\ntriplicate to confirm the results. \n\nResults \nThe researches on medicinal plants have been carried \n\nthroughout the world to explore the bioactive \n\ncompounds which could be used to make a preventive \n\nor treatment approach against various health \n\ncomplications. The ethnopharmacological applications \n\nof plants under study were depicted in Table 2. \n\nYields, TPC and TFC of plant extracts \nThe percentage yield of plant extracts varied from 5.94% \n\nto 28.47% (Table 3). Extracts of H. cordifolium had the \n\nhighest percentage yield (28.47%), followed by A. \n\ncatechu (23.0%), P. guajava (21.82%), and M. esculenta \n\n(19.02%). Noticeably all plant extracts were found to be \n\nin semi-solid inconsistency.  \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  53 \n\nTable 2: Medicinal plants selected understudy with their ethnopharmacological applications \n\nMedicinal plants Family Ethnopharmacological applications \n\nEclipta prostrata Asteraceae \n\nUsed as an anti-inflammatory, antivenom [25], anti-aging, hepatoprotective, anti-viral, \n\nantimicrobial agents. Bithiophenes and 5-(but-3-yne-1,2-diol)-5\u2032-hydroxy-methyl-\n\n2,2\u2032bithiophene isolated from this plant used as antibacterial and antihyperglycemic \n\n[26], [27]. \n\nShorea robusta Dipterocarpaceae \n\nUsed in the treatment of ulcer, cough, itching, leprosy, anthelmintic [28].  Antibacterial \n\nwound healing and anti-inflammatory activity due to the presence of polyphenols, \n\nflavonoids, and triterpenoids, etc. Ursolic acid extracted from this plant is responsible \n\nfor showing antibacterial activity [29]. \n\nSmallanthus sonchifolius Asteraceae \n\nUsed as a functional food, antioxidant, antimicrobial, prebiotic, growth promoter [30]. \n\nLeaves extract contains the compounds fluctuanin and enhydrin show antibacterial \n\nactivity [31]. \n\nHypericum cordifolium Hypericaceae \nTreatment of back pain and broken bones, an antidepressant [32]. Dermatological, \n\nneurological, and traumatological problems, antibacterial activity [33].", "start_char_idx": 12605, "end_char_idx": 15885, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a8198b02-f977-4523-9422-3757fd4bb356": {"__data__": {"id_": "a8198b02-f977-4523-9422-3757fd4bb356", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "837070a3-9bbc-44c7-a9f7-514c7fd7af15", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "fc2d483b3d0cd4bfab01c6ac0687a5f008deeef90f1b97b9c2491885482f6129", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "175b091c-7694-4599-b50e-556c08e5fa60", "node_type": "1", "metadata": {}, "hash": "ab828548ad3cb6062ca739652ae382adf1924c12d30120b5c31c0b078ef4f244", "class_name": "RelatedNodeInfo"}}, "text": "Mangifera indica Anacardiaceae \n\nUsed for gastric disorders, mouth sores, tooth pain, and dermatological disorders. [34] \n\nTreatment for diabetes, infertility, ethanolic extract of M. indica showed significant \n\nantibacterial activity. Methanolic extract displayed cytotoxicity against the pancreatic \n\ncancer cell line. Magniferin (5) from plant extract showed antimicrobial effect [35], [36]. \n\nMorus australis Moraceae \n\nTreatment for fever, protect the liver, improve eyesight, strengthen joints, lower blood \n\npressure [37]. Leaves contain 1-deoxynojirimycin known to have potential \u03b1- \n\nglucosidase inhibition activity. The piperidine alkaloid and glycoproteins from the \n\nextract of M. austrralis have been used for antidiabetic agents [38]. \n\nPsidium guajava Myrtaceae \n\nUsed for ulcers, wounds, toothache, anti-allergic effects, anti-cancer effects, and anti-\n\nhyperglycemia [39]. Used effectively in diabetes, diarrhea, dysentery, pain relief, cough, \n\ngastroenteritis, hypertension, caries. The hypoglycemic components in Psidium guajava \n\nmight be due to oleanolic acid, arjunolic acid, ursolic acid, and glucuronic acid [40]. \n\nChrysanthemum indicium Asteraceae \n\nUsed for hypertension, pneumonia, colitis, stomatitis, fever, neurological problems, \n\nheadache [41], antipyretic purpose, treatment of cephalgia, vertigo, and eye \n\ninflammations [42]. \n\nMyrica esculenta Myricaceae \n\nUsed for cough, anemia, asthma, chronic dysentery, fever, sores, tumors, nasal catarrh, \n\npiles, throat complaints, ulcers, and urinary discharges[43]. Used against different \n\ndisease conditions such as; antidiabetic, antiallergic, antimicrobial, anti-ulcer, anti-\n\nhypertensive, antioxidant, and higher phenolic and flavonoid compounds including \n\nmyricetin, myricanol, and myricanone have anti-inflammatory properties. [44]. \n\nUrtica ardens Urticaceae \n\nUsed for diabetes, diarrhea, excessive menstrual bleeding, urinary disorders, respiratory \n\nproblems, ulcers, asthma, rheumatism, high blood pressure [45]. Treatment for sprains, \n\nkidney stones, hemorrhoids, flu, fever, hepatoprotective, nephroprotective effect, etc. \n\n[46]. \n\nPterocarpus marsupium Fabaceae \nStomachache, cholera, dysentery, urinary complaints, tongue disease, toothache, and \n\ncough are all treated. [47]. Treatment of diabetes, jaundice, and an ulcer [48]. \n\nEupoterium adenophoium Asteraceae \n\nUsed for treatment of emetic, diaphoretic, stimulant, tonic, fever, cuts and wounds, \n\nanalgesic [49]. Used as an anti-inflammatory, blood coagulant, antimicrobial, antiseptic, \n\nand analgesic, antipyretic. Isomers of mono-caffeoylquinic acid present in E. \n\nadenophoium exhibit potent anti-inflammatory, anti-bacterium, and anti-obesity \n\nproperties [50]. \n\nZingiber officinale Zingiberaceae \n\nTreatment of diabetes, high blood pressure, cancer, stomachache, nausea, asthma, \n\nrespiratory disorders [51]. Treatment for diabetes, blood pressure, stomach ache, weight \n\nloss, diarrhea, and nausea. Geraniol present in Z. officinale shows potential anti-\n\ninflammatory and antioxidant effects [52]. \n\nAcacia catechu Fabaceae \n\nIt can be used to treat colds, coughs, ulcers, boils, and skin eruptions, bleeding masses, \n\nantipyretics, and acute and chronic wound healing. [53].", "start_char_idx": 15888, "end_char_idx": 19130, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "175b091c-7694-4599-b50e-556c08e5fa60": {"__data__": {"id_": "175b091c-7694-4599-b50e-556c08e5fa60", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a8198b02-f977-4523-9422-3757fd4bb356", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "27f1b524329aa0d07520f69c572d228be8393289bcaf2ea9e07c9a3615ac3b4b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b6df9a27-7070-4b78-8d3e-efe8ee2020f0", "node_type": "1", "metadata": {}, "hash": "5c83cb09b4080fc8ef34592142c534a8e63eb02d8a6fbc1855dc7a836cd50490", "class_name": "RelatedNodeInfo"}}, "text": "[53]. The key constituents of A. \n\ncatechu are catechin and taxifolin, which have antifungal, antiviral, antibacterial, anti-\n\ninflammatory, and antioxidant properties. [53]. \n\nSyzygium cumini Myrtaceae \n\nUsed for diabetes mellitus, constipation, stomachache, HIV, inflammation leucorrhoea, \n\nfever, strangury, and dermopathy [54], [55]. Ferulic acid and Catechins possess \n\nantioxidant properties [56]. Gallocatechins are used to treat diabetes. Quercetin isolated \n\nfrom S. cumini is used to treat diabetes and treat cytotoxicity. \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  54 \n\nTable 3: Physical characteristics and percentage yield of the crude extracts. \n\nMedicinal plants Local Name Dry weight of plant (gm) Percentage yield (%) \n\nHypericum cordifolium Arelu 40 28.46 \nAcacia catechu Khayr 50 23.0 \nPsidium guajava Guava 50 21.82 \nMyrica esculenta Kafal 50 19.02 \nSyzygium cumini Jamun 50 17.0 \n\nMangifera indica Mango 50 14.9 \n\nChrysanthemum indicium Godawari 50 13.44 \n\nZingiber officinale Ginger 50 12.5 \n\nSmallanthus sonchifolius Ground apple 50 11.16 \nPterocarpus marsupium Bijayasal 50 11.02 \n\nEupoterium adenophorum Banmara 50 10.42 \n\nShorea robusta Sal 50 9.1 \n\nEclipta prostrata Bhringraj 70 6.54 \nMorus australis Kimbu 34.8 6.03 \nUrtica ardens Sisnoo 50 5.94 \n\nTable 4: TPC of medicinal plants. \n\nMedicinal plants TPC (mg GAE/gm) \n\nAcacia catechu 55.21 \u00b1 11.09 \nUrtica ardens 50.01 \u00b1 5.0 \nMangifera indica 49.88 \u00b1 19.2 \nPsidium guajava 45.21 \u00b1 2.73 \nShorea robusta 45.21 \u00b1 4.15 \nEupoterium adenophorum 37.61 \u00b1 4.14 \nHypericum cordifolium 36.28 \u00b1 2.37 \nChrysanthemum indicium 32.95 \u00b1 4.43 \nSyzygium cumini 28.28 \u00b1 1.85 \nMyrica esculenta 23.21 \u00b1 4.42 \nPterocarpus marsupium 22.68 \u00b1 1.35 \nMorus australis 19.75 \u00b1 2.94 \nZingiber officinale 19.21 \u00b1 2.0 \nEclipta prostrata 18.95 \u00b1 1.24 \nSmallanthus sonchifolius 9.08 \u00b1 1.01 \n\nTable 5: TFC of medicinal plants.", "start_char_idx": 19125, "end_char_idx": 21050, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b6df9a27-7070-4b78-8d3e-efe8ee2020f0": {"__data__": {"id_": "b6df9a27-7070-4b78-8d3e-efe8ee2020f0", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "175b091c-7694-4599-b50e-556c08e5fa60", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "ae87df71ed4ce651b3c5acec77ced85e60824cf1b55648130531fb12d108ad7f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "54fd5bbb-fd12-4ae7-a058-3e3a55d95d1c", "node_type": "1", "metadata": {}, "hash": "ad0abe59740fecdf36622ecdc75c81bfa9a2b699ef7c66dda9cb9fa4cec69758", "class_name": "RelatedNodeInfo"}}, "text": "Medicinal plants TFC (mg QE/gm) \n\nEupoterium adenophorum 10.23 \u00b1 1.07 \n\nMorus australis 9.10 \u00b1 0.98 \n\nEclipta prostrata 8.67 \u00b1 0.57 \n\nAcacia catechu 8.34 \u00b1 0.77 \n\nZingiber officinale 7.78 \u00b1 0.71 \n\nPterocarpus marsupium 7.70 \u00b1 0.85 \n\nShorea robusta 7.68 \u00b1 0.71 \n\nMangifera indica 7.52 \u00b1 1.12 \n\nSmallanthus sonchifolius 7.40 \u00b1 0.83 \n\nMyrica esculenta 6.84 \u00b1 1.30 \n\nUrtica ardens 5.89 \u00b1 0.35 \n\nHypericum cordifolium 5.89 \u00b1 1.68 \n\nSyzygium cumini 5.72 \u00b1 0.52 \n\nPsidium guajava 5.26 \u00b1 1.15 \n\nChrysanthemum indicium 4.93 \u00b1 0.66 \n\nTPC of plant extracts was expressed in terms of gallic \n\nacid equivalent (mg GAE/gm dry weight of extract) and \n\nplaced in the order from higher to lower using a \n\ncalibration curve of gallic acid (y =0.0025x + 0.0413, R\u00b2 = \n\n0.981). TPC of plant extracts ranged from 55.21 \u00b1 11.09 to \n\n9.08 \u00b1 1.0 mg GAE/gm. Extract of A. catechu exhibited \n\nthe highest TPC, followed by U. ardens, M. indica, P. \n\nguajava, and S. robusta respectively (Table 4). \n\nSimilarly, TFC of plant extracts was expressed in terms \n\nof quercetin equivalent (mg QE/gm) and placed in the \n\norder from higher to lower using a calibration curve of \n\nquercetin (y = 0.0202x \u2013 0.972, R\u00b2 = 0.972). The extract of \n\nE. adenophorum showed the highest TFC (10.23 \u00b1 1.07 mg \n\nQE/gm), followed by M. australis and E. prostrata \n\nrespectively (Table 5).  \n\nAntioxidant activity \nFree radical scavenging activity was used to assess the \n\nantioxidant activity of plant extracts, and the resulting \n\ndegree of decolorization is stoichiometric in terms of the \n\nnumber of electrons captured from plant extracts.  \n\nThe results of antioxidant abilities of plant extracts were \n\ncompared with standard quercetin (IC50 2.28 \u00b5g/mL). \n\nAmong them, methanolic extract of A. catechu, M. \n\nesculenta, S.  cumini, and M. indica showed promising \n\nantioxidant properties with IC50 ranging 1.3-1.80 \u00b5g/mL \n\n(Table 6). \n\nEvaluation of antibacterial activity \nPlant extracts were examined for antibacterial activity \n\nagainst eight MDR bacteria and four ATCC bacterial \n\nspecies adopting the agar well diffusion technique. The \n\nextracts of M. australis, S. robusta, and M. indica showed \n\nthe largest ZoI i.e. 21 mm at 50 mg/mL towards E. coli \n\nATCC 25922 in agar plates. Meanwhile, only E. prostrata \n\nextract showed 7 mm of the ZoI against K. pneumoniae \n\nATCC 700603. The M. australis extract showed 22 mm of \n\nthe ZoI against S. aureus ATCC 25923, which was the \n\nhighest among the ZoI shown by plant extract. \n\nSimilarly, M. australis extract showed the highest ZoI \n\nagainst three MDR bacterial strains, K. pneumoniae, \n\nMRSA, and P. aeruginosa with 25 mm, 19 mm, and 20 \n\nmm, respectively (Table 8).", "start_char_idx": 21053, "end_char_idx": 23729, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "54fd5bbb-fd12-4ae7-a058-3e3a55d95d1c": {"__data__": {"id_": "54fd5bbb-fd12-4ae7-a058-3e3a55d95d1c", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b6df9a27-7070-4b78-8d3e-efe8ee2020f0", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "309317abb5f076ac529407863c329d2ad119af9f9e9c4b36815691c787d78c9a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0da3e16a-a219-4f42-96b5-6c231f044696", "node_type": "1", "metadata": {}, "hash": "431539256d0721957fefd6c437bf4b134a3d4aef76553105b07420536c623544", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  55 \n\nTable 6: IC50 values of plant extracts for antioxidant assay. \n\nMedicinal plants IC50 (\u00b5g/mL) \n\nSmallanthus sonchifolius 329.0 \u00b1 0.01 \nMorus australis 208.60 \u00b1 0.02 \nPterocarpus marsupium 38.50 \u00b1 0.04 \nShorea robusta 2.50 \u00b1 0.01 \nMangifera indica 1.80 \u00b1 0.06 \nSyzygium cumini 1.60 \u00b1 0.04 \nMyrica esculenta 1.50 \u00b1 0.03 \nAcacia catechu 1.30 \u00b1 0.05 \nQuercetin (Standard) 2.28  \nNote: only significant results were shown and placed in order from higher \nto lower IC50 value. \n\nThe extract of S. robusta and M. indica showed 17 mm of \n\nthe ZoI against MDR A. baumannii. Figure 1, presents \n\nZoI of plant extracts against ATCC strains E. coli and S. \n\naureus while Figure 2, presents ZoI of plant extracts \n\nagainst the MDR K. pneumoniae and Xanthomonas \n\nspecies. \n\nFigure 1. Antibacterial activity of plant extracts against ATCC \norganism E. coli and S. aureus: A) Neomycin; B) 50% DMSO; \nC) E. prostrata; D) P. marsupium; E) A. catechu: F) M. indica; \nG) S. robusta; H) C. indicium. \n\nAlthough some plant extracts exhibited potent \n\nantimicrobial activity towards some bacterial species, a \n\nhigher number of plant extracts had a minimum \n\nantibacterial effect. The MIC of plant extracts against \n\nATCC strains was between 0.012 mg/mL to 25 mg/mL \n\n(Table 9). Extracts of M. australis and H. cordifolium \n\nshowed a broad-spectrum antimicrobial activity against \n\nGram-positive and Gram-negative bacteria such as K. \n\npneumoniae, E. coli, and S. aureus. The most potent \n\nantibacterial activity (MIC = 0.012 mg/mL) was shown \n\nby extracts of M. australis, H. cordifolium, and P. guajava, \n\nand the least antibacterial activity (MIC = 25 mg/mL) \n\nwas observed in extracts of E. prostrata and S. cumini \n\nagainst ATCC strain of S.aureus. Regarding MDR \n\nstrains, the most potent antibacterial activity (MIC = \n\n0.012 mg/mL) was shown by the extracts of M. australis \n\nand H. cordifolium against K. pneumoniae (386), followed \n\nby M. australis against Xanthomonas species (4331) and P. \n\naeruginosa (484) (Table 10). \n\nFigure 2. Antibacterial activity of plant extracts against MDR \nK.pneumoniae and Xanthomonas species; A) Neomycin; B) 50% \nDMSO; C) H. cordifolium; D) S. cumini; E) M. australis: F) A. \ncatechu; G) M. indica; H) P. marsupium; I) M. esculenta. \n  \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  56 \n\nTable 7: Antibacterial activity of plant extracts against ATCC bacterial strains.", "start_char_idx": 23736, "end_char_idx": 26271, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0da3e16a-a219-4f42-96b5-6c231f044696": {"__data__": {"id_": "0da3e16a-a219-4f42-96b5-6c231f044696", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "54fd5bbb-fd12-4ae7-a058-3e3a55d95d1c", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "911b0bef63253e1d919f454c0d8b647ce345ff2eb618e1fa8c7afc4a0668284f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2454df20-eb6f-4445-bead-ee72c96d0cfd", "node_type": "1", "metadata": {}, "hash": "fc476c84ecf8668e6a19153a49159262f9b36b9c78c476d469afc7140da82880", "class_name": "RelatedNodeInfo"}}, "text": "Medicinal plants \n\nBacterial strains \n\nE. coli \nATCC 25922 \n\nK. pneumoniae \nATCC700603 \n\nS. Typhimurium \nATCC 14028 \n\nS. \naureus \nATCC \n25923 \n\nEupoterium adenophorum 12 - - 11 \nMorus australis 21 - - 22 \nEclipta prostrata 9.0 7.0 - 11 \nAcacia catechu 18 - - 15 \nZingiber officinale - - - - \nPterocarpus marsupium 12 - - 14 \nShorea robusta 21 - - 17 \nMangifera indica 21 - - 14 \nSmallanthus sonchifolius - - - - \nMyrica esculenta 16 - - 19 \nUrtica ardens - - - - \nHypericum cordifolium 10 - - 18 \nSyzygium cumini 17 - - 13 \nPsidium guajava 16 - - 15 \nChrysanthemum indicium 8.0 - - 12 \nNeomycin 22 10 15 20 \n50% DMSO - - - - \n\nDiameter of zone of inhibition in mm, well diameter = 6 mm, (-) = No antibacterial activity \n\n \n\nDiscussion \nIn developing health care, the search for new medicines \n\nwith better or enhanced therapeutic actions derived \n\nfrom medicinal plants with ethnobotanical significance \n\nhas become increasingly valuable [57,58]. Extraction is \n\nthe most important step in obtaining the plant's \n\nbioactive compounds, and the yield is determined by \n\nthe solvent and extraction method used [59]. In this \n\nstudy, methanol was used as a solvent with a  \n\nTable 8: Antibacterial activity of plant extracts against MDR bacterial strains. \n\nMedicinal plants \nBacterial strains \n\n2A 386 338 628 377 767 4331 484 \n\nEupoterium adenophorum - - - 13 - 15 11 - \n\nMorus australis - 25 19 14 - 15 - 20 \n\nEclipta prostrata - 10 - 16 - 10 9.0 - \n\nAcacia catechu - 14 - 12 - 14 - - \n\nZingiber officinale - - - - - - - - \n\nPterocarpus marsupium - - 13 12 - 17 11 - \n\nShorea robusta - - - 17 - - - - \n\nMangifera indica - - - 17 - 12 - - \n\nSmallanthus sonchifolius - - - 9.0 - - - - \n\nMyrica esculenta - - - 13 - 15 16 - \n\nUrtica ardens - - - - - - 9 - \n\nHypericum cordifolium - 20 12 16 - - - 20 \n\nSyzygium cumini - 16 16 14 - 17 - - \n\nPsidium guajava - - 12 14 - 17 - - \n\nChrysanthemum indicium - - 15 15 - 8.0 - - \n\nNeomycin 15 23 15 - - 11 - 10 \n\n50% DMSO - - - - - - - - \n\nAmikacin - - 23 20 - - 15 23 \n\nNitrofurantoin 22 18 16 - 17 16 - 15 \n\n(-) No antibacterial activity, 2A = E. coli, 338 = methicillin-resistant S. aureus (MRSA), 386 = K. pnemoniae, 628 = A. baumannii, 377 = \nC. freundii, 767 = Xanthomonas species, 4331 = M. morganii, 484 = P. aeruginosa \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al.", "start_char_idx": 26274, "end_char_idx": 28617, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2454df20-eb6f-4445-bead-ee72c96d0cfd": {"__data__": {"id_": "2454df20-eb6f-4445-bead-ee72c96d0cfd", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0da3e16a-a219-4f42-96b5-6c231f044696", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "811d865779a401e5965f7a329dbb3adb04b447a6565ad23ab1b36890805bdf1f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6673620d-a3a7-4e04-bd8a-40911169c6b8", "node_type": "1", "metadata": {}, "hash": "dd8eabf4174aa66d4a69eba0a3fc8d4829476ca8980e22d6674ff653d2d6c635", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  57 \n\npercentage yield of H. cordifolium being the highest \n\n(28.46 %) followed by A. catechu (23 %) (Table 3). The \n\nmethanolic extract of A. catechu showed the highest TPC, \n\nwhile the extract of E. adenophorum showed the highest \n\nTable 9: MIC of plant extracts against ATCC reference strains. \n\nMedicinal plants \n\nBacterial strains \n\nE. coli \nATCC 25922 \n\nK. pneumoniae \nATCC 700603 \n\nS. Typhimurium \nATCC 14028 \n\nS. aureus \nATCC 25923 \n\nEupoterium adenophorum - - - - \n\nMorus australis  3.125 6.25 - 0.012 \n\nEclipta prostrata 6.25 - - 25.0 \n\nAcacia catechu 0.39 - - 6.25 \n\nZingiber officinale - - - - \n\nPterocarpus marsupium 12.5 - - 1.56 \n\nShorea robusta 3.125 - - 12.5 \n\nMangifera indica 0.39 - - 12.5 \n\nSmallanthus sonchifolius - - - - \n\nMyrica esculenta 0.097 - - 1.56 \n\nUrtica ardens - - - - \n\nHypericum cordifolium 6.25 6.25 - 0.012 \n\nSyzygium cumini 0.39 - - 25.0 \n\nPsidium guajava 0.39 - - 0.012 \n\nChrysanthemum indicium 6.25 - - - \n\nNeomycin 0.39 3.12 0.78 0.39 \n\n50% DMSO - - - - \n\nDiameter of zone of inhibition in mm, well diameter = 6 mm, (-) = No antibacterial activity reported. Neomycin serves as positive control \nwhile 50% DMSO serves as a negative control for the test. The concentration of plant extracts expressed in mg/ml. \n\nTable 10: MIC of plant extracts against MDR bacterial strains.", "start_char_idx": 28623, "end_char_idx": 29947, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6673620d-a3a7-4e04-bd8a-40911169c6b8": {"__data__": {"id_": "6673620d-a3a7-4e04-bd8a-40911169c6b8", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2454df20-eb6f-4445-bead-ee72c96d0cfd", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "da8d8ba894948a37086e42af07c906557155ee633eec0cfd4d0014deca41deb7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "82a03242-3026-4f05-8efe-753c5505c519", "node_type": "1", "metadata": {}, "hash": "181205cc1ed10de988ebd667158f0fddc31221e06922a28a827c73c3e5a9758e", "class_name": "RelatedNodeInfo"}}, "text": "Table 10: MIC of plant extracts against MDR bacterial strains. \n\nMedicinal plants \n\nBacterial strains \n\n386 338 628 767 4331 484 \n\nEupoterium adenophorum 1.56 - - - - - \nMorus australis 0.012 0.19 3.12 0.05 - 0.05 \n\nEclipta prostrata 1.56 - 6.25 3.12 12.5 - \n\nAcacia catechu 0.78 - 6.25 1.56 - - \nZingiber officinale - - - - - - \n\nPterocarpus marsupium 0.39 1.56 3.12 0.39 12.5 - \n\nShorea robusta - - 6.25 - - - \nMangifera indica - - 3.12 0.78 - - \n\nSmallanthus sonchifolius - - 6.25 - - - \n\nMyrica esculenta 0.39 12.5 3.12 1.56 6.25 - \nUrtica ardens - - - - 12.5 - \nHypericum cordifolium 0.012 0.19 6.25 - - 0.78 \n\nSyzygium cumini 0.19 - 6.25 0.78 - - \n\nPsidium guajava - 3.12 3.12 1.56 - - \n\nChrysanthemum indicium - 1.56 6.25 1.56 - - \n\n50% DMSO - - - - - - \nNeomycin 0.78 6.25 - - 12.5 0.012 \nAmikacin - 3.12 3.12 - 6.25 0.78 \nNitrofurantoin 3.12 - - 3.12 - 0.78 \n\n(-) No antibacterial activity, 2A = E. coli, 338 = methicillin-resistant S. aureus (MRSA), 386 = K. pneumoniae, 628 = A. \nbaumannii, 377 = C. freundii, 767 = Xanthomonas species, 4331 = M. morganii, 484 = P. aeruginosa. Neomycin, Amikacin and \nNitrofurantoin were used as positive control and 50% DMSO as negative control for test. The concentration of plant \nextracts expressed in mg/ml. \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  58 \n\nTFC values of 55.21 \u00b1 11.09 mg GAE/gm and 10.23 \u00b1 \n\n1.07 mg QE/gm respectively (Table 4 and Table 5). A. \n\ncatechu had the highest free radical scavenging activity \n\nin the DPPH assay, followed by M. esculenta, S. cumini, \n\nand S. robusta. Flavonoid and phenolic compounds from \n\nplants have been shown to have free radical scavenging \n\nactivity and antioxidant properties, according to \n\nprevious research [60]. The methanolic extract of A. \n\ncatechu shows the IC50 of about 84.9\u2009\u00b1\u20091.9\u2009\u00b5g/mL while \n\n1.30 \u00b1 0.05 \u2009\u00b5g/mL in our study [19]. The difference \n\nmight be due to environmental variation, temperature, \n\nharvesting time, and temperature.  These antioxidant \n\nmechanisms defend humans from infections and \n\ndegenerative diseases by inhibiting and scavenging free \n\nradicals [61].", "start_char_idx": 29885, "end_char_idx": 32029, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "82a03242-3026-4f05-8efe-753c5505c519": {"__data__": {"id_": "82a03242-3026-4f05-8efe-753c5505c519", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6673620d-a3a7-4e04-bd8a-40911169c6b8", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "dd354f91d60c0c8969a4ba4c9f04d3157ef75c91fffee4d3c44bab18cc746380", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "caee2a05-8dcf-4485-abd3-5d08904967bc", "node_type": "1", "metadata": {}, "hash": "f65a1cf0f1e6cf254a6ea57f0d371e6c048391ca045fce405760f8636090e23e", "class_name": "RelatedNodeInfo"}}, "text": "The present study showed selected plant extracts \n\npossessed antibacterial activity; E. prostrata showed \n\npotential antibacterial activity against the ATCC strain \n\nof E. coli, S. aureus, and K. pneumoniae with ZoI ranging \n\nfrom 7 mm to 11 mm. Meanwhile, against MDR strains, \n\nthe extract of E. prostrata showed ZoI against \n\nAcinetobacter spp. (628), K. pneumoniae (386), Morganella \n\nmorganii (4331), and Xanthomonas spp. (767). Previous \n\nstudies also support the antibacterial and antifungal \n\nactivity of E. prostata (Chung et al., 2017; Khanna & \n\nKannabiran, 2008). Cherdtrakulki at et al. (2015) \n\nreported that bioactive compounds isolated from the \n\naerial parts of E. prostrata such as triterpenoids, 3-\n\nacetylaleuritolic acid, stigmasterol, a mixture of \n\ntriterpenoids, fatty esters, and aromatic components, \n\nhad effective antimicrobial activity \n\nagainst Corynebacterium diphtheria NCTC 10356, \n\nMorexella catarrhalis, Streptococcus pyogenes and \n\nSaccharomyces cerevisiae ATCC 2601. Another study \n\nsuggests the presence of alkaloids, cardiglycosides, \n\nphytosterol, beta-amyrin, polyacetylene, caffeic acid, \n\nstigmasterol, daucosterol on E. prostrata extracts and are \n\nfound to be effective against K. pneumoniae, S. \n\ndysenteriae, E. coli, S. Typhi, B. subtilis, P. aeruginosa, and \n\nS. aureus [26]. Recently, ecliprostins A, B, and C isolated \n\nfrom this plant showed MIC of  25.0, 6.25 and 25.0 \uf06dM, \n\nrespectively towards the growth of S. aureus [64]. \n\nM. australis extract showed a wide range of antibacterial \n\nactivity against the MDR strains of Acinetobacter spp. \n\n(628), methicillin-resistant S. aureus (MRSA) (338), K. \n\npneumoniae (386), P. aeruginosa (484), and Xanthomonas \n\nspp. (767) with MIC value of 3.12 mg/mL, 0.19 mg/mL, \n\n0.012 mg/mL, 0.05 mg/mL and 0.05 mg/mL \n\nrespectively. A similar kind of result was observed by \n\nWei et al. (2016) against a wide range of pathogens such \n\nas S. aureus, Fusarium roseum, S. faecalis,  B. cereus,  E. \n\ncoli,  K. pneumoniae, P. aeruginosa, Salmonella enterica \n\nserovar typhi,  C. freundii, Candida albicans, Microsporum \n\naudouinii, B. subtilis, Micrococus flavus,  and Salmonella \n\nabony due to presence of phytoconstituents such as that \n\nmulberrofuran, moracins, oxyresveratrol, morusin, and \n\nkuwanon C isolated from methanolic extract of Morus \n\nplant\u2019s root bark. Other plant extracts such as P. \n\nmarsupium, M. esculenta, H. cordifolium also exhibited \n\nantibacterial activity against MDR strains with varying \n\nMIC values (Table 9 and Table 10).  \n\nThe plant extracts might have a wide variety of \n\nphytochemicals that have different mechanisms of \n\naction for their antimicrobial activity [66]. By inhibiting \n\nenzymes and highly oxidizing compounds, phenol or \n\nhydroxylated phenol inhibits bacterial development, \n\nlikely through reaction with sulfhydryl groups or \n\nnonspecific interactions with proteins [67].", "start_char_idx": 32032, "end_char_idx": 34939, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "caee2a05-8dcf-4485-abd3-5d08904967bc": {"__data__": {"id_": "caee2a05-8dcf-4485-abd3-5d08904967bc", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "82a03242-3026-4f05-8efe-753c5505c519", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "9d02dfc7dcbdca0d92df1525caa743f97f4ada831a3d913aceb5221def11eadd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e6d89a4-776e-492c-b830-11c990a5566a", "node_type": "1", "metadata": {}, "hash": "4339445568b16c07c619504ee3c0027fbc283e03f9fe062bbb007339995d45bf", "class_name": "RelatedNodeInfo"}}, "text": "Antimicrobial effects are possibly due to flavonoid\u2019s \n\nability to bind to extracellular and soluble proteins, as \n\nwell as bacterial cell walls, inactivate enzymes, and \n\ndisrupt microbial membranes [68]. Tannins function as \n\nantimicrobials by binding to adhesins, inhibiting \n\nenzymes, depriving bacteria of their food, forming a \n\ncomplex with the cell wall, disrupting membranes, and \n\ncomplexing metal ions [69]. Terpenoids and essential \n\noils show antimicrobial activity by membrane \n\ndisruption by the lipophilic compounds.  Alkaloid acts \n\nas an antimicrobial agent by intercalating into the cell \n\nwall and DNA of parasites [10]. These results indicate \n\nthat Nepalese medicinal plants contain different \n\nphytochemicals that need to be explored further to \n\nacquire a future drug candidate against MDR \n\npathogens. \n\nConclusion \nMedicinal plants have long been used as traditional \n\nhealers for a range of infections, and they are also useful \n\nin the formulation of drugs to treat a variety of \n\nconditions. The leaves extract of E. andenophorum \n\nshowed the highest TFC (10.23 \u00b1 1.07 mg QE/gm) while \n\nbark extract of A. catechu showed a high TPC (55.21 \u00b1 \n\n11.09 mg GAE/gm). Morus australis showed a broad-\n\nspectrum antibacterial activity that might be a potential \n\nsource of the future drug to treat MDR-associated \n\ninfections. Similarly, other plant extracts such as E. \n\nprostrata, M. esculenta, P. marsupium, and H. cordifolium \n\nalso showed potential antibacterial activity against \n\nclinical isolates of MDR bacteria. Future studies are \n\nanticipated to examine the possibility of these plants in \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  59 \n\nethnomedicine and drug discovery to treat infections \n\ncaused by drug-resistant pathogens. \n\nAvailability of data and materials \nPlant specimen herbaria are kept in the National \n\nCollege, Kathmandu, and can be retrieved as needed. \n\nData supporting this manuscript are accessible upon \n\nappropriate request to the corresponding author. \n\nConflict of interests \nWe announce that none of the writers have a conflict of \n\ninterest in reporting these results. \n\nFunding statement \nNot applicable \n\nList of abbreviations \nAmerican type culture collection (ATCC); Minimum \n\ninhibitory concentrations (MIC); Multidrug-resistant \n\n(MDR); Optical density (OD); The inhibitory \n\nconcentration of drug/extract that gives half-maximal \n\nresponse (IC50); Total phenol contents (TPC); Total \n\nflavonoid contents (TFC); Zone of inhibition (ZoI); 2,2-\n\ndiphenyl-1-picrylhydrazyl (DPPH) \n\nReferences \n1. WHO. (2019). WHO global report on traditional and \n\ncomplementary medicine 2019. World Health \n\nOrganization. \n\nhttps://apps.who.int/iris/handle/10665/312342 \n\n2. Kirba\u011f SE, Zengin F, Kursat M. (2009). \nAntimicrobial activities of extracts of some plants. \n\nPakistan J. of Botany, 41(4), 2067-70.  \n\n3. Magiorakos, A.-P., Srinivasan, A., Carey, R. B., \nCarmeli, Y., Falagas, M. E., Giske, C. G., Harbarth, \n\nS., Hindler, J. F., Kahlmeter, G., Olsson-Liljequist, \n\nB., Paterson, D. L., Rice, L. B., Stelling, J., \n\nStruelens, M. J., Vatopoulos, A., Weber, J. T., & \n\nMonnet, D. L. (2012).", "start_char_idx": 34942, "end_char_idx": 38138, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e6d89a4-776e-492c-b830-11c990a5566a": {"__data__": {"id_": "9e6d89a4-776e-492c-b830-11c990a5566a", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "caee2a05-8dcf-4485-abd3-5d08904967bc", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "647c9119f320656cab8a9cd61b65dfdd8eba44fd09a7ebf2ff893b44c442b2c3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5dbfa9cc-94b9-4970-b0bf-4a8741d02265", "node_type": "1", "metadata": {}, "hash": "26f639e406b2f52e69c8408657ec90dc16f2421b74b25f04725805b8683b8386", "class_name": "RelatedNodeInfo"}}, "text": "(2012). Multidrug-resistant, \n\nextensively drug-resistant and pandrug-resistant \n\nbacteria: An international expert proposal for interim \n\nstandard definitions for acquired resistance. Clinical \n\nMicrobiology and Infection, 18(3), 268\u2013281. \n\nhttps://doi.org/10/bbqpzj \n\n4. Tavares, L. S., Silva, C. dos S. F. da, Souza, V. C., \nSilva, V. L. da, Diniz, C. G., & Santos, M. D. O. \n\n(2013). Strategies and molecular tools to fight \n\nantimicrobial resistance: Resistome, transcriptome, \n\nand antimicrobial peptides. Frontiers in \n\nMicrobiology, 4, 412. https://doi.org/10/ghf5qc \n\n5. Sommer, M. O., & Dantas, G. (2011). Antibiotics \nand the resistant microbiome. Current Opinion in \n\nMicrobiology, 14(5), 556\u2013563. \n\nhttps://doi.org/10/b6ttxr \n\n6. Lima, R., Del Fiol, F. S., & Balc\u00e3o, V. M. (2019). \nProspects for the Use of New Technologies to \n\nCombat Multidrug-Resistant Bacteria. Frontiers in \n\nPharmacology, 10. \n\nhttps://doi.org/10.3389/fphar.2019.00692 \n\n7. Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., \nMuzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. \n\nF., Aslam, M. A., Qamar, M. U., Salamat, M. K. F., \n\n& Baloch, Z. (2018). Antibiotic resistance: A \n\nrundown of a global crisis. Infection and Drug \n\nResistance, 11, 1645\u20131658. \n\nhttps://doi.org/10.2147/IDR.S173867 \n\n8. Tagliabue, A., & Rappuoli, R. (2018). Changing \nPriorities in Vaccinology: Antibiotic Resistance \n\nMoving to the Top. Frontiers in Immunology, 9, \n\n1068. https://doi.org/10.3389/fimmu.2018.01068 \n\n9. Mulani, M. S., Kamble, E. E., Kumkar, S. N., Tawre, \nM. S., & Pardesi, K. R. (2019). Emerging Strategies \n\nto Combat ESKAPE Pathogens in the Era of \n\nAntimicrobial Resistance: A Review. Frontiers in \n\nMicrobiology, 10, 539. https://doi.org/10/ghfddk \n\n10. Othman, L., Sleiman, A., & Abdel-Massih, R. M. \n(2019). Antimicrobial Activity of Polyphenols and \n\nAlkaloids in Middle Eastern Plants. Frontiers in \n\nMicrobiology, 10, 911. \n\nhttps://doi.org/10.3389/fmicb.2019.00911 \n\n11. Nascimento, G. G. F., Locatelli, J., Freitas, P. C., & \nSilva, G. L. (2000). Antibacterial activity of plant \n\nextracts and phytochemicals on antibiotic-resistant \n\nbacteria. Brazilian Journal of Microbiology, 31(4), \n\n247\u2013256. https://doi.org/10.1590/S1517-\n\n83822000000400003 \n\n12. Blanco, M. M., Costa, C. A. R. A., Freire, A. O., \nSantos, J. G., & Costa, M. (2009). Neurobehavioral \n\neffect of essential oil of Cymbopogon citratus in \n\nmice.", "start_char_idx": 38131, "end_char_idx": 40540, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5dbfa9cc-94b9-4970-b0bf-4a8741d02265": {"__data__": {"id_": "5dbfa9cc-94b9-4970-b0bf-4a8741d02265", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e6d89a4-776e-492c-b830-11c990a5566a", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "46e4ce75390496aa8e4098d79dac2fee0cba0e5db8f723ed33e75cef3a1cabe0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "57893fd0-9872-4b3d-b512-d30e18138d3c", "node_type": "1", "metadata": {}, "hash": "e917976bdc74d4b9ddf9d90c88e60a85f7ea6f6d9fe3b788aa728bf3257287b2", "class_name": "RelatedNodeInfo"}}, "text": "Phytomedicine, 16(2), 265\u2013270. \n\nhttps://doi.org/10.1016/j.phymed.2007.04.007 \n\n13. Bekoe, E. O., Kitcher, C., Gyima, N. A. M., \nSchwinger, G., & Frempong, M. (2018). Medicinal \n\nPlants Used as Galactagogues. Pharmacognosy - \n\nMedicinal Plants. https://doi.org/10/ghf3rm \n\n14. I\u015fcan, G., Ki\u0307ri\u0307mer, N., K\u00fcrkc\u00fco\u01e7lu, M., H\u00fcsn\u00fc Can \nBa\u015fer, & Demi\u0307rci\u0307, F. (2002). Antimicrobial \n\nScreening of Mentha piperita Essential Oils. Journal \n\nof Agricultural and Food Chemistry, 50(14), 3943\u2013\n\n3946. https://doi.org/10/cs7jf8 \n\n15. Ainsworth, E. A., & Gillespie, K. M. (2007). \nEstimation of total phenolic content and other \n\noxidation substrates in plant tissues using Folin\u2013\n\nCiocalteu reagent. Nature Protocols, 2(4), 875\u2013877. \n\nhttps://doi.org/10.1038/nprot.2007.102 \n\n16. Bhandari, S., Khadayat, K., Poudel, S., Shrestha, S., \nShrestha, R., Devkota, P., Khanal, S., & Marasini, B. \n\nP. (2021). Phytochemical analysis of medicinal \n\nplants of Nepal and their antibacterial and antibiofilm \n\nactivities against uropathogenic Escherichia coli. \n\nBMC Complementary Medicine and Therapies, \n\n21(1), 116. https://doi.org/10/gk45f5 \n\n17. Joubert, E., Manley, M., & Botha, M. (2008). \nEvaluation of spectrophotometric methods for \n\nscreening of green rooibos (Aspalathus linearis) and \n\ngreen honeybush (Cyclopia genistoides) extracts for \n\nhigh levels of Bio-active compounds. Phytochemical \n\nAnalysis, 19(2), 169\u2013178. https://doi.org/10/dv3t8n \n\n18. Brand-Williams, W., Cuvelier, M. E., & Berset, C. \n(1995). Use of a free radical method to evaluate \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  60 \n\nantioxidant activity. LWT - Food Science and \n\nTechnology, 28(1), 25\u201330. \n\nhttps://doi.org/10.1016/S0023-6438(95)80008-5 \n\n19. Aryal, B., Niraula, P., Khadayat, K., Adhikari, B., \nChhetri, D., Sapkota, B., Bhattarai, B., & Parajuli, N. \n\n(2021). Antidiabetic, Antimicrobial, and Molecular \n\nProfiling of Selected Medicinal Plants. Evidence-\n\nBased Complementary and Alternative Medicine, \n\n2021. https://doi.org/10/gjxhzt \n\n20. Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). \nMethods for in vitro evaluating antimicrobial \n\nactivity: A review. Journal of Pharmaceutical \n\nAnalysis, 6(2), 71\u201379. \n\nhttps://doi.org/10.1016/j.jpha.2015.11.005 \n\n21.", "start_char_idx": 40541, "end_char_idx": 42830, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "57893fd0-9872-4b3d-b512-d30e18138d3c": {"__data__": {"id_": "57893fd0-9872-4b3d-b512-d30e18138d3c", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5dbfa9cc-94b9-4970-b0bf-4a8741d02265", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "a9188fb0fde01ecc3d00fdf55457d838a8b8ffb51e4963075bc291a035b4e707", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "472b8714-424c-4134-bdf6-e66dcaee6faa", "node_type": "1", "metadata": {}, "hash": "5fa8a55cc07b325885b157620e4e855e2d5a45a2f4f01dc32590f43640518481", "class_name": "RelatedNodeInfo"}}, "text": "Marasini, B. P., Baral, P., Aryal, P., Ghimire, K. R., \nNeupane, S., Dahal, N., Singh, A., Ghimire, L., & \n\nShrestha, K. (2015, February 9). Evaluation of \n\nAntibacterial Activity of Some Traditionally Used \n\nMedicinal Plants against Human Pathogenic \n\nBacteria [Research Article]. BioMed Research \n\nInternational; Hindawi. \n\nhttps://doi.org/10.1155/2015/265425 \n\n22. Valgas, C., Souza, S. M. de, Sm\u00e2nia, E. F. A., & \nSm\u00e2nia Jr., A. (2007). Screening methods to \n\ndetermine antibacterial activity of natural products. \n\nBrazilian Journal of Microbiology, 38(2), 369\u2013380. \n\nhttps://doi.org/10/ftmw7n \n\n23. Kato, M., Sugiyama, K., Fukushima, T., Miura, Y., \nAwogi, T., Hikosaka, S., Kawakami, K., Nakajima, \n\nM., Nakamura, M., Sui, H., Watanabe, K., & Hakura, \n\nA. (2018). Negative and positive control ranges in \n\nthe bacterial reverse mutation test: JEMS/BMS \n\ncollaborative study. Genes and Environment, 40(1), \n\n7. https://doi.org/10.1186/s41021-018-0096-1 \n\n24. CLSI. (2015). Performance standards for \nantimicrobial susceptibility testing: 25th \n\ninformational supplement (M100-S23). Clinical and \n\nLaboratory Standards Institute. \n\n25. Mors, W. B., do Nascimento, M. C., Parente, J., da \nSilva, M. H., Melo, P. A., & Suarez-Kurtz, G. \n\n(1989). Neutralization of lethal and myotoxic \n\nactivities of South American rattlesnake venom by \n\nextracts and constituents of the plant Eclipta prostrata \n\n(Asteraceae). Toxicon, 27(9), 1003\u20131009. \n\nhttps://doi.org/10.1016/0041-0101(89)90151-7 \n\n26. Priya, K., John, P., Usha, P. T. A., Kariyil, B. J., \nUma, R., & Hogale, M. S. (2018). Phytochemical \n\nAnalysis of Eclipta prostrata L. (L.) Leaves. \n\nInternational Journal of Current Microbiology and \n\nApplied Sciences, 7(08), 1069\u20131075. \n\nhttps://doi.org/10.20546/ijcmas.2018.708.121 \n\n27. Chung, I.-M., Rajakumar, G., Lee, J.-H., Kim, S.-H., \n& Thiruvengadam, M. (2017). Ethnopharmacological \n\nuses, phytochemistry, biological activities, and \n\nbiotechnological applications of Eclipta prostrata. \n\nApplied Microbiology and Biotechnology, 101(13), \n\n5247\u20135257. https://doi.org/10.1007/s00253-017-\n\n8363-9 \n\n28. Kumar, D., Wani, T., Kumar, D., Prasad, R., Verma, \nP., Sardar, K., & Tandan, S. (2012). Analgesic \n\nactivity of the ethanolic extract of Shorea robusta \n\nresin in experimental animals. Indian Journal of \n\nPharmacology, 44(4), 493. \n\nhttps://doi.org/10.4103/0253-7613.99322 \n\n29. Liu, J. (1995). Pharmacology of oleanolic acid and \nursolic acid.", "start_char_idx": 42831, "end_char_idx": 45284, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "472b8714-424c-4134-bdf6-e66dcaee6faa": {"__data__": {"id_": "472b8714-424c-4134-bdf6-e66dcaee6faa", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "57893fd0-9872-4b3d-b512-d30e18138d3c", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "4ea0015d18006c49b6f2793812b23529a1f6d1f9e98592820ffe0b17b068b51d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "998841f4-38f3-4e14-9612-47d22a0e5df0", "node_type": "1", "metadata": {}, "hash": "555f3407c30b6e1f31267d94713f3f4a01df45a41a54f5c9e7f28459563511ec", "class_name": "RelatedNodeInfo"}}, "text": "(1995). Pharmacology of oleanolic acid and \nursolic acid. Journal of Ethnopharmacology, 49(2), \n\n57\u201368. https://doi.org/10.1016/0378-8741(95)90032-\n\n2 \n\n30. Hong, S. S., Lee, S. A., Han, X. H., Lee, M. H., \nHwang, J. S., Park, J. S., Oh, K.-W., Han, K., Lee, \n\nM. K., Lee, H., Kim, W., Lee, D., & Hwang, B. Y. \n\n(2008). Melampolides from the Leaves of \n\nSmallanthus sonchifolius and Their Inhibitory \n\nActivity of LPS-Induced Nitric Oxide Production. \n\nChemical and Pharmaceutical Bulletin, 56(2), 199\u2013\n\n202. https://doi.org/10.1248/cpb.56.199 \n\n31. Genta, S. B., Cabrera, W. M., Mercado, M. I., Grau, \nA., Catal\u00e1n, C. A., & S\u00e1nchez, S. S. (2010). \n\nHypoglycemic activity of leaf organic extracts from \n\nSmallanthus sonchifolius: Constituents of the most \n\nactive fractions. Chemico-Biological Interactions, \n\n185(2), 143\u2013152. \n\nhttps://doi.org/10.1016/j.cbi.2010.03.004 \n\n32. Paudel, M. (1970). Non-timber forest products from \ncommunity forestry practices, problems and \n\nprospects for livelihood strategy in Jumla. Banko \n\nJanakari, 17(2), 45\u201354. \n\nhttps://doi.org/10.3126/banko.v17i2.2155 \n\n33. Taylor, R. S. L., Manandhar, N. P., Hudson, J. B., & \nTowers, G. H. N. (1996). Antiviral activities of \n\nNepalese medicinal plants. Journal of \n\nEthnopharmacology, 52(3), 157\u2013163. \n\nhttps://doi.org/10.1016/0378-8741(96)01409-2 \n\n34. N\u00fa\u00f1ez-Sell\u00e9s, A. J. (2005). Antioxidant therapy: \nMyth or reality? Journal of the Brazilian Chemical \n\nSociety, 16(4), 699\u2013710. \n\nhttps://doi.org/10.1590/S0103-50532005000500004 \n\n35. Shah, K., Patel, M., Patel, R., & Parmar, P. (2010). \nMangifera Indica (Mango). Pharmacognosy Reviews, \n\n4(7), 42. https://doi.org/10.4103/0973-7847.65325 \n\n36. Jhaumeer Laulloo, S., Bhowon, M. G., Soyfoo, S., & \nChua, L. S. (2018). Nutritional and Biological \n\nEvaluation of Leaves of Mangifera indica from \n\nMauritius. Journal of Chemistry, 2018, 1\u20139. \n\nhttps://doi.org/10.1155/2018/6869294 \n\n37. Katsube, T., Imawaka, N., Kawano, Y., Yamazaki, \nY., Shiwaku, K., & Yamane, Y. (2006). Antioxidant \n\nflavonol glycosides in mulberry (Morus alba L.) \n\nleaves isolated based on LDL antioxidant activity. \n\nFood Chemistry, 97(1), 25\u201331. \n\nhttps://doi.org/10.1016/j.foodchem.2005.03.019 \n\n38. Hikino, H., Takahashi, M., Oshima, Y., & Konno, C. \n(1988).", "start_char_idx": 45227, "end_char_idx": 47490, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "998841f4-38f3-4e14-9612-47d22a0e5df0": {"__data__": {"id_": "998841f4-38f3-4e14-9612-47d22a0e5df0", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "472b8714-424c-4134-bdf6-e66dcaee6faa", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "50bce9c94ad9e3efa637861d7087726b4530f23acdada82d69a78f5d44c9822b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "20890ca0-ed39-402a-80c7-f3aae3bfbef7", "node_type": "1", "metadata": {}, "hash": "5b4ce34badca0731101f35155ca4578e7d17dc6c21dbaf1c83f8ff2323a5e506", "class_name": "RelatedNodeInfo"}}, "text": "(1988). Isolation and Hypoglycemic Activity of \n\nOryzabrans A, B, C and D, Glycans of Oryza sativa \n\nBran. Planta Medica, 54(01), 1\u20133. \n\nhttps://doi.org/10.1055/s-2006-962316 \n\n39. Heinrich, M., Ankli, A., Frei, B., Weimann, C., & \nSticher, O. (1998). Medicinal plants in Mexico: \n\nHealers\u2019 consensus and cultural importance. Social \n\nScience & Medicine, 47(11), 1859\u20131871. \n\nhttps://doi.org/10.1016/S0277-9536(98)00181-6 \n\n40. D\u00edaz-de-Cerio, E., Verardo, V., G\u00f3mez-Caravaca, \nA., Fern\u00e1ndez-Guti\u00e9rrez, A., & Segura-Carretero, A. \n\n(2016). Exploratory Characterization of Phenolic \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  61 \n\nCompounds with Demonstrated Anti-Diabetic \n\nActivity in Guava Leaves at Different Oxidation \n\nStates. International Journal of Molecular Sciences, \n\n17(5), 699. https://doi.org/10.3390/ijms17050699 \n\n41. Shunying, Z., Yang, Y., Huaidong, Y., Yue, Y., & \nGuolin, Z. (2005). Chemical composition and \n\nantimicrobial activity of the essential oils of \n\nChrysanthemum indicum. Journal of \n\nEthnopharmacology, 96(1\u20132), 151\u2013158. \n\nhttps://doi.org/10.1016/j.jep.2004.08.031 \n\n42. Lee, D. Y., Choi, G., Yoon, T., Cheon, M. S., Choo, \nB. K., & Kim, H. K. (2009). Anti-inflammatory \n\nactivity of Chrysanthemum indicum extract in acute \n\nand chronic cutaneous inflammation. Journal of \n\nEthnopharmacology, 123(1), 149\u2013154. \n\nhttps://doi.org/10.1016/j.jep.2009.02.009 \n\n43. Indra, D. B., & Uppe, D. (2004). Factors controlling \nmicropropagation of Myrica esculenta buch. Ham. ex \n\nD. Don: A high value wild edible of Kumaun \n\nHimalaya. African Journal of Biotechnology, 3(10), \n\n534\u2013540. https://doi.org/10.5897/AJB2004.000-2097 \n\n44. Gyawali, R., Adhikari, S., Gautam, S., Guragain, P., \nPokharel, S., Pradhan, N., Sijapati, S., & Shrestha, T. \n\nM. (2015). Antimicrobial and cytotoxic properties of \n\nselected medicinal plants from Kavrepalanchowk, \n\nNepal. Banko Janakari, 25(1), 15\u201319. \n\nhttps://doi.org/10.3126/banko.v25i1.13467 \n\n45. Taylor, K. (2009). Biological Flora of the British \nIsles: Urtica dioica L. Journal of Ecology, 97(6), \n\n1436\u20131458. https://doi.org/10.1111/j.1365-\n\n2745.2009.01575.x \n\n46. Ibrahim, M., Rehman, K., Razzaq, A., Hussain, I., \nFarooq, T., Hussain, A., & Akash, M. S. H.", "start_char_idx": 47483, "end_char_idx": 49756, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "20890ca0-ed39-402a-80c7-f3aae3bfbef7": {"__data__": {"id_": "20890ca0-ed39-402a-80c7-f3aae3bfbef7", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "998841f4-38f3-4e14-9612-47d22a0e5df0", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "d6fae9a4d42ba9b7ddf192905898d9953c001bce7262e8bce22367b7046e95f8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8671d30c-85ff-4030-b369-5a18010e2538", "node_type": "1", "metadata": {}, "hash": "160347147c83c1258d032585cefe8c570c3687cf85d494376d5368cf34cff0a2", "class_name": "RelatedNodeInfo"}}, "text": "(2018). \n\nInvestigations of Phytochemical Constituents and \n\nTheir Pharmacological Properties Isolated from the \n\nGenus Urtica: Critical Review and Analysis. Critical \n\nReviews in Eukaryotic Gene Expression, 28(1), 25\u2013\n\n66. \n\nhttps://doi.org/10.1615/CritRevEukaryotGeneExpr.2\n\n018020389 \n\n47. Mankani, K. L., Krishna, V., Manjunatha, B. K., \nVidya, S. M., Singh, S. J., Manohara, Y. N., \n\nRaheman, A.-U., & Avinash, K. R. (2005). \n\nEvaluation of hepatoprotective activity of stem bark \n\nof Pterocarpus marsupium Roxb. Indian Journal of \n\nPharmacology, 37(3), 165. \n\nhttps://doi.org/10.4103/0253-7613.16213 \n\n48. Jung, M., Park, M., Lee, H., Kang, Y.-H., Kang, E., \n& Kim, S. (2006). Antidiabetic Agents from \n\nMedicinal Plants. Current Medicinal Chemistry, \n\n13(10), 1203\u20131218. \n\nhttps://doi.org/10.2174/092986706776360860 \n\n49. Negi, A., Upadhyay, A., Semwal, A., & Kumar \nWahi, A. (2010). Pharmacognostical Studies on the \n\nLeaves of Eupatorium adenophorum Spreng. \n\nPharmacognosy Journal, 2(15), 1\u20137. \n\nhttps://doi.org/10.1016/S0975-3575(10)80071-9 \n\n50. Liu, B., Cao, L., Zhang, L., Yuan, X., & Zhao, B. \n(2016). Preparation, Phytochemical Investigation, \n\nand Safety Evaluation of Chlorogenic Acid Products \n\nfrom Eupatorium adenophorum. Molecules, 22(1), \n\n67. https://doi.org/10.3390/molecules22010067 \n\n51. Grzanna, R., Lindmark, L., & Frondoza, C. G. \n(2005). Ginger\u2014An Herbal Medicinal Product with \n\nBroad Anti-Inflammatory Actions. Journal of \n\nMedicinal Food, 8(2), 125\u2013132. \n\nhttps://doi.org/10.1089/jmf.2005.8.125 \n\n52. Singh, B. K., Tripathi, M., Chaudhari, B. P., Pandey, \nP. K., & Kakkar, P. (2012). Natural Terpenes Prevent \n\nMitochondrial Dysfunction, Oxidative Stress and \n\nRelease of Apoptotic Proteins during Nimesulide-\n\nHepatotoxicity in Rats. PLoS ONE, 7(4), e34200. \n\nhttps://doi.org/10.1371/journal.pone.0034200 \n\n53. Negi, B. S., & Dave, B. P. (2010). In Vitro \nAntimicrobial Activity of Acacia catechu and Its \n\nPhytochemical Analysis. Indian Journal of \n\nMicrobiology, 50(4), 369\u2013374. \n\nhttps://doi.org/10.1007/s12088-011-0061-1 \n\n54. Schoenfelder, T., Warmlin, C. Z., Manfredini, M. S., \nPavei, L. L., R\u00e9us, J. V., Trist\u00e3o, T. C., Fernandes, \n\nM. S., & Costa-Campos, L. (2010). Hypoglycemic \n\nand hypolipidemic effect of leaves from Syzygium \n\ncumini (L.) Skeels, Myrtaceae. In diabetic rats.", "start_char_idx": 49757, "end_char_idx": 52081, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8671d30c-85ff-4030-b369-5a18010e2538": {"__data__": {"id_": "8671d30c-85ff-4030-b369-5a18010e2538", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "20890ca0-ed39-402a-80c7-f3aae3bfbef7", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "ce3e035639326d0d320dbec1e4026b02395c8e3cf654009e24b38e8fe78032da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "457bdba5-736d-4058-8c5e-ae65819c7b0f", "node_type": "1", "metadata": {}, "hash": "cfe2050ef9a64e709055d7f45c53389c2e818439a21861ada40894f71d219751", "class_name": "RelatedNodeInfo"}}, "text": "Skeels, Myrtaceae. In diabetic rats. \n\nRevista Brasileira de Farmacognosia, 20(2), 222\u2013\n\n227. https://doi.org/10.1590/S0102-\n\n695X2010000200014 \n\n55. Ruan, Z. P., Zhang, L. L., & Lin, Y. M. (2008). \nEvaluation of the Antioxidant Activity of Syzygium \n\ncumini Leaves. Molecules, 13(10), 2545\u20132556. \n\nhttps://doi.org/10.3390/molecules13102545 \n\n56. Chagas, V. T., Fran\u00e7a, L. M., Malik, S., & Paes, A. \nM. de A. (2015). Syzygium cumini (L.) skeels: A \n\nprominent source of bioactive molecules against \n\ncardiometabolic diseases. Frontiers in \n\nPharmacology, 6. \n\nhttps://doi.org/10.3389/fphar.2015.00259 \n\n57. Hasan, M. M., Hossain, A., Shamim, A., & Rahman, \nM. M. (2017). Phytochemical and pharmacological \n\nevaluation of ethanolic extract of Lepisanthes \n\nrubiginosa L. leaves. BMC Complementary and \n\nAlternative Medicine, 17(1), 1\u201311. \n\nhttps://doi.org/10.1186/s12906-017-2010-y \n\n58. Kunwar, R. M., & Bussmann, R. W. (2008). \nEthnobotany in the Nepal Himalaya. Journal of \n\nEthnobiology and Ethnomedicine, 4, 1\u20138. \n\nhttps://doi.org/10.1186/1746-4269-4-24 \n\n59. Turkmen, N., Sari, F., & Velioglu, Y. S. (2006). \nEffects of extraction solvents on concentration and \n\nantioxidant activity of black and black mate tea \n\npolyphenols determined by ferrous tartrate and Folin-\n\nCiocalteu methods. Food Chemistry, 99(4), 835\u2013841. \n\nhttps://doi.org/10.1016/j.foodchem.2005.08.034 \n\n60. Khorasani Esmaeili, A., Mat Taha, R., Mohajer, S., \n& Banisalam, B. (2015, May 6). Antioxidant Activity \n\nand Total Phenolic and Flavonoid Content of \n\nVarious Solvent Extracts from In Vivo and In Vitro \n\nGrown Trifolium pratense L. (Red Clover) [Research \n\nArticle]. BioMed Research International; Hindawi. \n\nhttps://doi.org/10.1155/2015/643285 \n\n61. [61] Reznick, A. Z., Shehadeh, N., Shafir, Y., & \nNagler, R. M. (2006). Free radicals related effects \n\nand antioxidants in saliva and serum of adolescents \n\nwith Type 1 diabetes mellitus. Archives of Oral \n\n\n\nNepal J Biotechnol. 2 0 2 1  J u l ; 9 (1): 5 0 - 6 2      Shrestha et al. \n\n \n\n\u00a9NJB, BSN  62 \n\nBiology, 51(8), 640\u2013648. \n\nhttps://doi.org/10.1016/j.archoralbio.2006.02.004 \n\n62. Khanna, V. G., & Kannabiran, K. (2008). \nAntimicrobial activity of saponin fractions of the \n\nleaves of Gymnema sylvestre and Eclipta prostrata. \n\nWorld Journal of Microbiology and Biotechnology, \n\n24(11), 2737. https://doi.org/10.1007/s11274-008-\n\n9758-7 \n\n63.", "start_char_idx": 52045, "end_char_idx": 54427, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "457bdba5-736d-4058-8c5e-ae65819c7b0f": {"__data__": {"id_": "457bdba5-736d-4058-8c5e-ae65819c7b0f", "embedding": null, "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-133", "node_type": "4", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "58bdb7e116499f40c0e1a690df74a9813f2e38de24e35b09310a1b131006b3fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8671d30c-85ff-4030-b369-5a18010e2538", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "945169fcfbabfc578309938e0698558046235c7665e61f50c673ac4f68c9fdf8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e2ef026-70e6-48b6-9c99-a9de1defdfaa", "node_type": "1", "metadata": {}, "hash": "1735f23576211f974149b92ce5590bdc2f92be9619ace825c0ed34dca4716c42", "class_name": "RelatedNodeInfo"}}, "text": "Cherdtrakulkiat, R., Boonpangrak, S., Pingaew, R., \nPrachayasittikul, S., Ruchirawat, S., & \n\nPrachayasittikul, V. (2015). Bioactive triterpenoids, \n\nantimicrobial, antioxidant and cytotoxic activities of \n\nEclipta prostrata Linn. -. Journal of Applied \n\nPharmaceutical Science, 5(3), 046\u2013050. \n\n64. Yu, S.-J., Yu, J.-H., He, F., Bao, J., Zhang, J.-S., \nWang, Y.-Y., & Zhang, H. (2020). New antibacterial \n\nthiophenes from Eclipta prostrata. Fitoterapia, 142, \n\n104471. https://doi.org/10.1016/j.fitote.2020.104471 \n\n65. Wei, H., Zhu, J.-J., Liu, X.-Q., Feng, W.-H., Wang, \nZ.-M., & Yan, L.-H. (2016). Review of bioactive \n\ncompounds from root barks of Morus plants (Sang-\n\nBai-Pi) and their pharmacological effects. Cogent \n\nChemistry, 2(1). \n\nhttps://doi.org/10.1080/23312009.2016.1212320 \n\n66. Adeonipekun, P. A., Adeniyi, T. A., & Aminu, S. O. \n(2014). Investigating the Phytochemicals and \n\nAntimicrobial Activities of Shoot and Root of \n\nPycreus smithianus (Ridl.) C. B. Clarke (Family \n\nCyperaceae). Journal of Botany, 2014, 1\u20135. \n\nhttps://doi.org/10.1155/2014/761613 \n\n67. Bouarab-Chibane, L., Forquet, V., Lant\u00e9ri, P., \nCl\u00e9ment, Y., L\u00e9onard-Akkari, L., Oulahal, N., \n\nDegraeve, P., & Bordes, C. (2019). Antibacterial \n\nProperties of Polyphenols: Characterization and \n\nQSAR (Quantitative Structure\u2013Activity \n\nRelationship) Models. Frontiers in Microbiology, 10. \n\nhttps://doi.org/10.3389/fmicb.2019.00829 \n\n68. Tak\u00f3, M., Kerekes, E. B., Zambrano, C., Kotog\u00e1n, \nA., Papp, T., Krisch, J., & V\u00e1gv\u00f6lgyi, C. (2020). \n\nPlant Phenolics and Phenolic-Enriched Extracts as \n\nAntimicrobial Agents against Food-Contaminating \n\nMicroorganisms. Antioxidants, 9(2), 165. \n\nhttps://doi.org/10.3390/antiox9020165 \n\n69. Vu, T. T., Kim, H., Tran, V. K., Vu, H. D., Hoang, T. \nX., Han, J. W., Choi, Y. H., Jang, K. S., Choi, G. J., \n\n& Kim, J.-C. (2017). Antibacterial activity of tannins \n\nisolated from Sapium baccatum extract and use for \n\ncontrol of tomato bacterial wilt. PLOS ONE, 12(7), \n\ne0181499. \n\nhttps://doi.org/10.1371/journal.pone.0181499", "start_char_idx": 54428, "end_char_idx": 56469, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e2ef026-70e6-48b6-9c99-a9de1defdfaa": {"__data__": {"id_": "9e2ef026-70e6-48b6-9c99-a9de1defdfaa", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "457bdba5-736d-4058-8c5e-ae65819c7b0f", "node_type": "1", "metadata": {"identifier": "njb-133", "author": "Shresta, Shrimita; Bhattarai, Bibek Raj; Adhikari, Bikash; Rayamajhee, Binod; Poudel, Pramod; Khanal, Santosh; Marasini, Bishnu P.; Aryal, Babita; Bhandari, Sudip; Parajuli, Niranjan", "title": "Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants", "date": "2021-07-31", "file": "njb-133.pdf"}, "hash": "b774c9979fdb231b6ed41cc152c27ab7e309027aa88ecb6b9f5bcb0a25c1e658", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "142e0e03-0aa6-4c97-8028-b50d9cb79408", "node_type": "1", "metadata": {}, "hash": "238169df6ad612cda516fea451e7a7d4c43c69b4f9132ef12b4f729cb2f43665", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1 D e c ; 9 (2): 7-13 Research article  DOI: https://www.doi.org/10.54796/njb.v9i2.41908 \n\n\u00a9NJB, BSN 7 \n\nMicrobial and Physico-Chemical Quality Assessment of Rivers of \nKathmandu Valley \nSantosh Poudel1 , Akash Paudyal1, Bishnu Prasad Sharma1, Kamana Sharma1, Yubaraj Baral1, Shailaja Adhikari2, \n\nManju Shree Shakya Hada1 \n1Department of Microbiology, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal \n2Kathmandu Upatyaka Khanepani Ltd. (KUKL), Kathmandu, Nepal \n\nReceived: 05th Jun 2021; Revised: 13th Dec 2021; Accepted: 16th Dec 2021; Published online: 31st  Dec 2021 \n\nAbstract \nWater quality refers to the chemical, physical, biological characteristics of water. It is a measure of condition of water relative \nto requirement of one or more biotic species and or to any human need or purpose. The main objective of the study is to detect \nthe physio-chemical and microbiological parameters of water sample from the Bagmati river and its tributaries of Kathmandu \nvalley along with antibiotic susceptibility. In physico-chemical parameters, turbidity, temperature, pH, Electrical \nconductivity, Dissolved Oxygen, Biological Oxygen Demand, Ammonia, Alkalinity, Hardness, Chloride, Phosphate, Iron, \nNitrate, Total Dissolved solids, and color were analyzed. Iron and Turbidity was found to be above the World health \norganization and Nepal Standard guideline in all the samples (100%), while Ammonia was found to be above the WHO \nguideline in 10(90%) samples. Among 11 samples, 10(90%) showed a low Dissolved oxygen level. Most Probable Number \nmethod was followed for counting total load of coliform and fecal coliform. Escherichia coli was isolated from the sample and \nsubjected to Antibiotic susceptibility. Coliform was detected in all the samples and E. coli was identified as highly resistant \ntowards Gentamicin (81.8%) and sensitive towards Chloramphenicol (81.8%). High value of ammonia, turbidity and low \nvalue of Dissolved Oxygen in the lower belts of river was due to large inputs of wastewater and organic loads caused by \nanthropogenic activities. High value of Coliform in all the samples indicates bacterial contamination in river water. The \ncomparative study for the water quality variables in the urban areas showed that the main rivers and its tributaries were \nequally polluted.  \n\nKeywords: E. coli, Bagmati, physico-chemical parameters, Antibiotic Susceptibility, Most Probable Number method \n\n Corresponding author, email: Poudelsantosh550@gmail.com \n\nIntroduction \nWater is the main constituent of Earth's hydrosphere and \n\nthe fluids of all known living organisms. It is vital for all \n\nknown forms of life [1]. Among the type of surface water, \n\nRiver is the most accessible and widely available source \n\nof water to human beings. River water has various uses \n\nlike irrigation, drinking, and water hydropower, directly \n\nrelated to people. River water has an economic backbone \n\nin the upliftment of the country [2]. \n\nBagmati is one, with religion and geographical \n\nimportance. Various important religious shrines like \n\nGokarneshwor, Pashupatinath, Guhyeshwari, Teku \n\ndovan, and others lie on the river's bank [3]. The Bagmati \n\nRiver is considered the source of Nepalese civilization \n\nand urbanization. In contrast to the various large snow-\n\nfed rivers of the Himalayas, it is a river originating from \n\nBaghdwar and Shivapuri hill at an altitude of 2650m.", "start_char_idx": 48, "end_char_idx": 3490, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "142e0e03-0aa6-4c97-8028-b50d9cb79408": {"__data__": {"id_": "142e0e03-0aa6-4c97-8028-b50d9cb79408", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e2ef026-70e6-48b6-9c99-a9de1defdfaa", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "020fbf13db1ac794e05927e0375b264a6a5ea5e11675620f07bce0f7271a96de", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3e5d36a7-6212-46a0-8d15-235b239ef41c", "node_type": "1", "metadata": {}, "hash": "9321a324ce651361f2e55bd8f7208b22b94f1ae829a87df9a9edbb1fde9a00f1", "class_name": "RelatedNodeInfo"}}, "text": "Bagmati River is the principal river of the Kathmandu \n\nvalley river system. It drains the entire valley with its \n\nseven tributaries- Bishnumati River, Manohara River, \n\nDhobi River, Nakkhu River, Balkhu River, Hanumante \n\nRiver, and Tukucha River [4]. \n\nThe quality of the Bagmati River and its tributaries have \n\nbeen rapidly degrading. It contains large amounts of \n\nuntreated sewage, and high levels of pollution of the \n\nriver exist due primarily to the region's large population. \n\nMany residents of the Kathmandu valley empty personal \n\ngarbage and waste into the river [5]. In Kathmandu, \n\nurbanization and industrialization contribute to \n\ndeterioration in water quality with regional \n\nconsequences for the aquatic system and health of \n\ndownstream sub-basin user groups. Sewer lines for \n\ndomestic and industrial wastewater have been connected \n\nto the river [6]. These activities are responsible for serious \n\npollution and the production of foul odors near the \n\nriverside. River system water entering the core urban \n\narea is visibly black with filth stinks badly. In particular, \n\nthe Hanumante River, Dhobi River, Tukucha River, and \n\nBishnumati River are the most polluted [7]. A few \n\nkilometers of the uppermost section (High Mountain \n\nwith a catchment area of 17 km2) is only suitable for \n\ndrinking water supply [8]. The remaining sections are not \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0001-9327-8850\nmailto:Poudelsantosh550@gmail.com\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  8 \n\nused for potable purposes due to greater water quality \n\ndeterioration [9].  \n\nAlthough many wastewater treatments plants have been \n\nconstructed in the Kathmandu valley, only one of the \n\nGuheshwori wastewater treatment plant is currently \n\nfunctional. Various restoration project has been ongoing \n\nwithin Kathmandu valley focused on improving water \n\nquality and establishing minimum flow requirements \n\n[10]. \n\nAssessment of the Physico-chemical and microbiological \n\nparameters of the river water indicates the level of \n\npollution and provides progression to understand its \n\neffect on the aquatic life as well as on the human \n\npopulation [11]. Water is the principal vehicle for the \n\ntransmission of a wide range of communicable diseases. \n\nFrom the microbiological view, the presence of total \n\ncoliform and fecal coliform shows then water quality. \n\nColiform bacteria E. coli, which is a medically important \n\nbacteria causing many significant illnesses like \n\nBacteremia, cholangitis, UTI, travelers' diarrhea, neonatal \n\nmeningitis, pneumonia [12,13]. In addition, other bacteria \n\nlike Salmonella, Shigella, Pseudomonas, Proteus, and Vibrio \n\nin drinking water are major causes of water-borne \n\ndiseases [14]. \n\nVarious antibiotics are used for the treatment of the \n\ndiseases caused by E. coli. In recent years the number of \n\nantibiotic-resistant strains of E. coli is increasing by the \n\nhaphazard use of antibiotics [15]. Antimicrobial \n\nresistance is a global public health concern contributing \n\nto increased morbidity and mortality, particularly in low-\n\nincome countries. Studies on commensal bacteria are \n\nessential as they reflect the state of antimicrobial \n\nsusceptibility patterns in populations. Overpopulation of \n\nKathmandu valley and inappropriate use of \n\nantimicrobials signal significant rates of resistance \n\namong flora circulating within the community. Increased \n\nuse of antibiotics in agriculture, domestic, livestock, and \n\nthe hospital are likely to develop resistance by E. \n\ncoli strains.  \n\nThis study was carried out to analyze different Physico-\n\nchemical parameters and microbiological parameters of \n\nthe Bagmati River and its tributaries.", "start_char_idx": 3493, "end_char_idx": 7381, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3e5d36a7-6212-46a0-8d15-235b239ef41c": {"__data__": {"id_": "3e5d36a7-6212-46a0-8d15-235b239ef41c", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "142e0e03-0aa6-4c97-8028-b50d9cb79408", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "836b55bb5c3ac356e12aed59e34d3ebfc03cfb1c31bf9ee618a997c288eb7530", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0a42835c-63c3-4f5d-9343-77d37944582c", "node_type": "1", "metadata": {}, "hash": "9c58e03b111e3e1554ed5af2c46fec52379aa966919eb2e607ee606e24ea5d5f", "class_name": "RelatedNodeInfo"}}, "text": "Materials and Methods \nSampling site and sample \nA total of 11 samples were tested in the study. Water \n\nsamples were collected from four different Bagmati River \n\nsites (Sundarijal, Before Guheshwori treatment plant-\n\nGaurighat, After Guheshwori treatment plant-Gaurighat \n\nand Chovar) and seven sites of its tributaries in \n\nKathmandu valley (Bishnumati River-Teku dhovan, \n\nBalkhu River-Balkhu, Dhobi River-Baneshwor, Nakkhu \n\nRiver-Nakkhu, Tukuchu River-Tripureshwor, Mahohara \n\nRiver-Jadibuti, Hanumante River-Jadibuti). The sample \n\nwas collected in a sterile bottle of 500 mL capacity for \n\nmicrobiological analysis and a sample bottle of 500 mL \n\nfor physico-chemical analysis. The bottles were labeled \n\nwith time, place, and date of sample collection and \n\ntransported to the laboratory as soon as possible on the \n\nicebox. For Dissolved Oxygen (DO) and Biological \n\nOxygen Demand (BOD), two BOD bottles each of 300 mL \n\nwere taken at the site, processed at the lab. The study was \n\nconducted from November 2019 to January 2020 in \n\nKathmandu Upatyaka Khanepani Limited (KUKL) \n\nlaboratory, Mahankal Chaur, Kathmandu. \n\nSample Analysis \nAnalysis of Physico-chemical parameters \nThe standard method for examining water and \n\nwastewater [16] was followed to analyze the Physico-\n\nchemical parameters of water as Temperature (by \n\nmercury thermometer), pH (by digital pH meter), E.C. \n\n(by E.C. meter), Turbidity (by turbid-meter), Color and \n\nAmmonia (by colorimeter), Iron, Phosphate and Nitrate \n\n(by UV-visible spectrophotometer), Chloride, Alkalinity, \n\nand Hardness (by titration) and DO and BOD (by \n\nWinkler\u2019s method). \n\nAnalysis of microbiological parameters \nMicrobiological parameters analysis was carried out \n\nfollowing standard methods [16]. Enumeration of total \n\ncoliform count and fecal coliform were done by Most \n\nprobable number (MPN) method. \n\nIsolation of E.coli was done by enrichment in buffered \n\npeptone water and cultured in EMB (Eosin Methylene \n\nBlue)[17]. The colony was identified and confirmed by \n\nfollowing respective biochemical characteristics[18] and \n\nsubjected to antibiotic susceptibility using Kirby Bauer \n\ndisk diffusion method following CLSI guidelines[19]. \n\nThe antibiotics used were Ampicillin (10 \u00b5g), Gentamicin \n\n(10 \u00b5g), Cotrimoxazole (25 \u00b5g), Ciprofloxacin (5 \u00b5g), \n\nCeftriaxone (5 \u00b5g) and (Chloramphenicol (30 \u00b5g).  \n\nResults \nPhysical parameters assessment \nTemperature of sample ranges from 90C of Sundarijal \n\n(SL), Chobar (CHOB), and 16.50C of Hanumante (HAN) \n\nand Guheshwori Before treatment (GBT). Color was \n\nobserved highest in Tukucha (TUK) i.e., 40HU and lowest \n\nin Sundarijal (SL) i.e., 2.5HU. Only the water sample from \n\nSundarijal was clear while all other samples appeared \n\nturbid and hazy. SL sample was found to have lowest \n\nturbidity i.e., 2.7 NTU whereas Mahohara (MAN) sample  \n\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  9 \n\nhad the highest i.e., 759.32.  Only SL sample follows \n\nWHO and NDWQS guidelines (\u22645 NTU). Electrical \n\nconductivity of samples varied from SL 49.2 \u00b5S/cm being \n\nlowest and BAL 1109\u00b5S/cm being highest.", "start_char_idx": 7384, "end_char_idx": 10526, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0a42835c-63c3-4f5d-9343-77d37944582c": {"__data__": {"id_": "0a42835c-63c3-4f5d-9343-77d37944582c", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3e5d36a7-6212-46a0-8d15-235b239ef41c", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "5cfada16f54ab2870ee4b16155b206dfff361d278180a4bcafa8738bf773b0ae", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6aaf6af6-f020-45ce-8746-c89a15f59d49", "node_type": "1", "metadata": {}, "hash": "088ea7f9b7df29277c9b57dfff9e2ecc9f9163a2eae3235fb1da37547714fd5a", "class_name": "RelatedNodeInfo"}}, "text": "Out of 11 \n\nsamples HANU, TUK, BAL donot follow WHO \n\nguidelines with value (1039, 1090, 1109) \u00b5S/cm \n\nrespectively. Value of TDS was highest in TUK i.e. (371 \n\nmg/L) and lowest in SL (9 mg/L). All TDS value of \n\nsamples lies within the guideline of WHO (1000 mg/L). \n\nChemical parameter assessment \npH of the sample Manohara was found to be lowest i.e., \n\n7.22 and sample Dhobi(DHOBI) was highest i.e., 8.65 \n\nwhich doesn\u2019t follow WHO guidelines. Water in most of \n\nthe river samples were slightly alkaline. Dissolved \n\noxygen in the sample SL has highest i.e., 12.4 mg/L while \n\nBishnumati has lowest i.e.,0.77 mg/L. In the study \n\nperformed BOD of the sample Bishnumati has highest \n\ni.e., 354 mg/L and SL has lowest 8.9mg/L.  Ammonia of \n\nthe sample Sundarjal being lowest i.e., 0.2mg/L lies \n\nwithin the WHO guidelines while other sample don\u2019t \n\nfollow WHO guidelines. Sample Balkhu has highest \n\nvalue i.e., 90 mg/L. Highest value of   Total hardness was \n\nobtained from the sample Balkhu i.e., 344 mg/L and \n\nlowest value from Sundarijal i.e,.12 mg/L. All values \n\nwere within the NDWQS guidelines (\u2264500 mg/L). \n\nChloride value was highest in Tukucha i.e., 149.76mg/L \n\nand lowest in Sundarjal i.e., 1.92 mg/L. In the study \n\nperformed phosphate value was highest in Tukucha i.e., \n\n3.5 mg/L and lowest in Sundarijal i.e., 0.0 mg/L. All the \n\nvalues of iron don\u2019t follow the guidelines of WHO and \n\nNDWQS (\u22640.3). Iron value was highest in sample \n\nTable 1. Physical Parameters of water samples.", "start_char_idx": 10527, "end_char_idx": 12028, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6aaf6af6-f020-45ce-8746-c89a15f59d49": {"__data__": {"id_": "6aaf6af6-f020-45ce-8746-c89a15f59d49", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0a42835c-63c3-4f5d-9343-77d37944582c", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "4ff3d00e24efb5c145d4201bfc8da23e9e8e3164ec89ec9ac48cce46a4448eaf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5346f614-094e-4e0c-8609-5f8ca86511e9", "node_type": "1", "metadata": {}, "hash": "33a376458fda6ba09612ab5cb153afad45b3650a92d5a5744b2be5961afe007e", "class_name": "RelatedNodeInfo"}}, "text": "Iron value was highest in sample \n\nTable 1. Physical Parameters of water samples. (* Temperature during measurement) \nP\n\nh\ny\n\ns\nic\n\na\nl \n\nP\na\nra\n\nm\ne\nte\n\nr \n\nS\nu\n\nn\nd\n\na\nri\n\n j\na\nl \n\n(S\nL\n\n) \n\nG\nu\n\nh\ne\ns\nw\n\no\nri\n\n \n\nB\ne\nfo\n\nre\n T\n\nre\na\ntm\n\ne\nn\n\nt \n\n(G\nB\n\nT\n) \n\nG\nu\n\nh\ne\ns\nw\n\no\nri\n\n \n\nA\nft\n\ne\nr \n\nT\nre\n\na\ntm\n\ne\nn\n\nt \n\n(G\nA\n\nT\n) \n\nM\na\nn\n\no\nh\n\na\nra\n\n \n\nR\niv\n\ne\nr \n\n(M\nA\n\nN\n) \n\nH\na\nn\n\nu\nm\n\na\nn\n\nte\n \n\nR\niv\n\ne\nr \n\n(H\nA\n\nN\nU\n\n) \n\nT\nu\n\nk\nu\n\nc\nh\n\na\n \n\nR\niv\n\ne\nr \n\n(T\nU\n\nK\n) \n\nD\nh\n\no\nb\n\ni \n\nR\niv\n\ne\nr \n\n(D\nH\n\nO\nB\n\nI)\n \n\nN\na\nk\n\nk\nh\n\nu\n \n\nR\niv\n\ne\nr \n\n(N\nA\n\nK\n) \n\nB\na\nlk\n\nh\nu\n\n \n\nR\niv\n\ne\nr \n\n(B\nA\n\nL\n) \n\nB\nis\n\nh\nn\n\nu\n \n\nM\na\nti\n\n \n\n(B\nM\n\n) \n\nC\nh\n\no\nb\n\na\nr  \n\n(C\nH\n\nO\nB\n\n) \n\nA\np\n\np\ne\na\n\nra\nn\n\nc\ne\n\n \n\n \n\nC\nle\n\na\nr \n\nT\nu\n\nrb\nid\n\n \n\n \n\nT\nu\n\nrb\nid\n\n \n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\nT\nu\n\nrb\nid\n\n \n\n \n\nTemperature \n(\u00b0C) \n\n9\u00b0 19\u00b0 16.5\u00b0 12\u00b0 16.5\u00b0 16\u00b0 13\u00b0 11\u00b0 13\u00b0 15\u00b0 9\u00b0 \n\nColor \n(HU) \n\n2.5 25 30 35 25 40 20 35 25 15 25 \n\nTurbidity \n(NTU) \n\n2.7 52.59 295.12 759.32 122.66 179.78 186.71 261.33 112.03 305.80 15.57 \n\nE.C.* \n(\u00b5S/cm)  \n\n49.2 \n(9.1\u00b0) \n\n258.7 \n(13\u00b0) \n\n167.0 \n(18.6\u00b0) \n\n567 \n(13.0\u00b0) \n\n1039 \n(16\u00b0) \n\n1090 \n(14\u00b0) \n\n820 \n(13.9\u00b0) \n\n701 \n(14.5\u00b0) \n\n1109 \n(12.5\u00b0) \n\n986 \n(14\u00b0) \n\n983 \n(11.6\u00b0) \n\nTDS 9 89 57 143 367 371 300 162 354 349 268 \n\nTable 2.", "start_char_idx": 11947, "end_char_idx": 13255, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5346f614-094e-4e0c-8609-5f8ca86511e9": {"__data__": {"id_": "5346f614-094e-4e0c-8609-5f8ca86511e9", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6aaf6af6-f020-45ce-8746-c89a15f59d49", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "e9b2406ce5b7fb954329598b1028b8814d3597d4b32421209412017689f05908", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c7b46d55-d2c7-4186-b604-0a293861ce59", "node_type": "1", "metadata": {}, "hash": "d5910e03ed9bd1f4aecc6941b3b5e5d8d1538b6877f7bedd13feaaa9dfbc36d9", "class_name": "RelatedNodeInfo"}}, "text": "Chemical Parameters of water samples (* Temperature during measurement) \n\nP\nh\n\ny\ns\nio\n\n C\nh\n\ne\nm\n\nic\na\n\nl \n \n\nP\na\n\nra\nm\n\ne\nte\n\nr \n\nS\nu\n\nn\nd\n\na\nri\n\n \n\nja\nl \n\n(S\nL\n\n) \n\nG\nu\n\nh\ne\n\ns\nw\n\no\nri\n\n \n\nB\ne\n\nfo\nre\n\n T\nre\n\na\ntm\n\ne\nn\n\nt \n\n(G\nB\n\nT\n)  \n\nG\nu\n\nh\ne\n\ns\nw\n\no\nri\n\n \n\nA\nft\n\ne\nr \n\nT\nre\n\na\ntm\n\ne\nn\n\nt \n\n(G\nA\n\nT\n)  \n\nM\na\n\nn\no\n\nh\na\nra\n\n \n\nR\niv\n\ne\nr \n\n(M\nA\n\nN\n)  \n\nH\na\n\nn\nu\n\nm\na\n\nn\nte\n\n \n\nR\niv\n\ne\nr \n\n(H\nA\n\nN\nU\n\n) \n\nT\nu\n\nk\nu\n\nc\nh\n\na\n \n\nR\niv\n\ne\nr \n\n(T\nU\n\nK\n) \n\nD\nh\n\no\nb\n\ni \n\nR\niv\n\ne\nr \n\n(D\nH\n\nO\nB\n\nI)\n \n\nN\na\nk\n\nk\nh\n\nu\n \n\nR\niv\n\ne\nr \n\n(N\nA\n\nK\n) \n\nB\na\nlk\n\nh\nu\n\n \n\nR\niv\n\ne\nr \n\n(B\nA\n\nL\n) \n\nB\nis\n\nh\nn\n\nu\n \n\nM\na\nti\n\n \n\n(B\nM\n\n) \n\nC\nh\n\no\nb\n\na\nr  \n\n(C\nH\n\nO\nB\n\n) \n\npH* \n \n\n7.7 \n(9.1\u00b0) \n\n7.41 \n(13\u00b0) \n\n7.72 \n(18.6\u00b0) \n\n7.22 \n(13\u00b0) \n\n7.45 \n(16\u00b0) \n\n7.3 \n(14\u00b0) \n\n8.65 \n(13.9\u00b0) \n\n8.29 \n(14.6\u00b0) \n\n7.61 \n(13\u00b0) \n\n7.27 \n(14\u00b0) \n\n7.56 \n(11\u00b0) \n\nDO \n(mg/L) \n\n12.4 3.02 4.87 2.24 1.05 1.10 1.26 2.8 1.4 0.77 1.59 \n\nBOD \n(mg/L) \n\n8.9 66 48 160 279 261 242 187 291 354 213 \n\nAmmonia \n(mg/L) \n\n0.2 15 12 24 35 80 40 24 90 40 32.5 \n\nAlkalinity \n(mg/L) \n\n24 108 88 88 264 288 248 160 404 296 294 \n\nHardness \n(mg/L) \n\n12 128 56 80 264 152 312 148 344 176 178 \n\nChloride \n(mg/L) \n\n1.92 26.88 19.2 65.28 103.68 149.76 69.12 46.08 111.36 80.64 88.46 \n\nPhosphate \n(mg/L) \n\n0.0 0.4 0.2 1.0 2.0 3.5 2.5 0.5 2.0 2.5 2.5 \n\nIron \n(mg/L) \n\n0.4 1.6 6 23.2 8.8 4.4 6.4 3.2 6.4 15.2 4.8 \n\nNitrate \n(mg/L) \n\n0.2 0.9 2.1 3.7 5.7 5.7 4.8 1.0 4.6 5.3 2.1 \n\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.", "start_char_idx": 13256, "end_char_idx": 14711, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c7b46d55-d2c7-4186-b604-0a293861ce59": {"__data__": {"id_": "c7b46d55-d2c7-4186-b604-0a293861ce59", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5346f614-094e-4e0c-8609-5f8ca86511e9", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "f7994ea1fd73d744225f869ac35791cf61017a89155af1222aefa95f205380d9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "06596b90-b79d-4e32-992d-9fd66ff48a3e", "node_type": "1", "metadata": {}, "hash": "a62d5bafaa2b7dae7097a44deafaf117a7be1d7287184788f87463f3200b76d2", "class_name": "RelatedNodeInfo"}}, "text": "2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  10 \n\nManohara i.e., 23.2 and lowest in sample SL i.e., 0.4 \n\nmg/L. Hanumante and Tukucha sample have highest \n\nNitrate value i.e., 5.7 mg/L while Sundarijal sample has \n\nlowest i.e., 0.2 mg/L. \n\nTable 3. Total coliform count by MPN method \n\nSample \n\nNumber of tubes giving \npositive reactions \n\nMPN index \n(cfu/100mL) \n\n1 of \n50mL \neach \n\n5 of \n10mL \neach \n\n5 of \n1mL \neach \n\nSundarijal 1 5 5 >180 \nGuheshwori \nBefore \nTreatment 1 5 5 >180 \nGuheshwori \nBefore \nTreatment 1 5 5 >180 \nManohara 1 5 5 >180 \n\nHanumante 1 5 5 >180 \nTukucha 1 5 5 >180 \n\nDhobi 1 5 5 >180 \nNakkhu 1 5 5 >180 \n\nBalkhu 1 5 5 >180 \n\nBishnumati 1 5 5 >180 \n\nChobar 1 5 5 >180 \n\nMicrobiological analysis  \nAll 11 sample (100%) contain coliform above 180 per 100 \n\nmL.   \n\nAntibiotic Susceptibility \nTable 4. Susceptibility patterns of E. coli isolates. R= Resistant, \nI= Intermediate, S= Sensitive \n\n \n\nA\nm\n\np\nic\n\nil\nli\n\nn\n \n\n1\n0\n\n \u00b5\ng\n\n \n\nG\ne\nn\n\nta\nm\n\nic\nin\n\n \n\n1\n0\n\n \u00b5\ng\n\n \n\nC\no\n\ntr\nim\n\no\nx\n\na\nz\n\no\nle\n\n \n\n2\n5\n\n \u00b5\ng\n\n \n\nC\nip\n\nro\nfl\n\no\nx\n\na\nci\n\nn\n \n\n5\n \u00b5\n\ng\n \n\nC\ne\nft\n\nri\na\n\nx\no\n\nn\ne\n \n\n3\n0\n\n \u00b5\ng\n\n \n\nC\nh\n\nlo\nra\n\nm\np\n\nh\ne\nn\n\nic\no\n\nl \n\n3\n0\n\n \u00b5\ng\n\n \n\nSundarijal R R R S S S \n\nGBT S I S S S S \n\nGAT R I S S S S \n\nMAN R R S R I S \n\nHANU R R S I R S \n\nTUK R R R R I I \n\nDHOBI R R S S S S \n\nNAK S R S S S S \n\nBAL S R S S I S \n\nBM R R R R I S \n\nCHOB R R R I I R \n\n \n\nTable 4 showed that E.coli isolated from sites TUK, BM \n\nand CHOB were resistant against more than 50 percent of \n\nthe antibiotics testes while isolates from sites GBT, GAT, \n\nDHOBI, NAK and BAL were susceptible to more than 50 \n\npercent of the antibiotics tested. \n\n \nFigure 1. Green metallic sheen colony growth on M-Endo agar \n\n(E. coli).  \n\nFrom 11 isolates, 6 (54.4%) were found multi drug \n\nresistant (MDR) i.e., resistant to 3 or more class of \n\nantibiotics. Most of the MDR isolates (45.5%) were \n\nresistant to the Gentamicin in this study. \n\n \nFigure 2. Antibiotic Susceptibility by disk diffusion \n\nmethod \n\nDiscussion \nTurbidity in this study was recorded in range of 2.7NTU \n\nin Sundarijal to 759.32NTU in Manohara. The values of \n\nturbidity except Sundarijal were much higher compared \n\nTable 5.", "start_char_idx": 14674, "end_char_idx": 16866, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "06596b90-b79d-4e32-992d-9fd66ff48a3e": {"__data__": {"id_": "06596b90-b79d-4e32-992d-9fd66ff48a3e", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c7b46d55-d2c7-4186-b604-0a293861ce59", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "f6bb57efefa8ef4a34b52ea7b02c3524cb246527d6bc9141a47928baedca652c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b20dc123-6214-4931-98d9-8aff2a2c1050", "node_type": "1", "metadata": {}, "hash": "acfc45f3105c7c53f3542ec49cbbc9ea9e29c76187a6169c2634aeab0e982c18", "class_name": "RelatedNodeInfo"}}, "text": "Antibiotic Susceptibility of E. coli (N=11) \n\n Sensitive Intermediate Resistance \n\nAmpicillin \n(10 \u00b5g) \n\n27.27% \n(3) \n\n0 72.72% (8) \n\nGentamicin \n(10 \u00b5g) \n\n0 18.18% (2) 81.82% (9) \n\nCotrimoxazole \n(25 \u00b5g) \n\n63.6% (7) 0 36.4% (4) \n\nCiprofloxacin \n(5 \u00b5g) \n\n54.55% \n(6) \n\n18.2% (2) 27.27% (3) \n\nCeftriaxone \n(30 \u00b5g) \n\n45.5% (5) 45.5% (5) 9.1% (1) \n\nChloramphenicol \n(30 \u00b5g) \n\n81.9% (9) 9.1% (1) 9.1% (1) \n\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  11 \n\nto the previous study conducted i.e. 14.2NTU before \n\nmixing of tributaries of Kathmandu valley[20]. Discharge \n\nof industrial effluents such as alums and chemicals might \n\ncontribute to the high value of turbidity.  Tributaries of \n\nBagmati are major contributors to turbidness in Bagmati \n\nriver. \n\nThe pH value was found within the range of WHO \n\nstandards and NDWQS guidelines value except the \n\nsample of Dhobi river, which is found to be 8.65. It is \n\nhigher than the value reported in the previous study be \n\n8.20 [21]. High value of pH may be due to the increasing \n\nwaste discharge and industrial effluent along with \n\nmicrobial decomposition of organic matters. \n\nTemperature ranged from 90C to190C, while previous \n\nstudy conducted  [4] during summer season recorded a \n\nmaximum of 200 C. Since the study was conducted in \n\nwinter explains the low temperature recorded.  \n\nElectrical conductivity of the samples Hanumante, \n\nTukucha, and Balkhu were 1039 \u00b5S/cm, 1090\u00b5S/cm, and \n\n1109\u00b5S/cm respectively that do not lie within the \n\nguidelines of WHO standard 0-1000 \u00b5S/cm. In the \n\nprevious study [21], highest value was 889.59 \u00b5S/cm. \n\nHigher value indicates the presence of a higher amount \n\nof dissolved ions as well as plant nutrients in the water \n\nwhich might be due to the wash off of the fertilizer from \n\nagricultural lands. \n\nAlkalinity refers to the capability of water to neutralize \n\nacid. In natural water, there are many salts of weak acids \n\nsuch as silicates, borate-causing alkalinity. In this study, \n\nhighest alkalinity was of Balkhu 404mg/L. The alkalinity \n\nobserved in this study is higher than the result of \n\nprevious study 360 mg/L [20]. Samples were collected \n\nduring winter season, the flow and level of water were \n\nlow. Higher value of alkalinity might be due to increase \n\nconcentration of natural soil and minerals. \n\nThe ammonia concentration for Sundarijal was only 0.2 \n\nmg/L which was limit for surface water recommended \n\nby WHO [22]. But the concentration of ammonia for other \n\nsamples was a lot higher than the recommended value \n\nwhich might be due to more amount of municipal waste \n\ndumping in the river of Kathmandu valley. Highest \n\nammonia concentration was from Balkhu sample, 90 \n\nmg/L, and Tukucha sample, 80 mg/L. Similar results \n\nreported by [20], also had a higher value of ammonia in \n\nBalkhu river indicating that it was more polluted than \n\nother rivers.  \n\nThe highest value of chloride recorded was 149.76mg/L \n\nin Tukucha followed by Balkhu 111.36mg/L, indicating \n\nmost contaminated about chloride content.", "start_char_idx": 16867, "end_char_idx": 19926, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b20dc123-6214-4931-98d9-8aff2a2c1050": {"__data__": {"id_": "b20dc123-6214-4931-98d9-8aff2a2c1050", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "06596b90-b79d-4e32-992d-9fd66ff48a3e", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "669347922aa24fdbf0c56acceb71536f4018b87baaf7e9dc3ac161abeada2f88", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bf52f673-830e-4884-b324-2d4deac16afb", "node_type": "1", "metadata": {}, "hash": "2d6fe70dd821f263103e7d26d072c7a099f72045fb4fd093cdaf9e107eab27c4", "class_name": "RelatedNodeInfo"}}, "text": "Previous \n\nstudy [23] , presented a similar result being Teku and \n\nSundarighat as most contaminated sites[23] considering \n\nTukucha river and Balkhu river joins on Bagmati in Teku \n\nand Sundarighat respectively. High chloride \n\nconcentration in river is toxic to aquatic life and also \n\nincreases the potential corrosivity of water[24]. \n\nNitrate was recorded highest in Tukucha and \n\nHanumante i.e.5.7mg/L which is higher than the data \n\npreviously recorded [23], 3.95mg/L. Both studies \n\nshowed similarity in the increment of nitrate content \n\nfrom upstream to downstream[23]. Excess levels of \n\nnitrates can be considered to be a contaminant of river \n\nwaters. Most sources of excess nitrates come from human \n\nactivity. The source of excess nitrates can usually be \n\ntraced to agricultural activities, human wastes, or \n\nindustrial pollution. Rainwater can wash nitrates in the \n\nfertilizer into streams and rivers[25]. \n\nHighest phosphate value was 3.5 mg/L in Tukucha. \n\nSundarijal sample phosphate value was 0.0 mg/L and \n\nthat of Chobar 2.5 mg/L which are low compared to that \n\nof the data of similar research [23], 0.24 mg/L at \n\nSundarijal and 12.3 mg/L at Chobar [26]. Significant \n\nincrease was seen in the level of phosphate as the river \n\nenters an urban core area, which is from manmade \n\nsources such as septic systems, fertilizer runoff, and \n\nimproperly treated wastewater. \n\nAt Sundarijal, BOD level was found to be within the \n\nguideline by BBWMSIP. However, the level of BOD \n\nincreases with the increase in the organic waste in the \n\nriver. BOD indicated the pollution of organic waste \n\nresulting low level of dissolved oxygen. Highest BOD \n\nwas observed in Bishnumati. Previous study [26]  \n\nshowed a low level of BOD in Sundarijal but an \n\nincrement in the level of BOD towards downstream.    \n\nDissolved oxygen concentrations in the core urban areas \n\nwere significantly lower than that of Sundarijal showing \n\nthat the water is anoxic. As the river flows downstream, \n\nthe dissolved oxygen gets more reduced. Lowest \n\nconcentration was observed in Bishnumati of 0.77 mg/L. \n\nBacterial decomposition of incorporated organic matter \n\nwas most likely for low level of dissolved oxygen similar \n\nresearch [23,26] also observed low level of DO  in Bagmati \n\nriver.  \n\nIn this study, coliform was present in the entire river \n\nwater sample. The presence of a high amount of coliform \n\nmight be due to the fact; samples were taken from the \n\nriver before it was subjected to a treatment of \n\ndisinfection.  Previous studies on the river water from \n\nKathmandu valley showed the presence of various \n\nviruses [27], Human Enteric Viruses, Protozoa, and \n\nIndicators of Pathogens[28] except in the sample of \n\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  12 \n\nSundarijal. The water is contaminated by various \n\nanthropogenic activities as it flows downstream. \n\nAll 11 samples were positive for E. coli.  Identification of \n\nE. coli during the study indicates the fecal contamination \n\nof human origin in Bagmati River and its tributaries. \n\nAlthough there was a low detection rate of pathogen in \n\nSundarijal during a previous study[28] but the detection \n\nof E. coli from the Sundarijal suggest the contamination \n\nof water even upstream of the Bagmati river, which \n\ncannot be neglected.  \n\nMultiple drug-resistant (MDR) was 54.4% in this study. \n\nOther researchers have reported an increasing pattern of \n\nE. coli isolates against common antibiotics in Nepal[29], \n\n[30].", "start_char_idx": 19927, "end_char_idx": 23450, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bf52f673-830e-4884-b324-2d4deac16afb": {"__data__": {"id_": "bf52f673-830e-4884-b324-2d4deac16afb", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b20dc123-6214-4931-98d9-8aff2a2c1050", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "5a9b9e4aa634708493ee872dee967bd2c274fb43d2082e2364ee0e0ad055a6e2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ed59c02f-2c10-46cb-8741-c0c984a0d4bc", "node_type": "1", "metadata": {}, "hash": "c7e9cabbae07badd0854c76d59e15744ad87fa7e60a0d6f7b8eed57ce5f3f3f4", "class_name": "RelatedNodeInfo"}}, "text": "It is observed from the study that Gentamicin and \n\nAmpicillin were most resisted by 81.82% and 72.72% \n\nrespectively by the E. coli isolates, similar results of high \n\nresistance against \u03b2-lactamase(Ampicillin)[31] and \n\nAminoglycoside (Gentamicin) [30] were reported. \n\nAmong the antibiotics, Chloramphenicol was more \n\nsensitive to the isolates. A high rate of effectiveness by the \n\nChloramphenicol against MDR isolates of E. coli was \n\nreported in previous studies[31, 32]. \n\nConclusion  \nThe study showed the variation in water quality of the \n\nBagmati River and its tributaries. The quality of water is \n\nworse in the urban core areas compared to that of \n\nupstream. The presence of fecal coliform in river water is \n\nthe prime indication of a possible source of an outbreak \n\nfor waterborne diseases and water is not suitable for \n\ndrinking purposes without proper treatment. High \n\nresistance towards some antibiotics shows the threat to \n\nthe exposure of antibiotics resistance bacterial strains by \n\nthe population of Kathmandu valley. Immediate action is \n\nneeded to prevent further deterioration of the river and \n\namplify efforts to slow the emergence and spread of \n\nresistance. \n\nAuthor\u2019s Contribution  \nThe development of concept, preliminary work and \n\nlaboratory analysis, was done by SP, AP, BPS, KS and YB \n\nunder the guidance of MSSH and SA. All authors read \n\nand approved the final manuscript. \n\nCompeting Interests  \nThe authors declare that they have no conflicts of interest.  \n\nFunding  \nThis study was not funded by any agency or institution.  \n\nAcknowledgments  \nAll authors are grateful to the faculty and laboratory staff \n\nof Microbiology department of Tri-Chandra Multiple \n\nCampus for their continuous support in this research \n\nwork, and special mention to the Kathmandu Upatyaka \n\nKhanepani Limited(KUKL) and all the staffs for \n\nproviding laboratory and support.  \n\nEthical Approval and Consent  \nThis study was carried out with the approval from the \n\nconcerned authorities. \n\nData Availability   \nThe data can be made available upon request. \n\nReference \n1. [CIA.gov.  World - The World Factbook. [Internet] Available from: \n\nhttps://www.cia.gov/the-world-factbook/countries/world/ \n(accessed Jun. 03, 2020). \n\n2.  Government of Nepal Water resources of nepal- in the context of \nclimate change 2011 by Water and Energy commission \nsecretariate, Singh Durbar, Kathmandu, Nepal Available from: \nhttps://www.slideshare.net/BhimUpadhyaya/water-resources-\nof-nepal-in-the-context-of-climate-change-2011-by-wecs (accessed \nNov. 05, 2020). \n\n3. Tamrakar A, Parajuli RR. Conservation of Cultural Heritage: \nIssues along the Thapathali-Teku stretch of the Bagmati River in \nKathmandu, Nepal. Heritage. 2019 Sep;2(3):2228-42. \nhttps://doi.org/10.3390/heritage2030135 \n\n4. Paudyal R, Kang S, Sharma CM, Tripathee L, Sillanp\u00e4\u00e4 M. \nVariations of the physicochemical parameters and metal levels and \ntheir risk assessment in urbanized Bagmati River, Kathmandu, \nNepal. Journal of Chemistry. 2016 Jan \n1;2016.https://doi.org/10.1155/2016/6025905 \n\n5. Thakur JK, Neupane M, Mohanan AA. Water poverty in upper \nBagmati River basin in Nepal. Water Science. 2017 Apr 1;31(1):93-\n108.https://doi.org/10.1016/j.wsj.2016.12.001 \n\n6. United Nations Human Settlements Programme.  Global Atlas of \nExcreta, Wastewater Sludge, and Biosolids Management 2008  \nUN-Habitat.", "start_char_idx": 23451, "end_char_idx": 26836, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ed59c02f-2c10-46cb-8741-c0c984a0d4bc": {"__data__": {"id_": "ed59c02f-2c10-46cb-8741-c0c984a0d4bc", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bf52f673-830e-4884-b324-2d4deac16afb", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "729586a21e1198c6bc8f6f39b5e486c256ad32ecf8b018106c7a0e5130a414ab", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "300d4a4b-1c1d-432a-97b0-6ed595f5ebda", "node_type": "1", "metadata": {}, "hash": "11ed6b76b37e2532439d0e398cbf1d7e0332fdce4be1006a6182d46208bbd66d", "class_name": "RelatedNodeInfo"}}, "text": "[Internet] Available from: \nhttps://unhabitat.org/global-atlas-of-excreta- wastewater-\nsludge-and-biosolids-management (accessed Oct. 09, 2020). \n\n7. Davis JA. Water quality standards for the Bagmati River. Journal \n(Water Pollution Control Federation). 1977 Feb 1:227-34. \n\n8. Baniya B, Khadka N, Ghimire SK, Baniya H, Sharma S, Dhital YP, \nBhatta R, Bhattarai B. Water quality assessment along the \nsegments of Bagmati River in Kathmandu valley, Nepal. Nepal \nJournal of Environmental Science. 2019 Dec 31;7:1-\n0.https://doi.org/10.3126/njes.v7i0.34314 \n\n9. Regmi S. Wastewater Treatment in Kathmandu: Management, \nTreatment and Alternative. 2013 [thesis] [internet]. Available from: \nhttps://www.theseus.fi/handle/10024/59525 \n\n10. Nepali Time. Cleaning up the Bagmati 23 apr 2018 #754 [Internet] \nAvailable from: https://archive.nepalitimes.com/article/from-\nnepali-press/Cleaning-up-the-Bagmati,2185 (accessed Jun. 04, \n2020). \n\n11. Shah PK, Chaturwedi SB. Physicochemical and Bacteriological \nWater Analysis of Bagmati and Bishnumati River. Tribhuvan \nUniversity Journal. 2019 Jun 30;33(1):23-30. \nhttps://doi.org/10.3126/tuj.v33i1.28678 \n\n12. [Kim KS. Current concepts on the pathogenesis of Escherichia coli \nmeningitis: implications for therapy and prevention. Current \nopinion in infectious diseases. 2012 Jun 1;25(3):273-\n8.https://doi.org/10.1097/QCO.0b013e3283521eb0 \n\n13. Kaper JB, Nataro JP, Mobley HL. Pathogenic escherichia coli. \nNature reviews microbiology. 2004 Feb;2(2):123-\n40.https://doi.org/10.1038/nrmicro818 \n\n14.  Lee SH, Kang HJ, Park HD. Influence of influent wastewater \ncommunities on temporal variation of activated sludge \ncommunities. Water research. 2015 Apr 15;73:132-44.. \nhttps://doi.org/10.1016/j.watres.2015.01.014 \n\n\n\nNepal J Biotechnol. 2021Dec;9 (2): 7-13     Poudel et al.  \n\n\u00a9NJB, BSN  13 \n\n15. Fair RJ, Tor Y. Antibiotics and bacterial resistance in the 21st \ncentury. Perspectives in medicinal chemistry. 2014 Jan;6:PMC-\nS14459. https://doi.org/10.4137/PMC.S14459 \n\n16.  American Public Health Association, American Water Works \nAssociation, Water Pollution Control Federation, Water \nEnvironment Federation. Standard methods for the examination \nof water and wastewater. American Public Health Association.; \n1912. \n\n17.  Singh P, Prakash A. Isolation of Escherichia coli, Staphylococcus \naureus and Listeria monocytogenes from milk products sold \nunder market conditions at Agra region. Acta agriculturae \nSlovenica. 2008 Nov;92(1):83-8. \n\n18. Cheesbrough M. District laboratory practice in tropical countries, \nsecond edition, Dist. Lab. Pract. Trop. Countries, Second Ed., pp. 1\u2013\n434, Jan. 2006. https://doi.org/10.1017 /CBO9780511543470 \n\n19. CLSI C. Performance standards for antimicrobial susceptibility \ntesting. Clinical Lab Standards Institute.", "start_char_idx": 26836, "end_char_idx": 29637, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "300d4a4b-1c1d-432a-97b0-6ed595f5ebda": {"__data__": {"id_": "300d4a4b-1c1d-432a-97b0-6ed595f5ebda", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ed59c02f-2c10-46cb-8741-c0c984a0d4bc", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "94a47ba604341173d98a63047d5966bba0d0ccfcafca31cde7f43a4f5787e1a6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6dbbeccc-d6d0-487a-9073-3ac931d972e2", "node_type": "1", "metadata": {}, "hash": "c448f14eac93aec7d2b40a08d35bf576ea5ef75deb09d048df41c2482ebed9b4", "class_name": "RelatedNodeInfo"}}, "text": "CLSI C. Performance standards for antimicrobial susceptibility \ntesting. Clinical Lab Standards Institute. 2016;35(3):16-38. \n\n20.  Adhikari MP, Neupane MR, Kafle M. Physico-chemical \nparameterization and determination of effect of tributaries on \nenhancement of pollutants in bagmati river. Journal of Nepal \nChemical Society. 2019 Dec 31;40:36-\n43.https://doi.org/10.3126/jncs.v40i0.27276 \n\n21. Kannel PR, Lee S, Lee YS, Kanel SR, Pelletier GJ. Application of \nautomated QUAL2Kw for water quality modeling and \nmanagement in the Bagmati River, Nepal. Ecological modelling. \n2007 Apr 10;202(3-4):503-17.https://doi.org/10.1016 \n/j.ecolmodel.2006.12.033 \n\n22. Edition F. Guidelines for drinking-water quality. WHO chronicle. \n2011;38(4):104-8. \n\n23. Gautam R, Shrestha JK, Shrestha GK. Assessment of river water \nintrusion at the periphery of Bagmati River in Kathmandu Valley. \nNepal Journal of Science and Technology. 2013 Oct 14;14(1):137-\n46.https://doi.org/10.3126/njst.v14i1.8934 \n\n24. Water Resources. Chloride, Salinity, and Dissolved Solids | U.S. \nGeological Survey. U.S. Geological Survey [Internet] Mar 1 2019 \nAvailable from:  https://www.usgs.gov/mission-areas/water-\nresources/science/ chloride-salinity-and-dissolved-solids \n(accessed Dec. 11, 2020). \n\n25. US EPA. The Sources and Solutions: Agriculture [internet] \navailable from:  https://www.epa.gov/nutrientpollution \n/sources-and-solutions-agriculture (accessed Dec. 11, 2020). \n\n26. Shrestha N, Lamsal A, Regmi RK, Mishra BK. Current status of \nwater environment in Kathmandu Valley, Nepal. \n\n27. Haramoto E, Yamada K, Nishida K. Prevalence of protozoa, \nviruses, coliphages and indicator bacteria in groundwater and \nriver water in the Kathmandu Valley, Nepal. Transactions of the \nRoyal Society of Tropical Medicine and Hygiene. 2011 Dec \n1;105(12):711-6.https://doi.org/10.1016/j.trstmh.2011.08.004 \n\n28. Tandukar S, Sherchand JB, Bhandari D, Sherchan SP, Malla B, \nGhaju Shrestha R, Haramoto E. Presence of human enteric viruses, \nprotozoa, and indicators of pathogens in the Bagmati River, Nepal. \nPathogens. 2018 Jun;7(2):38. https://doi.org/10.3390 \n/pathogens7020038 \n\n29. Singh SD, Madhup SK. Clinical profile and antibiotics sensitivity \nin childhood urinary tract infection at Dhulikhel Hospital. \nKathmandu University Medical Journal. 2013;11(4):319-\n24.https://doi.org/10.3126/kumj.v11i4.12541 \n\n30. Parajuli NP, Maharjan P, Parajuli H, Joshi G, Paudel D, Sayami S, \nKhanal PR. High rates of multidrug resistance among \nuropathogenic Escherichia coli in children and analyses of ESBL \nproducers from Nepal. Antimicrobial Resistance & Infection \nControl. 2017 Dec;6(1):1-7.https://doi.org/10.1186/s13756-016-\n0168-6 \n\n31.", "start_char_idx": 29531, "end_char_idx": 32236, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6dbbeccc-d6d0-487a-9073-3ac931d972e2": {"__data__": {"id_": "6dbbeccc-d6d0-487a-9073-3ac931d972e2", "embedding": null, "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-16", "node_type": "4", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "43bb805f4295721daa73ed50b9b95ae3e5f4f33f6fec9aa2661af7d80670acf7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "300d4a4b-1c1d-432a-97b0-6ed595f5ebda", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "bff51b061c1bd53d3fea680cd7520fe128fd7fdf6019b4581fc861a47194719d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1d741777-fe31-472b-a070-c203933bc782", "node_type": "1", "metadata": {}, "hash": "a0951b467b416428aafcb42eb459ae132f8756f7cb5781ebb497f96c648cf269", "class_name": "RelatedNodeInfo"}}, "text": "Ansari S, Nepal HP, Gautam R, Shrestha S, Neopane P, Gurung G, \nChapagain ML. Community acquired multi-drug resistant clinical \nisolates of Escherichia coli in a tertiary care center of Nepal. \nAntimicrobial resistance and infection control. 2015 Dec;4(1):1-\n8.https://doi.org/10.1186/s13756-015-0059-2 \n\n32. AST AS. Physicochemical and bacteriological analysis of Bagmati \nriver in Kathmandu valley. Annals of Applied Bio-Sciences. \n2018;5(3). https://doi.org/10.21276/AABS.2213", "start_char_idx": 32237, "end_char_idx": 32716, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1d741777-fe31-472b-a070-c203933bc782": {"__data__": {"id_": "1d741777-fe31-472b-a070-c203933bc782", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6dbbeccc-d6d0-487a-9073-3ac931d972e2", "node_type": "1", "metadata": {"identifier": "njb-16", "author": "Poudel, Santosh; Paudyal, Akash; Sharma, Bishnu Prasad; Sharma, Kamana; Baral, Yubaraj; Adhikari, Shailaja; Shakya Hada, Manju Shree ", "title": "Microbial and Physico-Chemical Quality Assessment of Rivers of Kathmandu Valley", "date": "2021-12-30", "file": "njb-16.pdf"}, "hash": "ef8442a6550acd39da1d4e2de5403d8c3877133d3ba47a761bd79c2f171b450c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "304f3e3f-aaa2-4a5e-885e-72d4220cad2a", "node_type": "1", "metadata": {}, "hash": "dcec9a8f67a0a8a930ce8072072b80536bd54299c34363ecbcee435dee42af76", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2020 Dec;  8 (3): 111-115 DOI: https://doi.org/10.3126/njb.v8i3.33665 Research article \n\n\u00a9NJB, BSN 111 \n\nSelection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf \nRust and Crown Rust Diseases Using Molecular Markers \nResham B. Amgai1 , Shreejan Pokharel1, Sumitra Pantha2, Atit Parajuli2, Sudeep Subedi2, Shambhu P. Dhital1 \n1Biotechnology Division, Nepal Agricultural Research Council, PO Box No. 121, Lalitpur, Nepal \n2Agri Botany Division, Nepal Agricultural Research Council, Khumaltar, Lalitpur, Nepal \n\nArticle history:- Received: 7 Nov 2020; Revised: 21 Dec 2020; Accepted: 22 Dec 2020; Published online: 30 Dec 2020 \n\nAbstract \nBarley diseases are the major yield limiting factors for barley cultivation in Nepal. Stripe/Yellow rust (P. striformis f.sp. hordei \nand P. striformis f.sp. tritici), leaf rust (Puccinia hordei), and crown rust (P. coronata) are the major rust diseases in Nepal. \nPyramiding resistance genes against all these rust diseases are possible through molecular marker assisted breeding. Sweden \noriginated barley variety \u2018Bonus\u2019 is found resistant to stripe rust and having linked microsatellite markers for stripe rust and \ncrown rust resistance. Similarly, Nepalese hull-less barley variety \u2018Solu Uwa\u2019 and Nepalese awn-less barley landrace NPGR \nAcc# 2478 have linked microsatellite markers for leaf rust resistance. Therefore, one polymorphic sequence tagged sites (STS) \nmarker (ABG054) for stripe rust resistance, two polymorphic simple sequence repeats (SSR) markers (Bmac0144h and \nHVM049) for leaf rust and one polymorphic SSR marker (Bmag0006) for crown rust resistance were used to select the \nadvanced barley lines (at F8 stage) from above parents. Field screening of stripe rust resistance was also conducted. Among \n51 advanced and field disease resistance lines from Bonus/Solu Uwa cross, 10 pyramided lines for all three types of barley \nrust resistance were selected. Similarly, among 39 advanced and field disease resistance lines from Bonus/NPGR Acc#2478 \ncross, three pyramided lines were selected and advanced for further yield testing for general cultivation purpose. The chances \nof losing the desired gene are higher in late generation selection using molecular marker assisted selection (MAS), but the \nchances of getting agronomically superior varietal output is expected to increase.  \n\nKeywords: Rust, Pyramiding, Advanced lines, Barley, Marker Assisted Selection (MAS) \n\n Corresponding author, email: reshamamgain@yahoo.com \n\nIntroduction \nBarley diseases are the major yield limiting factors in \n\nNepal. Among many barley diseases, rust diseases are \n\ncritical from the crop production view. Strip/yellow rust \n\n(caused by Puccinia  striformis f.sp. hordei and P. striformis \n\nf.sp. tritici) is prevalent rust in the Nepalese barley field.\n\nPrasad et al. [1] also observed it as a major disease \n\ncausing a problem in the Nepalese barley field. However, \n\nleaf rust (caused by P. hordei) can be observed in some \n\nwarm barley cultivating areas. Crown rust of barley \n\n(caused by P. coronata) can be observed very sporadically \n\nonly. Any barley variety having resistance gene for all \n\nthree types of rust pathogen is highly sought in Nepalese \n\nbarley breeding program. \n\nSince, Nepalese barley germplasm has a high grain \n\nyielding capacity for hill and mountain regions of the \n\ncountry [2], adding rust resistance characteristics to them \n\nmay improve their yield and stability. Selection, \n\nidentification and incorporation of rust resistance genes \n\nis the only option for the development of rust resistance \n\nbarley varieties for Nepal.", "start_char_idx": 48, "end_char_idx": 3681, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "304f3e3f-aaa2-4a5e-885e-72d4220cad2a": {"__data__": {"id_": "304f3e3f-aaa2-4a5e-885e-72d4220cad2a", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1d741777-fe31-472b-a070-c203933bc782", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "14ad74cd0c3115529f2518545f077dcc150f75e62d4f9b50fcf768999ac90fc5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9ed246b9-79ef-40fb-bef8-cecdf5534dac", "node_type": "1", "metadata": {}, "hash": "a3bda6b0db83bea60bcaf7e19baf7ceacd55821884a37c13f1ad801b88874b30", "class_name": "RelatedNodeInfo"}}, "text": "Therefore, pyramiding major \n\nrust resistance genes for barley will be beneficial to \n\nfarmers. Molecular markers are highly preferred for gene \n\npyramiding program like this. \n\nThe sporadic nature of the crown rust occurrence in the \n\nNepalese barley field and overlapping of leaf rust and \n\nstripe rust in the disease screening field further pushed \n\nmolecular marker assisted selection (MAS) as the most \n\nviable option for gene pyramiding for rust resistance \n\nvarietal development. \n\nMaterials and Methods \nParent and Advanced lines selection \nA Swedish introduced variety \u2018Bonus\u2019 is the two-rowed \n\nstripe rust resistance barley variety for Nepal [2] and also \n\nhave linked microsatellite markers for stripe rust and \n\ncrown rust resistance. The polymorphic linked \n\nmicrosatellites are described in \u2018identification of \n\npolymorphism in parents\u2019 sub-heading. Similarly, \n\nNepalese hull-less barley variety \u2018Solu Uwa\u2019 and \n\nNepalese awn-less barley landrace \u2018NPGR Acc# 2478\u2019 has \n\nlinked microsatellite markers for leaf rust resistance. \n\nTherefore, crosses between Bonus with Solu Uwa and \n\nAcc #2478 will have a lot of chances of having pyramided \n\nlines. \n\nUse of marker assisted selection at early stage of barley \n\nbreeding such as in F2 and F3 is practically not feasible in \n\nour context due to cost, time and manpower shortage.  \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nmailto:reshamamgain@yahoo.com\nhttps://orcid.org/0000-0002-3651-4359\nmailto:reshamamgain@yahoo.com\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 111-115 Amgai et al. \n\n\u00a9NJB, BSN 112 \n\nSo, we have selected 51 advanced barley lines at F8 stage \n\nfrom \u2018Bonus\u2019 and \u2018Solu Uwa\u2019 crosses. Similarly, we have \n\nalso selected 39 advanced barley lines at F8 stage from \n\n\u2018Bonus\u2019 and \u2018Acc#2478\u2019 crosses to detect the pyramided \n\nlines (Table 1). All the selected lines showed the field \n\ndisease resistance. Similarly, they are forwarded to F8 \n\nbased on their superior agronomic characteristics \n\ncomparing to their parents. \n\nField Rust Evaluation \nTwo rows per line were sown at Khumatar, Lalitpur \n\nduring normal barley growing season (November to \n\nApril) in 2017. Spacing between each row was 20 cm and \n\nthe length of the row was 1.5m. Resistance and \n\nsusceptible parental lines were sown repeatedly after \n\nevery 15 advanced lines. A susceptible landrace \u2018Local \n\nJau\u2019 was used in two spreader rows around the disease \n\nscreening plots. Modified Cobb scale [3] was used for rust \n\nscoring at the heading stage for all three types of rust. \n\nIdentification of polymorphism among \nparents \nA series of microsatellite markers linked with rust \n\nresistance genes were screened to identify the \n\npolymorphic markers among the parents. Sequence \n\nTagged Sites (STS) marker ABG54, and Simple Sequence \n\nRepeats (SSR) markers Bmac144h, HVM49 and Bmag6 \n\nwere found polymorphic among the parents (Table 2). \n\nThese markers were used for the selection of advanced \n\nbarley lines. \n\nDNA extraction and PCR reaction \nModified CTAB method as described by Sul and Korban \n\n[4] was used to extract the genomic DNA of selected \n\nbarley advanced lines. PCR reaction mixture of 15 \u00b5l  \n\nTable 1. List of barley advanced breeding lines used for molecular marker assisted selection.", "start_char_idx": 3682, "end_char_idx": 7052, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9ed246b9-79ef-40fb-bef8-cecdf5534dac": {"__data__": {"id_": "9ed246b9-79ef-40fb-bef8-cecdf5534dac", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "304f3e3f-aaa2-4a5e-885e-72d4220cad2a", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "7a49f2fad1a9c7da41aa1036f3422892a9942e24613e69a8dc4db34653bf49cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "deac1395-e94f-4f54-a81c-c66cf0ae1ae5", "node_type": "1", "metadata": {}, "hash": "e69d02518ddab9f1ac7a0047df6702eed7aca18de11c3d5b7b34ba514718bdb6", "class_name": "RelatedNodeInfo"}}, "text": "List of barley advanced breeding lines used for molecular marker assisted selection. \n\nBonus/Solu \nUwa-3 \n\nBonus/Solu \nUwa-51 \n\nBonus/Solu Uwa-\n99 \n\nBonus/Solu Uwa-\n153 \n\nBonus/Acc#2478-\n201 \n\nBonus/Acc#2478-\n231 \n\nBonus/Solu \nUwa-6 \n\nBonus/Solu \nUwa-54 \n\nBonus/Solu Uwa-\n102 \n\nBonus/Solu Uwa-\n156 \n\nBonus/Acc#2478-\n202 \n\nBonus/Acc#2478-\n235 \n\nBonus/Solu \nUwa-9 \n\nBonus/Solu \nUwa-57 \n\nBonus/Solu Uwa-\n105 \n\nBonus/Solu Uwa-\n159 \n\nBonus/Acc#2478-\n204 \n\nBonus/Acc#2478-\n238 \n\nBonus/Solu \nUwa-12 \n\nBonus/Solu \nUwa-60 \n\nBonus/Solu Uwa-\n108 \n\nBonus/Acc#2478-\n162 \n\nBonus/Acc#2478-\n205 \n\nBonus/Acc#2478-\n244 \n\nBonus/Solu \nUwa-15 \n\nBonus/Solu \nUwa-63 \n\nBonus/Solu Uwa-\n111 \n\nBonus/Acc#2478-\n165 \n\nBonus/Acc#2478-\n206 \n\nBonus/Acc#2478-\n246 \n\nBonus/Solu \nUwa-18 \n\nBonus/Solu \nUwa-66 \n\nBonus/Solu Uwa-\n114 \n\nBonus/Acc#2478-\n168 \n\nBonus/Acc#2478-\n209 \n\nBonus/Acc#2478-\n248 \n\nBonus/Solu \nUwa-21 \n\nBonus/Solu \nUwa-69 \n\nBonus/Solu Uwa-\n117 \n\nBonus/Acc#2478-\n171 \n\nBonus/Acc#2478-\n210 \n\nBonus/Acc#2478-\n254 \n\nBonus/Solu \nUwa-24 \n\nBonus/Solu \nUwa-72 \n\nBonus/Solu Uwa-\n126 \n\nBonus/Acc#2478-\n174 \n\nBonus/Acc#2478-\n213 \n\nBonus/Acc#2478-\n257 \n\nBonus/Solu \nUwa-27 \n\nBonus/Solu \nUwa-75 \n\nBonus/Solu Uwa-\n129 \n\nBonus/Acc#2478-\n177 \n\nBonus/Acc#2478-\n216 \n\nBonus/Acc#2478-\n259 \n\nBonus/Solu \nUwa-30 \n\nBonus/Solu \nUwa-78 \n\nBonus/Solu Uwa-\n132 \n\nBonus/Acc#2478-\n180 \n\nBonus/Acc#2478-\n218 \n\nBonus/Acc#2478-\n268 \n\nBonus/Solu \nUwa-33 \n\nBonus/Solu \nUwa-81 \n\nBonus/Solu Uwa-\n135 \n\nBonus/Acc#2478-\n183 \n\nBonus/Acc#2478-\n222 \n\nBonus/Acc#2478-\n278 \n\nBonus/Solu \nUwa-36 \n\nBonus/Solu \nUwa-84 \n\nBonus/Solu Uwa-\n138 \n\nBonus/Acc#2478-\n186 \n\nBonus/Acc#2478-\n225 \n\nBonus/Solu \nUwa-39 \n\nBonus/Solu \nUwa-87 \n\nBonus/Solu Uwa-\n141 \n\nBonus/Acc#2478-\n189 \n\nBonus/Acc#2478-\n227 \n\nBonus/Solu \nUwa-42 \n\nBonus/Solu \nUwa-90 \n\nBonus/Solu Uwa-\n144 \n\nBonus/Acc#2478-\n192 \n\nBonus/Acc#2478-\n228 \n\nBonus/Solu \nUwa-45 \n\nBonus/Solu \nUwa-93 \n\nBonus/Solu Uwa-\n147 \n\nBonus/Acc# 2478-\n195 \n\nBonus/Acc#2478-\n229 \n\nBonus/Solu \nUwa-48 \n\nBonus/Solu \nUwa-96 \n\nBonus/Solu Uwa-\n150 \n\nBonus/Acc#2478-\n198 \n\nBonus/Acc#2478-\n230 \n\nTable 2.", "start_char_idx": 6968, "end_char_idx": 9030, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "deac1395-e94f-4f54-a81c-c66cf0ae1ae5": {"__data__": {"id_": "deac1395-e94f-4f54-a81c-c66cf0ae1ae5", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9ed246b9-79ef-40fb-bef8-cecdf5534dac", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "6fe08c2ba938aff6f2c1ecc401cedfd6f1822e7a21a62db47d29561a1fe42483", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "426e8152-8f7d-40cf-9e55-ac862d813a44", "node_type": "1", "metadata": {}, "hash": "78a20ed02460b2a15de65a2eb248126c454b7735c44181d546738cb6c46afb63", "class_name": "RelatedNodeInfo"}}, "text": "Polymorphism observed in parental lines for different molecular markers and disease characteristics \n\nParent \nField Stripe \n\nRust \nABG054 (Stripe \n\nRust QTL) \nBmac0144h (Leaf Rust-R \n\ngene) \nHVM049 (Leaf Rust-\n\nRph19) \nBmag0006 (Crown \n\nRust-Rpc1) \n\nBonus 0 1 0 0 1 \n\nSolu \nUwa \n\n10S 0 1 1 0 \n\nAcc#2478 60S 0 0 1 1 \n\nNote: Number in bracket is the linked resistance gene. Acc# = NPGR Accession Number", "start_char_idx": 9031, "end_char_idx": 9431, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "426e8152-8f7d-40cf-9e55-ac862d813a44": {"__data__": {"id_": "426e8152-8f7d-40cf-9e55-ac862d813a44", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "deac1395-e94f-4f54-a81c-c66cf0ae1ae5", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "6e586e57e02ed5c22ebfa10dc1b86385275da20ad1379d41af05c8595c1e5bed", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1949d3df-40c6-44fd-b249-0265dad7f55f", "node_type": "1", "metadata": {}, "hash": "beb7ced8b5fa0973333ae36e3b19fe3b3868233a711bb91da191c9b4ed6e6ac4", "class_name": "RelatedNodeInfo"}}, "text": "Acc# = NPGR Accession Number \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 111-115 Amgai et al. \n\n\u00a9NJB, BSN 113 \n\n volume was prepared using 1.5 \u00b5l (1 \u00b5M) for each \n\nprimer, 7.5 \u00b5l of PCR Master Mix (Promega Corporation, \n\nUSA), 2.5 \u00b5l water and 2 \u00b5l (100ng) DNA template. This \n\nPCR mixture was amplified as per the following protocol. \n\nFor Marker ABG54, Bmag6 and Bmac144h \n\nThirty cycles: denaturation 30 sec at 95oC, annealing 1 \n\nmin with temperature as per Table 3 and extension 2 min \n\nat 72oC.   \n\nFor Marker HVM49 \n\nTouch down PCR protocol was followed as 18 cycles of 1 \n\nmin denaturation at 94oC, 30 sec of touchdown protocol \n\nwith decreasing 1oC per 2 cycle from 64oC until 55oC as \n\nannealing and 1 min at 72oC for the extension. This  \n\ntouchdown cycle was followed by another 30 cycles of 1 \n\nmin denaturation at 94oC, 1 min annealing at 55oC and 1 \n\nmin extension at 72oC. The final extension was 7 min at \n\n72oC and the final holding is at 4oC.  \n\nThe PCR products were separated in 2% agarose gel in \n\n1XTAE buffer at 100V for one hour. Gels were stained \n\nwith ethidium bromide (0.1 \u00b5g/ml) and visualized under \n\nUV rays. The presence of a particular band size (Table 3) \n\nwas considered the presence of a particular linked gene. \n\nResults \nWe have observed stripe rust in susceptible parents; \n\nhowever, we had not observed leaf rust and crown rust \n\nin Khumaltar conditions (Table 2). This suggests that the \n\nuse of MAS techniques is very essential for pyramiding \n\nany resistance gene that cannot be screened in field \n\ncondition at that time. \n\nMany breeding lines showed the presence of one or more \n\ngenes for the rust resistance based on the particular \n\nmarker band (Figure 1-4).  \n\nHowever, the pyramided lines were very limited than \n\nour expectation for both types of crosses (Table 4 and \n\nTable 5).  \n\nFigure 1. Amplification of SSR marker HVM049 (105bp) in \nbarley advanced lines. (Note: Parents are underlined and bold; \nand selected lines are bold with asterisk mark) \n\nFigure 2. Amplification of SSR marker ABG054 (180bp) in \nbarley advanced lines from Bonus/Solu Uwa. (Note: Parents \nare underlined and bold; and selected lines are bold with \n\nasterisk mark) \n\nTable 3. List of molecular markers showing polymorphism and used in selection process \n\nMarker \nName \n\nForward Primer \n[5' \u2026 3'] \n\nReverse Primer \n[5'\u2026  3'] \n\nAnnealing \nTemperature \n\nResistance \nGene \n\nPCR Product \nSize \n\nChromosome \nNo. \n\nReference \n\nABG054 \nGTGCTTGG\nCGGTCGA\n\nCCAGT \n\nGATGTCCAAC\nGGTGGCTTGA \n\n55 \nStripe Rust \n\n(QTL) \n180bp* 4H [5] \n\nBmag0006 \nTTAAACCC\nCCCCCCTC\n\nTAG \n\nTGCAGTTACT\nATCGCTGATT\n\nTAGC \n58 \n\nCrown \nRust (Rpc1) \n\n174 3H [6] \n\nBmac0144h \nTACGTGTA\nCATACTCT\nACGATTTG \n\nACTTATTCTG\nCATCCTGGGT \n\n55 \nLeaf Rust \n(R-gene) \n\n179 1H [7] \n\nHVM049 \nCTCTATAG\nGCACGAA\nAAATTCC \n\nTTGCACATAT\nCTCTCTGTCA\n\nCA \n55 \n\nLeaf rust \n(Rph19) \n\n105 7H [8] \n\nNote: *=Field disease resistance data is used to identify the product size", "start_char_idx": 9403, "end_char_idx": 12332, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1949d3df-40c6-44fd-b249-0265dad7f55f": {"__data__": {"id_": "1949d3df-40c6-44fd-b249-0265dad7f55f", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "426e8152-8f7d-40cf-9e55-ac862d813a44", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "418d274d3fb54947884c8385a3cb3ff9aea957066ab6654dfb5270108d70d711", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "961c762a-5ef9-4972-a9dc-fd319d068475", "node_type": "1", "metadata": {}, "hash": "943225b32c72aa8b68d8553639ef16938274c957d5905f7f2acc04391a75f95a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2020 Dec; 8 (3): 111-115 Amgai et al. \n\n\u00a9NJB, BSN 114 \n\nFigure 3. Amplification of SSR marker HVM049 (105bp) in \n\nbarley advanced lines. (Note: Parents are underlined and bold; \n\nand selected lines are bold with asterisk mark) \n\nFigure 4. Amplification of SSR marker ABG054 (180bp) in \nbarley advanced lines from Bonus/Acc#2478. (Note: Parents \nare underlined and bold; and selected lines are bold with \nasterisk mark) \n\nDiscussion \nSelection on the late stage of the breeding may lead to \n\neroding many useful lines with the important genes that \n\nshowed the neutral effect in the previous season of field \n\ndisease screening. The leaf rust and crown rust could not \n\nbe screened in Khumaltar condition for all previous \n\nseasons, which ultimately lead us a few lines with leaf \n\nrust and crown rust resistance along with stripe rust \n\nresistance. But, the agronomic characteristics of our \n\nselected lines are superior and always safe from ending \n\nwith disease resistance but poor yielding varieties. \n\nDue to the less polymorphism between the parent Bonus \n\nand Acc#2478; we can select the lines with pyramided \n\nstripe rust and leaf rust resistance linked markers only. \n\nThe linked marker for crown rust resistance found in \n\n\u2018Bonus\u2019 is also found in Nepalese landrace \u2018Acc#1478\u2019 \n\n(Table 2). We identified linked markers for the leaf rust \n\nresistance gene in Nepalese local variety \u2018Solu Uwa\u2019 and \n\nlandrace \u2018Acc#1478\u2019 which support our observation of \n\nbarley field at Khumaltar and surroundings with \n\nnegligible infection from leaf rust.  \n\nHigher leaf rust resistance in Nepalese barley is also \n\nsupported by the observation of Tyrshkin [9] and \n\nHenderson [10].  Tyrshkin [9] also concluded that \n\nNepalese barley germplasm NB-3002 has one dominant \n\ngene for leaf rust resistance. \n\nTable 4. List of selected advanced breeding lines from crosses between Bonus and Solu Uwa with corresponding \nmolecular marker polymorphism. \n\nLine Field Stripe Rust ABG054 Bmac0144h HVM049 Bmag0006 \n\nBonus 0 1 0 0 1 \n\nSolu Uwa 10S 0 1 1 0 \n\nBonus/Solu Uwa-30 0 1 0 1 1 \n\nBonus/Solu Uwa-33 0 1 0 1 1 \n\nBonus/Solu Uwa-45 0 1 0 1 1 \n\nBonus/Solu Uwa-48 0 1 0 1 1 \n\nBonus/Solu Uwa-60 0 1 1 1 1 \n\nBonus/Solu Uwa-63 0 1 1 1 1 \n\nBonus/Solu Uwa-81 0 1 1 0 1 \n\nBonus/Solu Uwa-90 0 1 1 1 1 \n\nBonus/Solu Uwa-135 0 1 1 1 1 \n\nBonus/Solu Uwa-138 0 1 0 1 1 \n\nNote: 1 = Present, 0 = Absent \n\nTable 5. List of selected advanced breeding lines from Bonus \nand Acc#2478 cross with corresponding molecular marker \npolymorphism. \n\nLine Field Stripe Rust ABG054 HVM049 \n\nBonus 0 1 0 \n\nAcc#2478 60S 0 1 \n\nBonus/Acc#2478-186 0 1 1 \n\nBonus/Acc#2478-189 0 1 1 \n\nBonus/Acc#2478-209 0 1 1 \n\nNote: 1 = Present, 0 = Absent \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 111-115 Amgai et al. \n\n\u00a9NJB, BSN 115 \n\nSimilarly, we also observed that hull-less parent (\u2018Solu \n\nUwa\u2019) is less stripe rust susceptible than the hulled parent \n\n(\u2018Acc#2478\u2019) (Table 2) as Baniya et al.", "start_char_idx": 12337, "end_char_idx": 15289, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "961c762a-5ef9-4972-a9dc-fd319d068475": {"__data__": {"id_": "961c762a-5ef9-4972-a9dc-fd319d068475", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1949d3df-40c6-44fd-b249-0265dad7f55f", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "38c707e58d534b224561f721380b5fe0f2a619325ed312f9da69c320248bbdb5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b2869713-d24b-46f7-bf01-cedf76ecb6eb", "node_type": "1", "metadata": {}, "hash": "c9b6d76e7c1741e8edfe2f0d58703d58b3e26d88a5cfac075a42b925a3df2b91", "class_name": "RelatedNodeInfo"}}, "text": "[11] already \n\nconcluded for covered (hulled) barley and naked (hull-\n\nless) barley collection for Nepal. \n\nConclusion \nSelection in an early generation for marker assisted \n\nselection (MAS) program is considered a thumb rule; \n\nhowever, in the condition where laboratory resource is \n\npoor and costly than growing crops in the field, late \n\ngeneration selection for pyramided lines using molecular \n\ntechniques will be still competitive. On one hand, the \n\nchances of losing the desired gene (or marker) are high; \n\nbut in another hand, the chances of getting agronomical \n\nsuperior varietal output will also increase by late use of \n\nMAS techniques since early generation selection on MAS \n\nlargely depends on particular gene/marker rather than \n\ncrop performance itself.  \n\nAuthor\u2019s Contribution \nRBA selected parents and made the crosses. SuP, AP, SS \n\nadvanced the lines and maintain them. RBA, SuP did \n\ndisease scoring. RBA, ShP, SS did DNA extraction, PCR \n\nand gel electrophoresis. RBA did data analysis, wrote and \n\nfinalized the manuscript. All the authors read and \n\napproved the final manuscript.\n\nCompeting Interests \nNo competing interests were disclosed. \n\nFunding \nPart of this research is conducted under NG-NARC-Fund \n\n# 411. \n\nAcknowledgements \nNot Applicable. \n\nEthical Approval and Consent \nNot Applicable. \n\nReferences \n1. Prasad RC, Karki CB, Sharma S. Pathological Report on Barley. Hill\n\nCrops Proceedings. National Hill Crops Research Program, Kabre \nDolakha, Nepal; 1993. PP 52-78. \n\n2. Riley KW, Singh KM. Diversity and stability of barley in Nepal.\n1980 [cited 2020 Nov 5]. Available from: https://idl-bnc-\nidrc.dspacedirect.org/bitstream/handle/10625/6009/40369.pdf\n\n3. Peterson RF, Campbell AB, Hannah AE. A diagrammatic scale for\nestimating rust intensity of leaves and stem of cereals. Can J Res Sci.\n1948; 26:496-500. doi: https://doi.org/10.1139/cjr48c-033 \n\n4. Sul IW, Korban SS. A highly efficient method for isolating genomic\nDNA from plant tissues. Plant Tiss. Cult. Biotech. 1996; 2: 113-116. \n\n5. Rossi C. Testing the effectiveness of barley stripe rust resistance\nQTL detected in Mexico and the USA against a possible new race in \nPeru and mapping of genes conferring resistance to leaf rust and\nmildew in the same population [MS Thesis]. [Oregon]: Oregon State\nUniversity; 2005. 91p. \n\n6. Agrama HA, Dahleen L, Wentz M, Jin Y, Steffenson B: 2004. \nMolecular mapping of the crown rust resistance gene Rpc1 in\n\nBarley. Phytopathology. 2004; 94(8):858-861. \ndoi: 10.1094/PHYTO.2004.94.8.858 \n\n7. Jafary H, Albertazi G, Marcel TC, Niks RE. High diversity of genes\nfor non-host resistance of barley to heterologous rust fungi.\nGenetics. 2008; 178: 2327-2339. doi: 10.1534/genetics.107.077552\n\n8. Park RF, Poulsen D, Barr AR, Cakir M, Moody DB, Raman H and\nRead BJ. Mapping genes for resistance to Puccinia hordei in barley. \nAustralian J Agricultural Research. 2003; 54: 1323-1333.\nhttps://doi.org/10.1071/AR02244\n\n9. Tyryshkin LG: Genetic control of effective leaf rust resistance in \ncollection accessions of barley (Hordeum vulgare L).", "start_char_idx": 15290, "end_char_idx": 18367, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b2869713-d24b-46f7-bf01-cedf76ecb6eb": {"__data__": {"id_": "b2869713-d24b-46f7-bf01-cedf76ecb6eb", "embedding": null, "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-161", "node_type": "4", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "1bf8b51f0cb9b6ace9b2c26fcc0203150b0c53cb3cb54f06a3c90809d79936d8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "961c762a-5ef9-4972-a9dc-fd319d068475", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "ef529035744869775ac5f5d382467cbaa19c4731bf22e9e8afb87d8d0f6d963c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bb3a2846-9951-44ee-b9b3-e231db615ced", "node_type": "1", "metadata": {}, "hash": "4d63f27b992d9874a47752850a21d970bf6b658f5cca03bce079c578cf4ff946", "class_name": "RelatedNodeInfo"}}, "text": "Russian Journal\nof Genetics 2009, 45 (3): 376-378. \nhttps://doi.org/10.1134/S1022795409030181\n\n10. Henderson, MT. Studies of sources of resistance and inheritance of\nreaction to leaf rust (Puccinia anomala Rostr.) in barley [Ph.D.\nThesis]. [Minneapolis]: University of Minnesota, Minneapolis, 1945.\n\n11. Baniya BK, Riley RW, Dongol DMS, Sherchand KK.\nCharacterization of Nepalese hill crop landraces (Barley,\nBuckwheat, Finger Millet, Grain Amaranth, Foxtail, Proso and \nBarnyard Millets). National Hill Crops Research Program, Nepal \nAgriculture Research Council, Dolakha, Nepal; 1992. PP 7-17.", "start_char_idx": 18368, "end_char_idx": 18963, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bb3a2846-9951-44ee-b9b3-e231db615ced": {"__data__": {"id_": "bb3a2846-9951-44ee-b9b3-e231db615ced", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b2869713-d24b-46f7-bf01-cedf76ecb6eb", "node_type": "1", "metadata": {"identifier": "njb-161", "author": "Amgai, Resham B.; Pokharel, Shreejan; Pantha, Sumitra; Parajuli, Atit; Subedi, Sudeep; Dhital, Shambhu P.", "title": "Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers", "date": "2020-12-30", "file": "njb-161.pdf"}, "hash": "44b3bceed36e2b7a7d6f9f9f1fba56963e8b84dcd64026dc574a7fa8b8ee46ba", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "be1cd752-ec3f-4fab-af15-f4f3472374e2", "node_type": "1", "metadata": {}, "hash": "3290925ea14f4468c5b0bfcc84011d5db8b574c9c02ccaf731b285c635ea49cf", "class_name": "RelatedNodeInfo"}}, "text": "Special Issue   \nNepal J Biotechnol. 2 0 2 0  O c t ;  8 (2): 69-75  DOI: https://doi.org/10.3126/njb.v8i2.31893 \n\n \nResearch article \n\n  \n\n \n\u00a9NJB, BSN  69 \n\n \n \n\n \n\n \n\nComparative Assessment of Antibiotic Resistance in Lactic Acid \nBacteria Isolated from Healthy Human Adult and Infant Feces \nRasika Pawar1 , Vasudeo Zambare2 , Bela Nabar1   \n1Department of Microbiology, Smt Chandibai Himathmal Mansukhani College, Ulhasnagar, Thane, \n\nMaharashtra, India \n2Department of Life Sciences, School of Science, Sandip University, Nashik, Maharashtra, India \n\nArticle history:- Received: 20 Jun 2020; Revised: 23 Sep 2020; Accepted: 30 Sep 2020; Published online: 22 Oct 2020 \n\nAbstract \nLactic acid bacteria are normal inhabitants of the gastrointestinal tract of humans. Their occurrence in infant and \nadult feces is abundant. The current study assesses and compares the antibiotic resistance in lactic acid bacteria \nisolated from healthy human adult and healthy infant fecal samples. A total of 255 lactic acid bacteria isolates (126 \nfrom adult feces and 129 from infant feces) were isolated and characterized from 60 fecal samples. Lactobacillus spp., \nPediococcus spp. and Enterococcus spp. were included in the study. The study was done using the WHONET software \nfor the analysis of antibiotic susceptibility data of lactic acid bacteria. Most of the Lactobacillus and Pediococcus strains \nwere sensitive to vancomycin. Enterococcus strains showed resistance against vancomycin. Ampicillin, ciprofloxacin \nand cefuroxime resistance were significantly (p<0.05) higher in Lactobacillus strains isolated from adult fecal samples \nthan those isolated from infant fecal samples. A similar pattern was observed in Enterococcus strains with \nerythromycin, gentamycin and tobramycin resistance. Pediococcal isolates from adult feces showed significantly \nhigher resistance against tobramycin, ciprofloxacin, gentamycin, cefotaxime and cefuroxime in comparison with \ninfant fecal isolates. Antibiotic resistance was exhibited by lactic acid bacteria against most commonly used \nantibiotics and it was higher in strains isolated from adult fecal samples than in the strains isolated from infant fecal \nsamples. The increasing trend in antibiotic resistance from infant to adult might be due to food habits and antibiotic \nintakes. Thus, the widespread antibiotic resistance in different lactic acid bacteriamay pose a food safety concern as \nwell. \n\nKeywords: Lactic acid bacteria, Antibiotic resistance, Lactobacillus, Feces, Fecal microbes  \n\n Corresponding author, email: belanabar23@gmail.com \n\nIntroduction \nThe lactic acid bacteria (LAB) originate from a \n\ntaxonomically diverse group of microorganisms, \n\nwhich are non-sporing rods and cocci, usually non-\n\nmotile that ferment carbohydrates and form lactic \n\nacid. Lactic acid bacteria contain the genera namely \n\nLactobacillus, Lactococcus, Pediococcus, Streptococcus, \n\nEnterococcus, Oenococcus, Leuconostoc, \n\nCarnobacterium, Vagococcus, Tetragenococcus, and \n\nWeissella [1]. The microflora of humans and animal \n\ngut is complex and it is primarily dominated by \n\nlactic acid bacteria. There is high density and rich \n\ndiversity of microorganisms in the gut, and the \n\nmicroflora complexity increases from the upper \n\ngastrointestinal tract to the colon [2]. The human gut \n\ncontains more than a thousand bacterial species and \n\nsome of them start to colonize the gut during infancy \n\n[3]. Soon after the birth of a newborn infant, the gut \n\nflora begins to develop and microbes start to \n\ncolonize the small intestine and large intestine.", "start_char_idx": 52, "end_char_idx": 3646, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "be1cd752-ec3f-4fab-af15-f4f3472374e2": {"__data__": {"id_": "be1cd752-ec3f-4fab-af15-f4f3472374e2", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bb3a2846-9951-44ee-b9b3-e231db615ced", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "e30bd7edb1538156e5c3a10558c74013220def68ac913805588f50c722ed4757", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "af60d5d6-c670-4780-b156-15ba63d11098", "node_type": "1", "metadata": {}, "hash": "d48ec1c89253ca29be96ec9051717c338cb93f41493204aec5cc89e0a0adb06c", "class_name": "RelatedNodeInfo"}}, "text": "Aerobic and facultative anaerobic bacteria \n\n(Enterobacteria, Enterococci and Streptococci) are the \n\nearly colonizers in the human gut. After they \n\ncolonize, they create anaerobic environment in the \n\ngut. This helps anaerobic bacteria \n\n(Bifidobacteria, Bacteroides and Clostridia) to start with \n\ntheir colonization majorly in the large intestine [4]. \n\nThe development of complex, diverse and stable \n\nmicroflora continues from infancy to one year of age. \n\nAfter a year it is similar to adults and it is stable [4]. \n\nMany factors are governing the development, \n\ndiversity, composition and colonization gut \n\nmicroflora of infants, out of which mother\u2019s gut \n\nmicroflora, food and environment are the deciding \n\nones [5]. During birth, an infant is exposed to the \n\nmother\u2019s vaginal microflora and also to fecal \n\nmicroflora, and with this exposure colonization of \n\nthe gut in infants begins [6]. Infant gut microflora is \n\naffected by colostrum and later by breast milk. After \n\nthe introduction of formula and solid foods, \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nmailto:belanabar23@gmail.com\nhttps://orcid.org/0000-0003-3739-3752\nhttps://orcid.org/0000-0001-9064-8289\nhttps://orcid.org/0000-0002-4370-3510\nmailto:belanabar23@gmail.com\nmailto:belanabar23@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  70 \n\ncomplexity and diversity is generated in the gut \n\nmicroflora of infants. Microbes present in the \n\nenvironment and those present directly on the skin \n\nof the infant also enter the gut and create a complex \n\nniche [7]. Colonization of the gut with diverse \n\nmicroflora creates continuous impacts on the \n\nimmune system; and in this process, it strengthens \n\nthe immune system [8]. \n\nOver the past few decades, there has been a huge \n\ninterest developed in LAB physiology and genetics, \n\ninvolving their increasing importance as starter \n\ncultures in different industrial fermentation \n\nprocesses and also as probiotics. Since probiotics are \n\ndirectly administered in humans and animals it is \n\nvery necessary to determine the level of antibiotic \n\nresistance. This is a part of the assessment of the \n\nsafety of the probiotic cultures which are \n\nadministered as therapeutics.  \n\nIn the past 60 years, approximately 10 million tons of \n\nantibiotics have been utilized and released into the \n\nenvironment. As presented in the reports of \n\nEuropean Commission there is a huge probability of \n\nthe spread of antibiotic resistance in the biosphere \n\n[9]. Hence,there is a very strong selective pressure in \n\nthe development of antibiotic resistance in bacterial \n\nstrains [10]. \n\nLactic acid bacteria dominate the gastrointestinal \n\ntract of humans. They are present in large amounts \n\nin the gut and are also added or sometimes \n\nadditionally consumed along with the regular diet. \n\nHence, it is speculated that the presence of antibiotic \n\nresistance in lactic acid bacteria used as probiotics \n\ncan be dangerous. Probiotics are generally \n\nadministered to maintain microbial balance during \n\ngastrointestinal tract infections such as diarrhea.  \n\nThey are administered as therapeutic agents along \n\nwith antibiotics. If probiotics harbor antibiotic-\n\nresistant genes, it could be beneficial in sustaining \n\nthe antibiotics during the treatment; however, there \n\nis a risk of antibiotic-resistant probiotic strains to \n\ntransfer the resistance genes to the pathogenic \n\nbacteria. This could complicate the treatment of a \n\npatient with an antibiotic-resistant bacterial \n\ninfection or disease. Additionally, there is the \n\npossibility of the transfer of antibiotic resistance \n\nfrom beneficial lactic acid bacteria, in the food chain.", "start_char_idx": 3649, "end_char_idx": 7507, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "af60d5d6-c670-4780-b156-15ba63d11098": {"__data__": {"id_": "af60d5d6-c670-4780-b156-15ba63d11098", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "be1cd752-ec3f-4fab-af15-f4f3472374e2", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "7f5fefa7d5b4cf4e3f3a7fed8ea5a266a0da94374390f4ae169045997552582d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c35898a7-8c28-4639-b4e6-cef76b3cb420", "node_type": "1", "metadata": {}, "hash": "9a4bc79b4a21d4dfd44ae1a55735ec35c10bcf9953e6ae9d2c9b06dc12da6fe4", "class_name": "RelatedNodeInfo"}}, "text": "Therapy with any antibiotic, particularly long term \n\nand especially oral administration is liable to alter \n\nthe balance of antibiotic-resistant to sensitive \n\norganisms in the intestine [11]. \n\nCertain strains of these genera are more commonly \n\nused in the food and especially dairy industries or as \n\nprobiotics [12]. The World Health Organization \n\n(WHO) has established a program known as the \n\nAntimicrobial Resistance Monitoring (ARM) \n\nprogram for monitoring antimicrobial resistance. \n\nWHO has also devised an electronic format \n\nWHONET, freely available to download. A special \n\nfocus of antimicrobial susceptibility test results is \n\navailable on windows-based database software, \n\ndeveloped for the management and analysis of \n\nmicrobiology data [13]. This study aimed to \n\ndetermine the antibiotic susceptibility of lactic acid \n\nbacteria (using WHONET software) isolated from \n\nadult and infant feces to various groups of \n\nantibacterial agents that are mainly isolated from the \n\nfeces of breastfed infants. Also, the comparative \n\nassessment was done to determine the isolates that \n\nare more resistant to antibiotics.  \n\nMaterials and Methods \nSample collection and ethics statement  \nThirty healthy adult human volunteers (from \n\nMumbai and Suburbs, India) aged between 25 and \n\n30, who were not suffering from any chronic disease, \n\nhad not taken antibiotics, proton pump inhibitors, \n\nbismuth compounds, Histamine H2-receptor, \n\nnonsteroidal anti-inflammatory drugs within the \n\nprevious 6 months, were selected for the study. \n\nSimilarly, fecal samples were also collected from \n\nthirty healthy infants aged between 3 months to 9 \n\nmonths. Infants who were exclusively breast-fed, \n\nhealthy and free from acute or chronic disease were \n\nselected in the study. The study protocol was \n\napproved by an independent ethical committee and \n\nperformed in compliance with the US Code of \n\nFederal Regulations on Good Clinical Practices (21 \n\nCFR 10.90, 50, 56 and 812) and the World Medical \n\nAssociation Declaration of Helsinki (1996 \n\namendment) [14]. All adult volunteers and parents \n\nof infants signed informed consent before samples \n\nwere collected. \n\nIsolation of lactic acid bacteria from the \nfecal sample \nFecal samples were collected in sterile \n\npolypropylene containers and processed \n\nimmediately as follows. A 0.5 g portion of feces was \n\ntaken from mid sample, added in 4.5 ml of sterile  \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  71 \n\nsaline solution, and completely homogenized. A \n\ndilution series (10\u20131 to 10\u20137) was made and 100 \u00b5l  \n\n aliquots of each dilution were inoculated on the \n\nagar plates by spread plating. Rogosa SL agar (Hi-\n\nMedia, Mumbai, India) was used to isolate LAB and \n\nthe plates were incubated micro-aerobically for 3 \n\ndays at 37\u00b0C. Kenner fecal (KF) agar was used for the \n\nisolation of Enterococcus and incubated aerobically at \n\n37\u00b0C for 24 h [15].  \n\n Enumeration and selection of bacterial \nisolates \n After incubation, the plates that showed discrete \n\ncolonies were selected and the colonies were \n\ncounted. The total count of Lactic acid bacteria in \n\nfeces was expressed as colony-forming units/g (wet \n\nweight). From each fecal sample, 10-20 colonies of \n\nLAB were randomly selected. A provisional \n\nidentification of genera was made based on Gram\u2019s \n\nstaining, and catalase reaction using 3% (v/v) H2O2 \n\non single colonies. Putative Lactobacilli colonies \n\n(Gram-positive, catalase test-negative, rod-shaped) \n\nwere chosen and further purified using MRS agar. \n\nSimilarly, putative colonies of Enterococci and \n\nPediococci (Gram-positive, catalase test-negative, \n\ncocci, able to grow at 10oC and 45oC, and in 18% \n\nNaCl and at pH 4.4) from KF agar plates were \n\npurified by re-streaking on the MRS agar.", "start_char_idx": 7510, "end_char_idx": 11347, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c35898a7-8c28-4639-b4e6-cef76b3cb420": {"__data__": {"id_": "c35898a7-8c28-4639-b4e6-cef76b3cb420", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "af60d5d6-c670-4780-b156-15ba63d11098", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "0eabc294de499a279e0993d73b6e1242d945e1dfc9e881d2a683e0eab2a154ee", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5dd5c0dc-3dc5-4312-9894-e21e200c2812", "node_type": "1", "metadata": {}, "hash": "e42f8e9305ff1ecbd99cb19df4dd514437ed6a42e136819d8becfff57e8f77ab", "class_name": "RelatedNodeInfo"}}, "text": "The \n\ncultures were stored in MRS broth with 15% glycerol \n\nat \u201320\u00b0C [15]. \n\nAntibiotic resistance \n The antibiotic resistance/susceptibility patterns of \n\nisolated strains of lactic acid bacteria were studied \n\nusing the Kirby-Bauer disk diffusion method \n\n(according to the CLSI document M2-A9 \n\nsuggestions) [16]. The antibiotics used in this study \n\nwere penicillin (10 \u00b5g), ampicillin (10 \u00b5g), \n\nvancomycin (30 \u00b5g), cefuroxime (30 \u00b5g), cefotaxime \n\n(30 \u00b5g), ciprofloxacin (5 \u00b5g), gentamycin (10 \u00b5g), \n\ntobramycin (10 \u00b5g), erythromycin (15 \u00b5g) and \n\nchloramphenicol (30 \u00b5g). The culture densities were \n\nadjusted to McFarland 1.5; they were spread on MRS \n\nagar plates. Antibiotic discs (Hi-Media, Mumbai, \n\nIndia) were placed on the surface of the agar plates, \n\nwhich were incubated at 37\u00b0C for 24 h. The \n\ndiameters of the clearance zones around the discs \n\nwere measured and the result (the average of 2 \n\nreadings) was expressed as susceptible, \n\nintermediate, or resistant according to the standard \n\ndisc diffusion method [16]. The experiment was \n\ndone in triplicates. Microsoft Excel (2013) was used \n\nto obtain data in the appropriate format for BacLink \n\n2019, used to format data to be used in WHONET \n\n2019, which automatically calculates the % \n\nresistance using a data analysis tool. \n\nStatistical analysis \nThe data was analyzed to check the significant \n\ndifference between groups using Student\u2019s T-test \n\nwith a probability level of 0.05 (P\u2009<\u20090.05) using \n\nMicrosoft Excel (2013). \n\nResults  \nIsolation of lactic acid bacteria from the \nfecal sample \nA total of 255 LAB isolates were isolated from 30 \n\nhuman adult and 30 human infant fecal samples. \n\nOut of the 255 isolates, 126 isolates were from the \n\nadult fecal sample, and 129 from the infant fecal \n\nsample, the results are presented in Table 1. The \n\nisolates were identified phenotypically and \n\ncharacterized. Based on the characters, the LAB \n\nisolates were characterized as mesophilic \n\nhomofermentative cocci, able to grow at 10oC and \n\n45oC as Enterococcus (81 isolates). Homofermentative \n\ncocci in tetrads, unable to grow in 18% NaCl, and \n\nshowing growth at pH 4.4 were characterized as \n\nPediococcus (84 isolates). Lactobacilli (90 isolates) \n\nwere represented as catalase-negative, slender \n\ngram-positive rods. All strains grew at 4oC and 6.5% \n\nNaCl concentration. \n\nAntibiotic resistance of lactic acid \nbacteria \nData of diameter of zone of clearance in mm of LAB \n\nisolated from adult and infant feces was entered in \n\nMicrosoft Excel and via BacLink software \n\nincorporated into WHONET software (Table 2).  \n\nTable 1. Count of LAB isolates in the adult and infant fecal samples. \n\nSample source Number of samples \n\n(n = 60) \n\nLAB isolates \n\n(n = 255) \n\nLactobacillus spp. \n\n(n=90) \n\nPediococcus spp. \n\n(n=84) \n\nEnterococcus spp. \n\n(n=81) \n\nAdult feces 30 126 46 41 39 \n\nInfant feces 30 129 44 43 42 \n\n*LAB= Lactic Acid Bacteria \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  72 \n\n \nFigure 1. Antibiotic resistance pattern of Pediococcus spp. \n\n(a), Lactobacillus spp. (b) and Enterococcus spp.", "start_char_idx": 11348, "end_char_idx": 14495, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5dd5c0dc-3dc5-4312-9894-e21e200c2812": {"__data__": {"id_": "5dd5c0dc-3dc5-4312-9894-e21e200c2812", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c35898a7-8c28-4639-b4e6-cef76b3cb420", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "890e39a0713b0ed96a27d020c1dfe3b03197c100e68bb9e62e8fa1543480869d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6a2d5090-da14-4982-b8a9-69074acbbaa4", "node_type": "1", "metadata": {}, "hash": "7ae906c432de1f04b0812c12dfa35762f9e3a5b415354b238e1ac1884fd39a42", "class_name": "RelatedNodeInfo"}}, "text": "(a), Lactobacillus spp. (b) and Enterococcus spp. (c) isolated \n\nfrom adults and infant feces, respectively [AMP-\n\nAmpicillin, CHL-Chloramphenicol, CIP-Ciprofloxacin, \n\nCTX-Cefotaxime, CXM-Cefuroxime, ERY-Erythromycin, \n\nGEN-Gentamicin, PEN-Penicillin G, TOB-Tobramycin, \n\nVAN-Vancomycin]. All experiments were performed in \n\ntriplicates and the error bar represents the standard \n\ndeviation of independent performs experiments (n=3). \n\nPediococcus spp. isolated from adult feces was \n\ncomparatively more resistant to antibiotics than \n\nthose isolated from infant feces. Significantly higher \n\nresistances (P < 0.05) were found against ampicillin \n\n(7.3%), cefotaxime (22.0%), cefuroxime (36.6%), \n\npenicillin (12.2%) gentamycin (26.8%), erythromycin \n\n(19.5%), tobramycin (29.3%) and ciprofloxacin \n\n(26.8%) from isolates from adult feces than those \n\nisolated from infant feces, 7.0%, 9.3%, 0.0%, 11.6%, \n\n9.3%, 0.0%, 2.3% and 20.9% respectively (Figure 1a). \n\nAll the isolates from both adult and infant samples \n\nwere sensitive to vancomycin and chloramphenicol. \n\nPediococcus spp. were intrinsically resistant to high \n\nlevels of glycopeptides and penicillin. Resistance to \n\nerythromycin was also reported and was due to a \n\nplasmid with an erythromycin resistance methylase \n\nB [erm(B)] gene [17]. \n\nDiscussion \nTo develop probiotics for human or animal \n\nconsumption, it is necessary to distinguish strains \n\nharboring antibiotic resistance genes from other \n\nstrains because of potential risk for the \n\ndissemination of resistance genes. In this study, it \n\nwas demonstrated that strains isolated from infants \n\nwere more sensitive than those isolated from adult \n\nfeces. Lactobacilli and Pediococciare widely used as \n\nprobiotics and promoters for biological growth. \n\nLactobacilli are reported to be resistant to several \n\nantibiotics [18]. In the present study, Lactobacillus \n\nspp. isolated from adult feces were more resistant to \n\nantibiotics than those isolated from infant feces. \n\nSignificantly higher resistance was found against \n\ncefuroxime (26.1%) and ciprofloxacin (32.6%) from \n\nisolates from adult feces than those isolated from \n\ninfant feces, 4.5%, and 2.3% respectively. \n\nLactobacillus spp. isolated from feces also showed \n\nmoderate resistance to cefotaxime (13.0%), penicillin \n\n(10.9%), chloramphenicol, (10.9%), gentamycin \n\n(23.9%), erythromycin (13.0%) and tobramycin \n\n(26.1%). Whereas those isolated from infant feces \n\nshowed comparatively lesser resistance 4.5%, 11.4%, \n\n9.1%, 20.5% and 9.1%, respectively (Figure 1b). \n\nResistance to gentamycin and ciprofloxacin was \n\nearlier documented [19, 20]. Concerning cell wall \n\nsynthesis inhibitors, Lactobacilli are reported to be \n\nresistant to oxacillin and cephalosporins (cefoxitin \n\nand ceftriaxone) [21].   \n\nThey were also found to show resistance to \n\naminoglycosides (neomycin, kanamycin, \n\n0\n\n10\n\n20\n\n30\n\n40\n\n50\n%\n\n R\ne\nsi\n\nst\na\n\nn\nt\n\nAntibiotics\n\n[a] Pediococcus spp.\n\nAdult\n\n0\n\n10\n\n20\n\n30\n\n40\n\n50\n\n%\n R\n\ne\nsi\n\nst\na\n\nn\nt\n\nAntibiotics\n\n[b] Lactobacillus spp.", "start_char_idx": 14446, "end_char_idx": 17499, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6a2d5090-da14-4982-b8a9-69074acbbaa4": {"__data__": {"id_": "6a2d5090-da14-4982-b8a9-69074acbbaa4", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5dd5c0dc-3dc5-4312-9894-e21e200c2812", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "9916bd83f0b01475fab8047df6e9445a7702acf902f3d87a10d3d94a92d07d71", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "95f3ecda-4193-4afd-870e-d3083443d7e0", "node_type": "1", "metadata": {}, "hash": "3a9d6a88bb9df383f186998338e8df60942f706e7c0a0e19dea66bb138cf30fb", "class_name": "RelatedNodeInfo"}}, "text": "Adult\n\nInfant\n\n0\n\n10\n\n20\n\n30\n\n40\n\n50\n\n%\n R\n\ne\nsi\n\ns\nta\n\nn\nt\n\nAntibiotics\n\n[c] Enterococcus spp.\n\nAdul\n\nt\n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  73 \n\nstreptomycin, and gentamicin) [22]. There are many \n\nspecies of Lactobacilli which contain intrinsic \n\nresistance to vancomycin, erythromycin and \n\ntetracycline. The matter of concern is that since \n\nLactobacilli are added to infant food, they can act as \n\nreservoirs of antibiotic resistance genes, which could \n\nbe transferable [23]. \n\nEnterococcus spp. also followed a similar pattern \n\nwhere the antibiotic resistance associated with adult \n\nfecal samples was higher than those isolated from \n\ninfant feces. Adult fecal isolates were 30.8% resistant \n\nto erythromycin, 20.5% resistant to tobramycin, and \n\n41% resistant to gentamycin. This was significantly \n\nhigher (P < 0.05) than infant fecal isolates, which \n\nwere sensitive to erythromycin, 9.5% resistant to \n\ntobramycin, and 4.8% to gentamycin.  Higher \n\nresistance was also found against vancomycin \n\n(30.8%), ciprofloxacin (33.3%), ampicillin (28.2%), \n\ncefuroxime (20.5%) and cefotaxime (28.2%); \n\nhowever, it was not statistically significant in \n\ncomparison to infant fecal isolates which showed \n\n11.9%, 9.5%, 21.4%, 9.8% and 26.2% resistance \n\nagainst above-mentioned antibiotics, respectively. \n\nAll the isolates (fecal and adult) were susceptible to \n\nchloramphenicol. Infant isolates were 11.9% \n\nresistant to penicillin; this was higher than adult \n\nisolates, which showed 10.3% resistance (Figure 1c). \n\nEnterococci showed intrinsic and acquired \n\nresistance against many antibiotics [24, 25]. Such \n\nintrinsic resistance was reported inlincosamides, \n\nnalidixic acid penicillin, polymyxins, quinupristin\u2013\n\ndalfopristin, monobactams, and low levels of \n\naminoglycosides. Resistance to high levels of \n\naminoglycosides, high levels of trimethoprim, and \n\nhigh levels of clindamycin, chloramphenicol, \n\ntetracyclines, penicillins (due to \u03b2-\n\nlactamase), fluoroquinolones, macrolides (e.g. \n\nerythromycin), glycopeptides and oxazolidinones \n\n(linezolid) were acquired [26-27].  Acquired \n\nresistance is a major threat in treatment, such a trait \n\nwas found to be transferred to other Enterococci in \n\nthe gut [28]. Vancomycin resistance is especially \n\nimportant as vancomycin is the last drug option for \n\ntreating diseases caused by multidrug resistance \n\nEnterococci [29].  \n\nApart from probiotic use, Pediococci are also widely \n\nused for the fermentation of meat and vegetables \n\nand also in cheese production [30]. According to the \n\nEFSA\u2019s FEEDAP Panel [31] (European Food Safety \n\nAuthority Panel on Additives and Products or \n\nSubstances used in Animal Feed), the bacterial \n\ncultures which are used for the production of animal \n\nfeed should be susceptible to antibiotics used in \n\ntreating humans bacterial infections. Hence, it is \n\nextremely necessary to distinguish antibiotic \n\nsusceptible and resistant strains. This also \n\nemphasizes the importance of safe source or niche of \n\na selection of strains used as probiotics. The results \n\nof the study indicate that infant feces could be a \n\nbetter source for isolation of LAB cultures intended \n\nto be used as probiotics. \n\nApart from being used traditionally as starter \n\ncultures in dairy products, LAB are also used for the \n\nproduction of animal feed.", "start_char_idx": 17501, "end_char_idx": 20902, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "95f3ecda-4193-4afd-870e-d3083443d7e0": {"__data__": {"id_": "95f3ecda-4193-4afd-870e-d3083443d7e0", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6a2d5090-da14-4982-b8a9-69074acbbaa4", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1f8030aee267adf0d7a5949842c1b29f750285b641ea974fdb865f30faa5c470", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5ab4c67a-b30b-489e-8c6e-58e8e8f788bd", "node_type": "1", "metadata": {}, "hash": "c603a2ecd343a8fd5d459f4637395651775351da6d95120dc3dfd7834d34aa47", "class_name": "RelatedNodeInfo"}}, "text": "They also belong to \n\nnormal flora of the human gut and confer health \n\nbenefits to the host. During the process of food \n\nmanufacturing and passage of food through gut, \n\nthere is a possibility of antibiotic resistance, carried \n\nby LAB getting transferred to human pathogenic \n\nbacteria [32]. Hence, it is imperative to select strains \n\nTable 2. Percent antibiotic resistance in target microorganisms isolated from adult and infant fecal samples. \n\nExpressed in percentage (%) \n\nMechanism of \nAction  \n\nAntibiotic Lactobacillus spp. Pediococcus spp. Enterococcus spp. \n\nAdult Infant Adult Infant Adult Infant \n\nCell Wall \nInhibitors \n\nAmpicillin 10.9 11.4 7.3 7.0 28.2 21.4 \nCefotaxime 13.0 4.5 22.0 9.3 28.2 26.2 \nCefuroxime 26.1 4.5 36.6 0.0 20.5 9.8 \nPenicillin 10.9 11.4 12.2 11.6 10.3 11.9 \nVancomycin 0.0 0.0 0.0 0.0 30.8 11.9 \n\nProtein \nSynthesis \nInhibitor \n\nChloramphenico\nl 10.9 \n\n9.1 0.0 0.0 0.0 0.0 \n\nErythromycin 13.0 9.1 19.5 0.0 30.8 0.0 \nGentamycin 23.9 20.5 26.8 9.3 41.0 4.8 \nTobramycin 26.1 6.8 29.3 2.3 20.5 9.5 \n\nDNA \nSynthesis \nInhibitor \n\nCiprofloxacin 32.6 2.3 26.8 20.9 33.3 9.5 \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  74 \n\nthat have low resistance against antibiotics for \n\nhuman and animal use. From the results of the \n\nantibiotic susceptibility in the current study, \n\nobtained from a broad range of antibiotics, it was \n\nfound that the isolated strains of Lactobacillus, \n\nPediococcus and Enterococcus were resistant to \n\nvarious antibiotics. However, antibiotic resistance \n\nwas lesser in strains obtained from infant fecal \n\nsamples than adult fecal samples.  \n\nConclusion \nLactobacillus, Pediococcus and Enterococcus as LAB \n\nwere isolated from the human fecal samples \n\nexhibiting more antibiotic resistance from adult fecal \n\nisolate than the infant. The development of antibiotic \n\nresistancein LAB can be attributed to the long term \n\nexposure of antibiotic as therapeutic agents as well \n\nas food habits which pose food safety concerns. \n\nThus, it is essential to see safety measure during \n\nantibiotic uptake in day to day life. In addition to \n\nthis, the low antibiotic-resistant strains from infant \n\ncould be the choice of strain to avoid the risk of \n\ntransferof LAB linked antibiotic resistance to human \n\npathogenic bacteria. \n\nAuthors Contribution \nRP has made a substantial contribution to data \n\nanalysis and its interpretation. VZ contributed in \n\ndesigning the experiments. BN contributed to data \n\ninterpretation and all authors RP, VZ and BN \n\ncontributed equally to drafting and reviewing of the \n\nmanuscript followed by final approval from BN. \n\nCompeting Interests \nNo competing interests were disclosed. \n\nFunding \n The author(s) declared that no grants were involved \n\nin supporting this work. \n\nAcknowledgements \nRSP is thankful to Principal, Smt CHM College and \n\nVZ is thankful to Sandip University for providing \n\nthe laboratory facilities and chemicals.", "start_char_idx": 20903, "end_char_idx": 23883, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5ab4c67a-b30b-489e-8c6e-58e8e8f788bd": {"__data__": {"id_": "5ab4c67a-b30b-489e-8c6e-58e8e8f788bd", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "95f3ecda-4193-4afd-870e-d3083443d7e0", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "aeb0812c12c874f8432401a412b34e37044c5e59ea8bf193a59ea5609085e283", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "88cd00e8-8cc3-40e9-9ed9-d596d7984e4e", "node_type": "1", "metadata": {}, "hash": "203ce39329e1c7160e1e40d670df21523c479610682766e544ce9c9a8ba169c2", "class_name": "RelatedNodeInfo"}}, "text": "Ethical Approval and Consent \nThe study protocol was approved by an \n\nindependent ethical committee and performed in \n\ncompliance with the US Code of Federal Regulations \n\non Good Clinical Practices (21 CFR 10.90, 50, 56, and \n\n812) and the World Medical Association Declaration \n\nof Helsinki (1996 amendment). All adult volunteers \n\nand parents of infants signed informed consent \n\nbefore sample collection. \n\nReferences  \n1. Holzapfel WH, Haberer P, Geisen R, Bj\u00f6rkroth J, Schillinger \n\nU. Taxonomy and important features of probiotic \nmicroorganisms in food and nutrition. The American Journal \nof Clinical Nutrition. 2001 Feb 1;73(2):365s-73s. \nhttps://doi.org/10.1093/ajcn/73.2.365s \n\n2. Thursby E, Juge N. Introduction to the human gut microbiota. \nBiochemical Journal. 2017 Jun 1;474(11):1823-36. \nhttps://doi.org/10.1042/BCJ20160510 \n\n3. Srikumar S, Fanning S. The Therapeutic Potential of the \u201cYin-\nYang\u201d Garden in Our Gut. InRole of Microbes in Human \nHealth and Diseases 2018 Dec 4. IntechOpen.  \n\n4. Vael C, Desager K. The importance of the development of the \nintestinal microbiota in infancy. Current Opinion in \nPediatrics. 2009 Dec 1;21(6):794-800. \nhttps://doi.org/10.1097/MOP.0b013e328332351b \n\n5. Milani C, Duranti S, Bottacini F, Casey E, Turroni F, Mahony \nJ, Belzer C, Palacio SD, Montes SA, Mancabelli L, Lugli GA. \nThe first microbial colonizers of the human gut: composition, \nactivities, and health implications of the infant gut microbiota. \nMicrobiology and Molecular Biology Reviews. 2017 Dec \n1;81(4). https://doi.org/10.1128/MMBR.00036-17 \n\n6. Makino H, Kushiro A, Ishikawa E, Muylaert D, Kubota H, \nSakai T, Oishi K, Martin R, Amor KB, Oozeer R, Knol J. \nTransmission of intestinal Bifidobacterium longum subsp. \nlongum strains from mother to infant, determined by \nmultilocus sequencing typing and amplified fragment length \npolymorphism. Applied and Environmental Microbiology. \n2011 Oct 1;77(19) :6788-93. https://doi.org \n/10.1128/AEM.05346-11 \n\n7. van den Elsen LW, Garssen J, Burcelin R, Verhasselt V. \nShaping the gut microbiota by breastfeeding: the gateway to \nallergy prevention? Frontiers in Pediatrics. 2019 Feb 27;7:47. \nhttps://doi.org/10.3389/fped.2019.00047 \n\n8. Walker WA. Role of nutrients and bacterial colonization in the \ndevelopment of intestinal host defense. Journal of Pediatric \nGastroenterology and Nutrition. 2000 Jan 1;30:S2-7. \nhttps://doi.org/10.1097/00005176-200000002-00002 \n\n9. Feedap P. Opinion of the scientific panel on additives and \nproducts or substances used in animal feed on the updating \nof the criteria used in assessment of bacteria for resistance to \nantibiotics of human and veterinary importance. European \nFood Safety Authority Journal. 2005;223:1-2. \nhttps://doi.org/10.2903/j.efsa.2005.223 \n\n10. Bronzwaer SL. European Antimicrobial Resistance \nSurveillance System. A European study on the relationship \nbetween antimicrobial use and antimicrobial resistance. \nEmergency and Infectious Diseases. 2002;8:278-\n82. https://doi.org/10.3201/eid0803.010192 \n\n11. Yoon MY, Yoon SS. Disruption of the gut ecosystem by \nantibiotics. Yonsei Medical Journal.", "start_char_idx": 23886, "end_char_idx": 27017, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "88cd00e8-8cc3-40e9-9ed9-d596d7984e4e": {"__data__": {"id_": "88cd00e8-8cc3-40e9-9ed9-d596d7984e4e", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5ab4c67a-b30b-489e-8c6e-58e8e8f788bd", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "7939bd87a833ad9d45e9a0221faac67549d5c8f369cc2be643ca9f80aafd6510", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "992b04c2-3a74-49e0-88ad-57559bbba034", "node_type": "1", "metadata": {}, "hash": "0e1541457b62d40ea24503144d6d12cb8c94e473c5c02c340ef6499381345588", "class_name": "RelatedNodeInfo"}}, "text": "Disruption of the gut ecosystem by \nantibiotics. Yonsei Medical Journal. 2018 Jan 1;59(1):4-12. \nhttps://doi.org/10.3349/ymj.2018.59.1.4 \n\n12. Heller KJ. Probiotic bacteria in fermented foods: product \ncharacteristics and starter organisms. The American Journal \nof Clinical Nutrition. 2001 Feb 1;73(2):374s-\n9s.  https://doi.org/10.1093/ajcn/73.2.374s \n\n13. World Health Organization:WHONET software. Available \nfrom: https://whonet.org/ \n\n14. World Medical Association Declaration of Helsinki (1996) 48th \nGeneral Assembly, Somerset West, Republic of South Africa, \n1-3. \n\n15. Shioiri, T, Yahagi, K, Nakayama, S.  The effects of symbiotic \nfermented milk beverage containing Lactobacillus casei strain \nShirota and transgalactosylated oligosaccharides on \ndefecation frequency, intestinal microflora, organic acid \nconcentrations, and putrefactive metabolites of sub-optimal \n\nhttps://doi.org/10.3389/fped.2019.00047\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8 (2) [Special Issue]: 69-75    Pawar et al. \n \n\n \n\u00a9NJB, BSN  75 \n\nhealth state volunteers: a randomized placebo-controlled \ncross-over study. Bioscience Microflora 2006; 25, 137\u2013146. \n\n16. Clinical Laboratory Standards Institute. M2-M9. Performance \nstandardsfor antimicrobial disk susceptibility tests; approved \nstandard 2006; Ninth edition PA. CLS.  \n\n17. Charteris WP, Kelly PM, Morelli L, Collins JK. Antibiotic \nsusceptibility of potentially probiotic Lactobacillus species. \nJournal of Food Protection. 1998 Dec;61(12):1636-43. \nhttps://doi.org/10.4315/0362-028X-61.12.1636 \n\n18. Klare I, Konstabel C, Werner G, Huys G, Vankerckhoven V, \nKahlmeter G, Hildebrandt B, M\u00fcller-Bertling S, Witte W, \nGoossens H. Antimicrobial susceptibilities of Lactobacillus, \nPediococcus and Lactococcus human isolates and cultures \nintended for probiotic or nutritional use. Journal of \nAntimicrobial Chemotherapy. 2007 May 1;59(5):900-12. \nhttps://doi.org/10.1093/jac/dkm035 \n\n19. Dec M, Urban-Chmiel R, St\u0119pie\u0144-Py\u015bniak D, Wernicki A. \nAssessment of antibiotic susceptibility in Lactobacillus isolates \nfrom chickens. Gut Pathogens. 2017 Dec 1;9(1):54. \nhttps://doi.org/10.1186/s13099-017-0203-z \n\n20. Fukao M, Tomita H, Yakabe T, Nomura T, Ike Y, Yajima N. \nAssessment of antibiotic resistance in probiotic strain \nLactobacillus brevis KB290. Journal of Food Protection. 2009 \nSep;72(9):1923-9. https://doi.org/10.4315/0362-028X-\n72.9.1923 \n\n21. Shazali N, Foo HL, Loh TC, Choe DW, Rahim RA. Prevalence \nof antibiotic resistance in lactic acid bacteria isolated from the \nfeces of broiler chicken in Malaysia. Gut Pathogens. 2014 Dec \n1;6(1):1. https://doi.org/10.1186/1757-4749-6-1 \n\n22.", "start_char_idx": 26945, "end_char_idx": 29583, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "992b04c2-3a74-49e0-88ad-57559bbba034": {"__data__": {"id_": "992b04c2-3a74-49e0-88ad-57559bbba034", "embedding": null, "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-170", "node_type": "4", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "1289ef259062b9e777939cc6f614f4b55b467feccfa8f7decba25c2cf0f36bf4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "88cd00e8-8cc3-40e9-9ed9-d596d7984e4e", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "09713e5209ce49f0b24225f44715d89f06d8cb2e3c294e6f9cdc738d37b68ad1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "db33f70b-303c-499f-8253-3257e5e4240a", "node_type": "1", "metadata": {}, "hash": "019b605526dcb3f643b26937f470c824fd7f8ae22f545087f4018cf338415037", "class_name": "RelatedNodeInfo"}}, "text": "Coppola R, Succi M, Tremonte P, Reale A, Salzano G, \nSorrentino E. Antibiotic susceptibility of Lactobacillus \nrhamnosus strains isolated from Parmigiano Reggiano cheese. \nLe Lait. 2005 May 1;85(3):193-204. \nhttps://doi.org/10.1051/lait:2005007 \n\n23. Zhou JS, Pillidge CJ, Gopal PK, Gill HS. Antibiotic \nsusceptibility profiles of new probiotic Lactobacillus and \nBifidobacterium strains. International Journal of Food \nMicrobiology. 2005 Feb 1;98(2):211-7. \nhttps://doi.org/10.1016/j.ijfoodmicro.2004.05.011 \n\n24. Mathur S, Singh R. Antibiotic resistance in food lactic acid \nbacteria\u2014a review. International Journal of Food \nMicrobiology. 2005 Dec 15;105(3):281-95. \nhttps://doi.org/10.1016/j.ijfoodmicro.2005.03.008 \n\n25. Miller WR, Munita JM, Arias CA. Mechanisms of antibiotic \nresistance in Enterococci. Expert Review of Anti-infective \nTherapy. 2014 Oct 1;12(10):1221-36. \nhttps://doi.org/10.1586/14787210.2014.956092 \n\n26. Hollenbeck BL, Rice LB. Intrinsic and acquired resistance \nmechanisms in Enterococcus. Virulence, 3 (5), 421\u2013433. \nhttps://doi.org/10.4161/viru.21282 \n\n27. Kimiran-Erdem A, Arslan EO, Yurudu NO, Zeybek Z, \nDogruoz N, Cotuk A. Isolation and identification of \nenterococci from seawater samples: assessment of their \nresistance to antibiotics and heavy metals. Environmental \nMonitoring and Assessment. 2007 Feb 1;125(1-3):219-28. \nhttps://doi.org/10.1007/s10661-006-9506-0 \n\n28. Rathnayake IU, Hargreaves M, Huygens F. Antibiotic \nresistance and virulence traits in clinical and environmental \nEnterococcus faecalis and Enterococcus faecium isolates. \nSystematic and Applied Microbiology. 2012 Jul 1;35(5):326-33. \nhttps://doi.org/10.1016/j.syapm.2012.05.004 \n\n29. Ahmed MO, Baptiste KE. Vancomycin-resistant Enterococci: a \nreview of antimicrobial resistance mechanisms and \nperspectives of human and animal health. Microbial Drug \nResistance. 2018 Jun 1;24(5):590-606. \nhttps://doi.org/10.1089/mdr.2017.0147 \n\n30. Reuter G. Present and future of probiotics in Germany and in \nCentral Europe. Bioscience and Microflora. 1997;16(2):43-51. \nhttps://doi.org/10.12938/bifidus1996.16.43 \n\n31. EFSA Panel on Additives and Products or Substances used in \nAnimal Feed (FEEDAP). Guidance on the assessment of \n\nbacterial susceptibility to antimicrobials of human and \nveterinary importance. EFSA Journal. 2012 Jun;10(6):2740. \nhttps://doi.org/10.2903/j.efsa.2012.2740 \n\n32. Schj\u00f8rring S, Krogfelt KA. Assessment of bacterial antibiotic \nresistance transfer in the gut. International journal of \nmicrobiology. 2011 Oct;2011. https:// \ndoi.org/10.1155/2011/312956", "start_char_idx": 29584, "end_char_idx": 32167, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "db33f70b-303c-499f-8253-3257e5e4240a": {"__data__": {"id_": "db33f70b-303c-499f-8253-3257e5e4240a", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "992b04c2-3a74-49e0-88ad-57559bbba034", "node_type": "1", "metadata": {"identifier": "njb-170", "author": "Pawar, Rasika; Zambare, Vasudeo; Nabar, Bela", "title": "Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces", "date": "2020-10-22", "file": "njb-170.pdf"}, "hash": "4c56fef0ed15b165d66358db2ea8a3d749d48667284cd3b30ac2d6e70e345125", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a4ce3f19-0aa9-4ecd-a9ce-3c3116102b3d", "node_type": "1", "metadata": {}, "hash": "c1a8a08380594f0e329061c38ab220ddad51adfee65762aeddd34bce576c2147", "class_name": "RelatedNodeInfo"}}, "text": "Special Issue   \nNepal J Biotechnol. 2 0 2 0  O c t ;  8(2): 76-81  DOI: https://doi.org/10.3126/njb.v8i2.31889 \n\n \n\nResearch article \n  \n\n\u00a9NJB, BSN  76 \n\n \n \n \n \nStudy of the Impact of Organic Manures and Biofertilizers on \ngrowth of Phaseolus aureus Roxb. \nChhaya Bhalshankar   \n\nNew Arts Commerce and Science College, Shevgaon, Dist.-Ahmednagar, M.S. (India) \n\nArticle history:- Received: 15 Jun 2020; Revised: 20 Sep 2020; Accepted: 1 Oct 2020; Published online: 22 Oct 2020 \n\nAbstract  \nWeeds are wild plants growing where they are not wanted, and they compete with the cultivated crop for \nnutrition. Though they are seen as agricultural waste throughout the year, they are rich sources of nutrients. \nThey grow in abundance during the rainy season, but as the season ends these biomasses get wasted. In the \npresent investigation, Tephrosia hamiltonii Drumm belonging to family Fabaceae, and Achyranthes aspera L. \nbelonging to the family Amaranthaceae were used as a nutrient source to develop crop Phaseolus aureus Roxb. \nWeed manures, Vermicompost and Compost, were prepared by using weeds T. hamiltonii Drumm and A. aspera \nL. in 1:1 proportion. Chemical analysis of weed and weed manures were done before administering it into the \nsoil. Neem cake was also used as one of the organic manures. In the experiment, a single dose of biofertilizers \nAzotobacter and Phosphate solubilizing bacteria were mixed with Weed Vermicompost, Weed Compost and \nNeem cake; and in one of the treatments, only biofertilizers were used indouble dose. Treatments were given \nto the crop as ATVB, ATCB, NCB, BioD, NPK, and Control in a randomized block design of experimental plot \nsize 1.5X 1.5 m. The use of chemical pesticides or fertilizers was completely avoided except for NPK treatment \nplots. Single plant analysis of pulse crop P. aureus Roxb. was done. Observations were recorded in the forms of \nfresh weight and dry weight of root, stem, leaves, leaf (4th number), and legumes. Total fresh yield (kg ha-1), \nDM (kg ha-1) increase over control, and Nitrogen efficiency ratio were recorded. Results showed that %DM (an \nincrease over control) and DM kg ha-1 recorded highest in ATVB treatment and the highest N efficiency ratio \nwas in BioD. The present investigation emphasized reducing the input cost of the farm products along with \nprotection of the environment and natural resources. \n\nKeywords: Biofertilizer, Neemcake, Organic agriculture, P. aureus, Weed compost, Weed Vermicompost. \n Corresponding author, email: bhalshankarchhaya@gmail.com \n\nIntroduction \nRecent agricultural trends are focused on both \n\nreducing the usage of inorganic fertilizers by using \n\norganic manure and applying biofertilizers such as \n\nvermicompost and phosphatic biofertilizers [1]. \n\nMicrobial activities play a key role in agriculture \n\nbecause they are significant in the movement and \n\navailability of minerals required for plant growth \n\nand ultimately lower the use of chemical fertilizers \n\n[2]. The maintenance of nutrients in the soil is most \n\nimportant for healthy plant growth [3]. Biofertilizers \n\nenhance soil health and crop yield. They improve \n\nfertility of soil, nutrient uptake, decomposition of \n\ncrop residue, and microbial diversity of soil. They \n\nalso reduce the requirement of chemical fertilizers \n\n[4]. The use of excessive chemical fertilizer, however, \n\ncauses hazardous effects on the soil, leading to \n\nserious problems; thus, biofertilizers are important \n\nalternative sources of nutrients. They are \n\nbiologically active microorganisms, like bacteria, \n\nalgae, fungi; they can provide nutrients to crops [5, \n\n6].", "start_char_idx": 52, "end_char_idx": 3687, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a4ce3f19-0aa9-4ecd-a9ce-3c3116102b3d": {"__data__": {"id_": "a4ce3f19-0aa9-4ecd-a9ce-3c3116102b3d", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "db33f70b-303c-499f-8253-3257e5e4240a", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "34396909862c52b10abf6e8b97aa57109bc8cbd3d786f4a04ddd3b7961d667fc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "462c9e37-4e38-4def-8576-fcf19d7437ba", "node_type": "1", "metadata": {}, "hash": "04048a6353840d696c59f880a69eb0ccfb30bdaa172c3a204a004d7a221b407b", "class_name": "RelatedNodeInfo"}}, "text": "Among Biofertilizers, beneficial bacteria are \n\nAzotobacter, Azospirillum, Rhizobium, symbiotic fungi \n\nMycorrhizae; they are essential in crop production. \n\nBiofertilizers improve plants\u2019 resistance to an \n\nunfavorable environment [7]. The biological manure \n\nhelps to increase crop yields, and also plays a vital \n\nrole in the nutrient accessibility in soil by improving \n\nthe physical, chemical, and biological structure of \n\nsoil, and it enhances the utilization of applied \n\nfertilizers [8]. In developing countries, residue \n\nmanagement is very important as the amount of \n\nnutrients in crop residue is several times higher than \n\nthe quantities of these nutrients applied as high cost \n\nfertilizer [9]. \n\nWeed plants compete with the agricultural crops; \n\nthey cause a tremendous reduction in crop yields \n\nand increase their production costs. Several \n\nscientists have estimated such losses in crop yields \n\nin different parts of India. A very broad-based \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-7518-5754\nmailto:bhalshankarchhaya@gmail.com\nmailto:bhalshankarchhaya@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8(2) [Special Issue]: 76-81    Bhalshankar  \n\n\u00a9NJB, BSN  77 \n\naverage of these estimates show that weeds reduced \n\nproductivity of wheat by 15-30%, rice by 30-35%, and \n\nmaize, sorghum, pulses and oilseeds by 18-85% \n\neach. Many cases of complete crop failure due to \n\nweeds particularly in upland rice and vegetable \n\ncrops were recorded [10, 11, 12]. T. hamiltonii Drumm. \n\nand A. aspera L. are the weed plants used in the \n\npresent study. The present study emphasized \n\nconversion and utilization of weeds beneficially by \n\nusing them for the preparation of compost and \n\nvermicompost. Neem cake is a residue left after the \n\nextraction of neem oil and used as an organic \n\nfertilizer. With the utilization of these organic \n\nmanuresalong with biofertilizers like Azotobacter \n\nand Phosphate solubilizing bacteria for the \n\ncultivation of Pulse crop P. aureus Roxb. belonging to \n\nthe family Fabaceae. \n\nWe can minimize the cost of production, increase \n\noutput per hectare by using organic manures like \n\ncompost, vermicompost prepared from weed \n\nbiomass, Neem cake, and biofertilizers for the \n\nproduction of crops and for sustainable agriculture. \n\nMaterial and Methods \nThe experiment was conducted during March 2008. \n\nA summer variety of P. aureus Roxb. was cultivated \n\nat college campus of New Arts, Commerce and \n\nScience College, Shevgaon District Ahmednagar, \n\n(Maharashtra), India. Shevgaon extends between \n\n19013 North latitude to 19035 North latitude and \n\nbetween 75001 East longitudes to 75037 East \n\nlongitude. \n\nWeed collection and preparation of \nmanures \nThe fresh vegetation of weeds i.e. Aghada (A. aspera \n\nL.) and Unhali (T. hamiltonii Drumm.) were collected \n\nfrom different localities and chopped into small \n\npieces (2-3 cm) by locally available iron cutter. Equal \n\namount (6944+6944 kg ha-1) 1:1 proportion mixture \n\nof weed pieces were used for the preparation of \n\ncompost and vermicompost. To prepare compost \n\nthis material was placed into pit (90x90x90 cm) and \n\nthen added cow dung, soil and weed plant material \n\nlayer by layer and sprinkled with water per \n\nrequirement. Finally, the compost pit was sealed \n\nwith dung-mud mixture to prevent loss of heat and \n\nmoisture. After partial decomposition first turning \n\nwas given after 15 days for homogeneous \n\ndecomposition, subsequent turnings were given \n\nafter every 15 days interval. Sufficient water was \n\nsprinkled to maintain moisture.", "start_char_idx": 3688, "end_char_idx": 7414, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "462c9e37-4e38-4def-8576-fcf19d7437ba": {"__data__": {"id_": "462c9e37-4e38-4def-8576-fcf19d7437ba", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a4ce3f19-0aa9-4ecd-a9ce-3c3116102b3d", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "7e7a41e600a73660924d75f8ad9abadae1f43f772fa4d51bf2b7eb79eeece6ea", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f6c24e6e-b912-4678-b2b4-9cb0eb0db5cc", "node_type": "1", "metadata": {}, "hash": "8326bfe670877d6d1cb4abcd51c99e61e1d346d7aaee28931049a6aa23814fc1", "class_name": "RelatedNodeInfo"}}, "text": "Sufficient water was \n\nsprinkled to maintain moisture. Finally, amorphous, \n\ndark brown, well fermented compost was obtained \n\nwithin 70 days.  Fresh weight of Compost obtained \n\nfrom pit was 33 kg. Same procedure was applied for \n\nvermicomposting, only with the addition of the \n\nworms in the pits after 15 days (Worms\u2019 variety \n\nEudriluseugeniae and Iceniafoetida).  Identification of \n\nearthworms was done by the method prescribed in \n\nFauna of India and Adjacent countries [13]. The \n\nprepared vermicompost was used for field trials. \n\nFresh weight of vermicompost obtained from pit \n\nwas 32 kg. The uniformly mixed samples (100 g) \n\nwere collected immediately from the pit for nutrient \n\nanalyses. Chemical analyses of weeds and weed \n\nmanures and Neem cake were done using oven \n\ndried and pulverized powder of samples. All the \n\nmanures compost, vermicompost and neemcake \n\n(1000 kg ha-1)were mixed with biofertilizer \n\nAzotobacter and phosphate solubilizing bacteria at \n\nthe rate 25 kg ha-1 (recommended dose); and only \n\nBiofertilizer double dose treatment 50 kg ha-1 in two \n\nsplit doses were applied to appropriate plots except \n\nchemical fertilizer (NPK) plots. The Mung (P. aureus \n\nRoxb.) Variety \u201cRaj Biotech\u201d Balwan R.J. Biotech, Pvt \n\nLtd. Siddharth Arcade, Station Road, Aurangabad \n\nwas sown in the research plots of size 1.5 x1.5 m. at \n\nthe rate of 20 kg ha-1. \n\nApplication of Inorganic Fertilizers \nThe inorganic fertilizers were supplied to the \n\nexperimental plots as Nitrogen (N), Phosphorus (P) \n\nand Potassium (K) through urea, single super \n\nphosphate (SSP) and muriate of potash at the rate of \n\n25 kg N, 50 kg P and \u20180\u2019 K kg ha-1 (25:50:0) only for \n\nfertilizer treatment plots. Entire amount of P2O5 and \n\nK2O and N was applied at the time of sowing. The \n\ncrop supplemented with irrigation during periods of \n\ngrowth and whenever necessary weeding was done. \n\nUse of insecticides and pesticides was completely \n\navoided. \n\nSeeds were planted in rows at a distance 30 cm x 10 \n\ncm. Soil was murum so the crop was grown under \n\nfrequent irrigation after each 8-10 days. Sample from \n\neach plot was brought into laboratory chopped into \n\n3-4 cm pieces. Measured amount of biomass was \n\nkept in digital electrical oven separately in pre-\n\nweighted tray at 95\u00b150C for 48 hours or more till \n\nconstant weight. Weight of dried samples were \n\nreported as DM. Results were used to calculate \n\n%DM, DM Kgha-1, increase over control and \n\nNitrogen efficiency ratio of crop.\n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8(2) [Special Issue]: 76-81    Bhalshankar  \n\n\u00a9NJB, BSN  78 \n\nResults \n\n \n\n \n\n \n\n  \n\nTable 1. Analyses of weeds administered in experimental plots through compost and vermicompost weed manures. \nHere,weeds used were A. aspera L. and T. hamiltonii Drumm.", "start_char_idx": 7360, "end_char_idx": 10150, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f6c24e6e-b912-4678-b2b4-9cb0eb0db5cc": {"__data__": {"id_": "f6c24e6e-b912-4678-b2b4-9cb0eb0db5cc", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "462c9e37-4e38-4def-8576-fcf19d7437ba", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "2f547e640c5dd5ca326a6518a22bb91ad316bd2b805c96a30a39b73c1a00643a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b327bfc7-c783-47f5-b61c-846f0ffca47c", "node_type": "1", "metadata": {}, "hash": "e6c680f23f48f6b5eaca0ddc17be41499f1cafde80a38b1d66b046f7d04d6ea8", "class_name": "RelatedNodeInfo"}}, "text": "Kg plot-1   (Plot size 1.5 m X 1.5m) \n\n  Fresh weight DM Nitrogen % \n\nC:N Weed Name \nKg \n\nplot-1 \nKg \nha-1 \n\n% \nDM Kg ha-1 % N Kg ha-1 Ash P K C \n\nAchyranthes 1.56 6944 19.29 1339.50 2.03 27.19 17.43 0.123 0.43 10.11 4.99 \n\nTephrosia 1.56 6944 22.40 1555.46 1.94 30.18 18.57 0.115 0.51 10.77 5.54 \n\nTable 2.Analyses of weed manure and Neem cake amendment along with biofertilizer. Here, ATVB=Achyranthes, \nTephrosia vermicompost mixed with Biofertilizer single dose, ATCB=Achyranthes, Tephrosia compost along with \nBiofertilizer single dose, NC=Neem cake along with Biofertilizer single dose \n\nTreat ments                 \n \n\nFresh            \n weight       \nKg plot-1 \n\nFresh \nweight \nKg ha-1 \n\nDM N % \n\n% kg hect-1 % N Kg hect-1 P  K Ca \n\nATVB                2.00 8889 67.21 5974.30 0.42 25+4.485 0.13 0.14        3.6 \nATCB                2.06 9169 65.07 5966.27 0.50 30+4.485 0.12 0.16        4.3 \n\nNCB                  0.23 1000 97.94 0979.40 1.96 19+4.485 0.81 0.48        0.9 \n\n(Amount of Nitrogen fixed by single dose of biofertilizer is 4.485 kg ha-1 as according to N balance method[14]). These \nvalues added in N kg ha-1 of other treatments and amount of N kg ha-1 fixed by Azotobacter biofertilizer double dose \nwas 8.97 kg ha-1). \n\nTable 3. C:N ratio of organic amendments. Here, ATV=Achyranthes, Tephrosia vermicompost, ATC=Achyranthes, \nTephrosia compost, NC=Neem cake    \n\nTreatments \n% \n\nC:N \nAsh C N \n\nATV 32.00 18.56 0.42 44.56 \n\nATC 36.50 21.17 0.50 42.36 \n\nNC 74.93 43.46 1.96 22.17 \n\nTable 4. Fresh wt and DM analyses of Single Plant of Phaseolus (at 56 DAS). Here, ATVB=Achyranthes, Tephrosia \nvermicompost mixed with Biofertilizer single dose, ATCB=Achyranthes, Tephrosia compost mixed with Biofertilizer \nsingle dose, NC=Neem cake along with Biofertilizer single dose. BioD=Biofertilizer double dose, NPK=Inorganic \nfertilizer, CON=Control.", "start_char_idx": 10151, "end_char_idx": 12015, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b327bfc7-c783-47f5-b61c-846f0ffca47c": {"__data__": {"id_": "b327bfc7-c783-47f5-b61c-846f0ffca47c", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f6c24e6e-b912-4678-b2b4-9cb0eb0db5cc", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "dd9c029219f5ab93de4f56135449fe61a3d14439ce90e38b25efeecd33819285", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1fa0c576-0a33-43fb-8137-9c8be8f249a7", "node_type": "1", "metadata": {}, "hash": "2bf3f74043d8cd4b0251ca6a6b79ceb514f113fea82c5f923063589b54cfcc07", "class_name": "RelatedNodeInfo"}}, "text": "(DAS=Days after sowing) \n\nTreatment Plant Fresh wt in gm   DM in gm     \n\n  Root Stem Leaves \n4th \nleaf \n\nTotal \nplant Legume Root Stem Leaves \n\n4th \nleaf \n\nTotal \nplant Legume \n\nATVB 0.49 4.89 11.29 3.59 22.55 5.83 0.21 1.44 2.99 0.82 7.99 2.71 \n\nATCB 0.48 3.93 09.79 2.57 18.53 4.21 0.19 1.17 2.77 0.60 6.47 3.03 \n\nBioD 0.60 5.29 12.87 3.92 24.37 5.44 0.24 1.40 3.25 0.90 8.30 3.49 \n\nNCB 0.39 2.75 06.79 1.66 12.71 2.65 0.13 0.74 1.80 0.43 4.20 1.67 \n\nNPK 0.51 3.70 08.38 2.15 15.09 2.63 0.20 1.08 2.21 0.53 4.76 1.60 \nCON 0.19 1.82 03.49 1.14 07.26 1.72 0.08 0.47 1.02 0.27 2.33 1.41 \n\nS.E 0.06 0.53 1.37 0.44 2.61 0.68 0.02 0.16 0.34 0.10 0.95 0.36 \n\nC.D. 0.12 1.13 2.92 0.94 5.56 1.45 0.05 0.33 0.73 0.21 2.02 0.76 \n \nTable 5.   Increase over control of total biomass and Nitrogen efficiency ratio (73DAS). It is the ratio of the crop Nitrogen \nuptake to the total input of Nitrogen fertilizer. \n\nTreatment \n\n  FRESH WT    %      DRY WT %  N Efficiency Ratio \n\nFW  \nKg ha-1 \n\nIncrease \nOver Con \n\nIncrease \nOver Con \n\n DM \n Kg ha-1 \n\nIncrease \nover con \n\nIncrease \nover con Input N   Fresh     Dry \n\nATVB 4685 1676 55.71 1660 695 72.02 29.485 56.85 23.57 \nATCB 4312 1303 43.31 1505 540 55.96 34.485 37.79 15.66 \nBioD 4136 1127 37.44 1409 444 46.01 8.970 125.60 49.50 \nNCB 3480 0471 15.64 1150 185 19.17 23.485 20.04 7.88 \n\nNPK 3380 0371 12.33 1066 101 10.47 25.000 14.84 4.04 \nCON 3009 0000 00.00 965 000 00.00 0.00 00.00 00.00 \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8(2) [Special Issue]: 76-81    Bhalshankar  \n\n\u00a9NJB, BSN  79 \n\nAnalyses of weeds were done on dry matter basis. \n\nObservations of weed analyses are recorded in the  \n\nTable 1. Fresh weightswere used, 1.56 kg plot -1 (6944 \n\nkg ha-1) of each weed, for preparations of manures. \n\n% DM of Tephrosia weed was higher (22.4), followed  \n\nby Achyranthes (19.29). The DM kg ha-1of Tephrosia \n\nweed was higher (1555.46) followed by Achyranthes \n\nweed (1339.50).", "start_char_idx": 12016, "end_char_idx": 13941, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1fa0c576-0a33-43fb-8137-9c8be8f249a7": {"__data__": {"id_": "1fa0c576-0a33-43fb-8137-9c8be8f249a7", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b327bfc7-c783-47f5-b61c-846f0ffca47c", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "ccc92c197349f9891a77a8f29d4c878ef8a232299c2d609329724701fb5834f1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d626b4fd-81cc-4241-a07e-975077feef7e", "node_type": "1", "metadata": {}, "hash": "755354cf8367695c35a2b533769205d8a956197df112619559471aa9abe8deb5", "class_name": "RelatedNodeInfo"}}, "text": "% N was higher in Achyranthes (2.03) \n\nfollowed by Tephrosia (1.94) (Tephrosia weed was \n\ncollected from comparatively non fertile land and \n\nAchyranthes from fertile land with ample domestic \n\nwaste nearby. So, Nitrogen percent of Tephrosia was \n\nless than Achyranthes though it is leguminous weed). \n\nN kg ha-1of Tephrosia weed was higher (30.18) it was \n\nfollowed by Achyranthes (27.19). % Ash of Tephrosia \n\nweed was higher (18.57); it was followed by \n\nAchyranthes (17.43). % Pwas higher in Achyranthes \n\n(0.123) followed by Tephrosia (0.115). % K was higher \n\nin Tephrosia (0.51) followed by Achyranthes (0.43). % \n\nCof Tephrosia weed was higher (10.77); it was \n\nfollowed by Achyranthes (10.11). C:N ratio of \n\nTephrosia weed was higher (5.54), and it was \n\nfollowed by Achyranthes (4.99). \n\nAnalyses of Achyranthes and Tephrosia weed manure \n\nand Neemcake were done; it is presented in Table 2. \n\nFresh weight of weed compost (ATC) was \n\nadministered at the rate of 2.06 kg plot-1 ( 9169 kg ha-\n\n1) and weed Vermicompost (ATV) was added at the \n\nrate of  2.00 kg plot-1 (8889 kg ha-1). Fresh weight of \n\nNeem cake (NC) was used at the rate of 0.23 kg plot-\n\n1 (1000 kg ha-1). All the manures treatment was \n\nmixed with single dose of biofertilizer i.e. 25 kg ha-1. \n\nDouble dose of biofertilizer 50kg ha-1was given to \n\nthe biofertilizer treatment (BioD). % DM of ATV (on \n\n211th day) was 67.21 %; it was followed by ATC (on \n\n211th day) was 65.07% and Neemcake 97.94%.  DM \n\nkg ha-1 was highest in Vermicompost (5974.30) \n\nfollowed by Compost (5966.27) and lowest in \n\nNeemcake (979.40). % N and N kg ha-1 was highest \n\nin ATCB (0.5%, 30 kg) followed by ATVB (0.42%, 25 \n\nkg) and NCB (1.96%, 19 kg). Single dose of \n\nbiofertilizer fixed 4.485  Nkg ha-1; so, input  of N was \n\n29.485 for ATVB, 34.485 for ATCB and 23.485 for \n\nNCB.  %Phosphorus recorded highest in Neem cake \n\n(0.81) and % Potassium in Neemcake (0.48); and %Ca \n\nwas highest in ATC (4.3). \n\nAmount of Nitrogen fixed by single dose \n\n(recommended dose) of biofertilizer was 4.485 kg ha-\n\n1 and amount of N kg ha-1 fixed by Azotobacter \n\nbiofertilizer double dose was 8.97 kg ha-1 in \n\naccording to N balance method [14]. As per Table 3, \n\n% ash % C and % N were highest in NC (74.93, 43.46 \n\nand 1.96, respectively) followed by ATC (36.5, 21.17, \n\n& 0.5) and lowest in ATV (32, 18.56 & 0.42). C:N ratio \n\nwas highest in ATV (44.56), followed by ATC (42.36) \n\nlowest in Neemcake (22.17).  \n\nIn Table 4, Analyses of Fresh weight and Dry weight \n\nof single plantare presented. Fresh weight of root \n\nwas highest in BioD (0.60) followed by NPK, ATVB, \n\nATCB, NCB and lowest in the CON (0.19), \n\nstatistically significant in all the treatments.", "start_char_idx": 13942, "end_char_idx": 16655, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d626b4fd-81cc-4241-a07e-975077feef7e": {"__data__": {"id_": "d626b4fd-81cc-4241-a07e-975077feef7e", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1fa0c576-0a33-43fb-8137-9c8be8f249a7", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "f0fbc72e6d3e7e96abc858d5289df90793897ee0cc9fcc512e7ec0e1ea7be115", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9d808ee1-7713-4318-ab6a-0cb797ceb637", "node_type": "1", "metadata": {}, "hash": "d90ce53c197c1e2c400cb9b2fcf7aeeb758820e3868966f9b53630abfd12a9fd", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nfresh weight of stem leaves and 4th leaf and total \n\nplant was highest in BioD followed by ATVB, ATCB, \n\nNPK, NCB and lowest in the CON, FW of stem and \n\ntotal plant statistically not significant in NCB. FW of \n\nlegume was highest in ATVB (5.83) followed by \n\nBioD, ATCB, NCB, NPK and lowest in CON (1.72), \n\nstatistically significant in all the treatments except in \n\nNCB and NPK. The DM of root was highest in BioD \n\n(0.24) followed by ATVB, NPK, ATCB, NCB and \n\nlowest in the CON (0.08), statistically significant in \n\nall the treatments. DM of stemwas highest in ATVB \n\n(1.44) followed by BioD, ATCB, NPK, NCB and \n\nlowest in CON (0.47), statistically significant in all \n\nthe treatments except in NCB. DM of leaves, 4th leaf \n\nand total plant was highest in BioD followed by \n\nATVB, ATCB, NPK, NCB and lowest in CON \n\ntreatments, statistically not significant in NCB for 4th \n\nleaf and total plant. DM of legume was highest in \n\nBioD (3.49) followed by ATCB, ATVB, NCB, NPK \n\nand lowest in CON (1.41), statistically not significant \n\nin NCB and NPK treatments.  \n\nIn Table 5, Percent increase over control and \n\nnitrogen efficiency ratio is presented. The percent \n\nincrease over control in Phaseolus for fresh weight \n\nwas found highest in ATVB (55.71) followed by \n\nATCB (43.31), BioD (37.44), NCB (15.64), and \n\nminimum in NPK (12.33). Similarly, dry matter \n\npercentage (DM%) was found maximum with the \n\ntreatment ATVB (72.02) followed by ATCB (55.96), \n\nBioD (46.01), NCB (19.17) and minimum in NPK \n\n(10.47).DM kg ha-1 recorded highest in ATVB (1660) \n\nfollowed by ATCB, BioD, NCB, NPK and lowest in \n\nCON (965), statistically significant in ATVB, ATCB, \n\nBioD, but statistically not significant in NPK and \n\nNCB. The nitrogen efficiency ratio for fresh weight \n\nwas found highest in BioD (125.60) followed by \n\nATVB (56.85), ATCB (37.79), NCB (20.04) and lowest \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8(2) [Special Issue]: 76-81    Bhalshankar  \n\n\u00a9NJB, BSN  80 \n\nin NPK (14.84). Similarly, the nitrogen efficiency \n\nratio for Dry matter (DM) was found highest in BioD \n\n(49.50) followed by ATVB (23.57), ATCB (15.66), \n\nNCB (7.88) and lowest in NPK (4.04). Highest Fresh \n\nweight and DM kg ha-1 was recorded in Treatment \n\nATVB. \n\nDiscussion \nAzotobacter treated seedlings of knolkhol showed the \n\nhighest whole plant weight [15]. Biofertilizers such \n\nas Azotobacter, Azospirillum, PSB, and a mixture of \n\nAza + Azo + PSB were administered to crops which \n\nshowed the increased plant fresh weight, dry weight \n\n[16]. Similar results showing fresh weight and dry \n\nweight of BioD treatment was recorded highest at 56 \n\nDAS. Combined inoculation of soybean by \n\nsymbiotic bacteria improved the dry weight of \n\nsoybean [17]. Vermicompost and phosphate \n\nbiofertilizer showed improved growth and yield in \n\nAnise (Pimpinella anisum L) [1]. Vermicompost and \n\nPSB when applied together was found helpful in \n\ndeveloping production and yield in anise [18]. \n\nAzotobacter increases the production of agriculture \n\ncrop plants by 10-12%. Azotobacter can also improve \n\ngrowth and grain yield in wheat crops.", "start_char_idx": 16657, "end_char_idx": 19778, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9d808ee1-7713-4318-ab6a-0cb797ceb637": {"__data__": {"id_": "9d808ee1-7713-4318-ab6a-0cb797ceb637", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d626b4fd-81cc-4241-a07e-975077feef7e", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "c3b47822a55c67578fd7fe51198ed0e3f2683d10a02c775b1888130334082e49", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "66817ab3-e2ce-474a-9bdc-2d67dc2d2d10", "node_type": "1", "metadata": {}, "hash": "1e7b18718462438f56166778ef0c8801764efca092ce910994c529cd2cbe4633", "class_name": "RelatedNodeInfo"}}, "text": "Azotobacter can also improve \n\ngrowth and grain yield in wheat crops. Azotobacter \n\nact as one of the vital biofertilizers in the case of rice \n\nand some cereals could be applied by seed dipping \n\nand seedling root dipping methods [19]. Maize \n\nhybrid seed priming with Azotobacter showed the \n\nhighest grain yield (7.01 ton/ha) and DM \n\naccumulation (2019 gr /m2) in treatment compound \n\nSC-434 [20]. Panchgavyawas found to contribute to \n\nbetter growth and yield of Pisum sativum as \n\ncompared to NPK [21]. \n\nIn biochemical analyses of the total biomass of plant, \n\nNitrogen, and total crude protein was recorded \n\nhighest in ATVB [22]. The findings of the present \n\nexperiment showed that Fresh weight and Dry \n\nweight was recorded highest in Biofertilizer double \n\ndose at 56 DAS. But at harvesting 73 DAS maximum \n\nFresh and Dry Yield was recorded highest in Weed \n\nvermicompost + Biofertilizer Azotobacter and \n\nPhosphate solubilizing bacteria treatment (ATVB). \n\nConclusion \nThe results of this investigation concluded that weed \n\nvermicompost, weed compost along with a single \n\ndose of biofertilizer and biofertilizer double dose can \n\neffectively be used as a nutrient source to increase \n\ncrop yield and soil fertility. Weed manures and \n\nNeem Cake with biofertilizers worked more \n\nefficiently as compared to the chemical fertilizers \n\n(NPK) to improving the quality of the crop; it could \n\nreduce the input cost of the farm produce as well in \n\naddition to protecting the environment and natural \n\nresources.  \n\nAuthor\u2019s Contribution \nCB is responsible for all the data collection, \n\nconceptualization, writing \u2013 original draft \n\npreparation, review &editing the final draft of the \n\nmanuscript. CB read and approved the final \n\nmanuscript. \n\nCompeting Interests \nNo competing interests \n\nFunding \nThe author(s) declared that no grants supported this \n\nwork. \n\nAcknowledgments \nI express my deep sense of gratitude to my respected \n\nteacher and research guide Late Capt. Dr. Bharati \n\nJadhav (retired professor), Department of Botany; \n\nDr. Babasaheb Ambedkar Marathwada University, \n\nAurangabad. I express my deep sense of gratitude to \n\nmy respected institute Ahmednagar Jilha Maratha \n\nVidya Prasarak Samaj\u2019s Management for their \n\ncontinuous encouragement. \n\nEthical Approval and Consent \nNot applicable. \n\nReferences: \n1.  Darzi MT, Hadi MH, Rejali F. Effects of vermicompost and \n\nphosphate biofertilizer application on yield and yield \ncomponents in Anise (Pimpinella anisum L.). Iranian Journal of \nMedicinal and Aromatic Plants. 2011;26(4). \n\n2.  Verma S, Singh A, Pradhan SS, Singh RK, Singh JP. Bio-\nefficacy of organic formulations on crop production-A review. \nInternational Journal of Current Microbiology and Applied \nSciences. 2017;6(5):648-65. https:// doi.org/ 10.20546/ ijcmas. \n2017.605.075 \n\n3.  Devi V, Sumathy VJ. Production of biofertilizer from fruit \nwaste. European journal of pharmaceutical and medical \nresearch. 2017;4(9):436-43. \n\n4.  Singh M, Dotaniya ML, Mishra A, Dotaniya CK, Regar KL, \nLata M. Role of biofertilizers in conservation agriculture. \nInConservation Agriculture 2016 (pp. 113-134). Springer, \nSingapore.https://doi.org/10.1007/978-981-10-2558-7_4 \n\n5.  Javorekov\u00e1 S, Makov\u00e1 J, Medo J, Kov\u00e1csov\u00e1 S, Charousov\u00e1 I, \nHor\u00e1k J. Effect of bio-fertilizers application on microbial \ndiversity and physiological profiling of microorganisms in \narable soil. Eurasian Journal of Soil Science. 2015;4(1):54. \nhttps://doi.org/10.18393/ejss.07093 \n\n6.", "start_char_idx": 19709, "end_char_idx": 23206, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "66817ab3-e2ce-474a-9bdc-2d67dc2d2d10": {"__data__": {"id_": "66817ab3-e2ce-474a-9bdc-2d67dc2d2d10", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9d808ee1-7713-4318-ab6a-0cb797ceb637", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "248b03db50694f5e0712495f46d8826ed2f65930d67a7f62d79374f289393881", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "08a6e170-6f04-4a2e-9cf3-4521d7ef9d5f", "node_type": "1", "metadata": {}, "hash": "0ca44d82194d2b4170ae3a4acf9b93387cdc186fb9691b968cf98185d32a9c7d", "class_name": "RelatedNodeInfo"}}, "text": "Rao DL, Balachandar D, Thakuria D. Soil biotechnology and \nsustainable agricultural intensification. Indian Journal of \nFertilisers. 2015 Oct;11:87-105. \n\n\n\nNepal J Biotechnol. 2 0 2 0  O c t ; 8(2) [Special Issue]: 76-81    Bhalshankar  \n\n\u00a9NJB, BSN  81 \n\n7.  Sneha S, Anitha B, Sahair RA, Raghu N, Gopenath TS, \nChandrashekrappa GK, Basalingappa KM. Biofertilizer for \ncrop production and soil fertility. Acad J Agric Res. \n2018;6(8):299-306.  \n\n8.  Mercy S, Mubsira BS, Jenifer I. Application of different fruit \npeels formulations as a natural fertilizer for plant growth. Int \nJ SciTechnol Res. 2014 Jan 25;3(1):300-7. \n\n9.  Singh H. Effect of crop residue management on microbial \nbiomass accumulation in the soil. Current science (Bangalore). \n1993;65(6):487-8. \n\n10. Mukhopadhyay SK. Emerging problems and advances in \nweed management. InPresidential Address, Agriculture \nSection, Indian Science Congress held at Baroda 1992. \n\n11. Jain NK, Choubey SD. Weed control in Soyabean. Ind. J. Weed \nSci. 1969;1(1):5-9. \n\n12. Friesen GH, Korwer GR. Weed management for dry land \ncrops. Weed Res. 1983;23:365-71. https://doi.org/10.1111 \n/j.1365-3180.1983.tb00560.x \n\n13. Julka JM, India. ZS of.The fauna of India and the adjacent \ncountries. Calcutta: Zoological Survey of India; 1988. \n\n14. Peoples MB, Herridge DF. Nitrogen fixation by legumes in \ntropical and subtropical agriculture. InAdvances in agronomy \n1990 Jan 1 (Vol. 44, pp. 155-223). Academic Press. \nhttps://doi.org/10.1016/S0065-2113(08)60822-6 \n\n15.  Sharma JP, Rattan P, Kumar S. Response of vegetable crops to \nuse of integrated nutrient management practices. ISABB \nJournal of Food and Agricultural Sciences. 2012 Jan 31;2(1):15-\n9. \n\n16.  Patel HD, Krishnamurthy R, Azeez MA. Effect of biofertilizer \non growth, yield and bioactive component of Plumbago \nzeylanica (Lead Wort). Journal of Agricultural Science. \n2016;8(5). https://doi.org/10.5539/jas.v8n5p141 \n\n17. Rosas SB, Rovera M, Andres JA, Correa NS. Effect of \nphosphorous solubilizing bacteria on the rhizobia-legume \nsimbiosis. InFirst International Meeting on Microbial \nPhosphate Solubilization 2007 (pp. 125-128). Springer, \nDordrecht. https://doi.org/10.1007/978-1-4020-5765-6_17 \n\n18. Darzi MT, Seyedhadi MH, Rejali F. Effects of the application \nof vermicompost and phosphate solubilizing bacterium on \nthe morphological traits and seed yield of anise (Pimpinella \nanisumL.). Journal of Medicinal Plants Research. 2012 Jan \n31;6(2):215-9. https://doi.org/10.5897/JMPR11.949 \n\n19. Shridhar BS. Review: Nitrogen fixing microorganisms. Int J \nMicrobiol Res. 2012;3(1):46-52. \n\n20.", "start_char_idx": 23208, "end_char_idx": 25822, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "08a6e170-6f04-4a2e-9cf3-4521d7ef9d5f": {"__data__": {"id_": "08a6e170-6f04-4a2e-9cf3-4521d7ef9d5f", "embedding": null, "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-171", "node_type": "4", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "4ecf17405dc1b39deaf3ccf8222258db6735ec027ecacf14caa89849493b1989", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "66817ab3-e2ce-474a-9bdc-2d67dc2d2d10", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "3a9a74e4250ce010bcb703dac196512bb0a582b21a3b2aaec89de591226fa9b5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b4876ed0-e5bc-476e-b785-3b88fc3f1eca", "node_type": "1", "metadata": {}, "hash": "c589c723d1ae846a20d2e4c7fade9c5a9785e5f12ae9ef6dfbb20987a4b6cd03", "class_name": "RelatedNodeInfo"}}, "text": "2012;3(1):46-52. \n\n20. Sharifi RS, Khavazi K. Effects of seed priming with Plant \nGrowth Promoting Rhizobacteria (PGPR) on yield and yield \nattributes of maize (Zea mays L.) hybrids. Journal of Food, \nAgriculture & Environment. 2011;9(3/4 part 1):496-500. \n\n21. Bhusare BP, Kulkarni AA (2017) Studies on Comparative \nEffect of NPK and Panchagavya on Growth and Yield of \nPisumsativum L. Flora and Fauna 23:341\u2013344. \n\n22.  Bhalshankar CK, Ahire MD. Integrated Nutrient Management \nof Phaseolus Aureus Roxb. by Using Weed Manures and \nBiofertilizers [Internet]. Scholar World-International Refereed \nMultidisciplinary Journal of Contemporary Research; 2016 \n[cited 2020 Jan 2]. Available from: http://irmjcr. \nscholarsworld.net/index.html#Archive?archiveId=62317036\n09671680", "start_char_idx": 25800, "end_char_idx": 26572, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b4876ed0-e5bc-476e-b785-3b88fc3f1eca": {"__data__": {"id_": "b4876ed0-e5bc-476e-b785-3b88fc3f1eca", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "08a6e170-6f04-4a2e-9cf3-4521d7ef9d5f", "node_type": "1", "metadata": {"identifier": "njb-171", "author": "Bhalshakar, Chhaya", "title": "Study of the Impact of Organic Manures and Biofertilizers on growth of Phaseolus aureus Roxb.", "date": "2020-10-22", "file": "njb-171.pdf"}, "hash": "dfd684a5eaf8f4081ca18f07f4b802737456a28fac328113666237c97410d0f8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e4a1547d-3963-498d-85a5-f14a8a4a5f10", "node_type": "1", "metadata": {}, "hash": "d8f752f93632b3b28faa47b6660018c572b2d67869e97f47b72642d6bfabfd74", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68 DOI: https://doi.org/10.3126/njb.v8i1.30209 \n  Review article \n\n\u00a9NJB, BSN  54 \n\n \nRegeneration Technique of Bamboo Species through Nodal \nSegments: A Review \nMeena Maiya Suwal, Janardan Lamichhane, Dhurva Prasad Gauchan     \n\nDepartment of Biotechnology, School of Science, Kathmandu University, Dhulikhel, Kavre, Nepal. \n\nArticle history:- Received: 23 Nov 2018; Revised: 7 Jul 2020; Accepted: 21 Jul 2020; Published online: 31 Jul 2020 \n\nAbstract \nMicropropagation is an alternative technique to propagate at large scale plants to meet global plant \ndemand. Various researchers have worked on the micropropagation technique to regenerate bamboo \nspecies by using nodal segments from years. Contamination, browning, necrosis, and acclimatization \nwith physiological stress are the extreme problems of the micropropagation technique. But, many \nnumbers of papers have been published on micropropagation of the bamboo species through nodal \nsegments as explants. The proliferation of the bamboo shoots is dependent on the season of collection, \nsize of explants, the position of explants, diversity of plants, concentration and combination of plant \ngrowth regulators, most adequate culture medium, environmental condition of the equipment, \nhandling, and individual species. Bamboo is a monocarpic fast-growing, tall perennial grass and having \nthe high potential to generate economic and social benefits. It helps to maintain land patterns and \ncontrol soil erosion. The long life cycle of the bamboo produces a huge amount of seeds but \nunfortunately, mostly, they are non-viable. So, bamboos are propagated from vegetative by cutting and \nair layering. However, these methods are only for a small scale and they also tend to destroy large \nmother plant stocks and difficult to be transported. So, the in vitro propagation technique is useful to \n\nobtain large progenies from desired genotypes. Mostly, BAP and TDZ growth hormones are widely \nused for shoot multiplication and IBA, NAA and IAA are used for root initiation as per developed \nprotocols in tissue culture for large scale production. This review intends to explore an overview of the \nrecent literature reports to summarize the importance of micropropagation by using nodal segments of \nbamboo species and factors influencing it. \n\nKeywords: Micropropagation, Nodal segments, Bamboo species, Plant Growth Regulators, Shooting \nand Rooting \n\n     Corresponding author, email: gauchan@ku.edu.np \n\nIntroduction \nBamboo is an attractive plant species for various \n\npurposes due to its versatile utilization with high \n\neconomic potentiality and its accelerated growing \n\ncapacity at short period [1, 2]. It is perennial strong \n\nwoody giant grass [1-5] having a unique complex \n\nbranching system of rhizomes with root. It belongs \n\nto subfamily Bambusoideae of the Poaceae family \n\nand subsequently divided into three tribes [3, 4, 5, 6]. \n\nIt can grow from approximately 75 to 400mm per \n\nday [7]. It is distributed up to 4000 m. a. s.l. in \n\ntemperate to the zone of all continents except Europe \n\n[8]. It prefers to grow at a minimum of 100 cm annual \n\nrainfall with high atmospheric humidity in steep \n\nhillsides, road embankments, gullies, or on the \n\nbanks of ponds and streams [9]. It occupies 3% of the \n\nforest in the tropical, subtropical, and temperate \n\nzone of the world but while, in Asia; it covers 10 % \n\nof the forest. There are 120 genera and 1641 species \n\nfound in the world [10, 11 and 12]. Tropical Asia has \n\nrich bamboo diversity with up to 60 genera and 1000 \n\nspecies [13]. However, Nepal has only 12 genera and \n\n65 species of bamboos distributed in the tropical, \n\nsubtropical, and temperate zone [14].", "start_char_idx": 48, "end_char_idx": 3789, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e4a1547d-3963-498d-85a5-f14a8a4a5f10": {"__data__": {"id_": "e4a1547d-3963-498d-85a5-f14a8a4a5f10", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b4876ed0-e5bc-476e-b785-3b88fc3f1eca", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "9e95b1c11239939221e893605b6eeb08c21d5b13bc44cccc6fbd8b1b6aeab6a4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d02a859e-a1c8-4013-831f-5ab099cafe42", "node_type": "1", "metadata": {}, "hash": "78725531ab872ec2e1d604727869cec11650f371a645fc107fe1ab759dff7ffb", "class_name": "RelatedNodeInfo"}}, "text": "Since the ancient period, bamboo species are widely \n\nused as a renewable source due to its versatile nature \n\n[15, 16]. According to Hsiung (1988), more than 4000 \n\ntraditional uses and 1500 commercial applications \n\nhave been known to bamboo. It\u2019s multipurpose \n\npotentiality from cradles to coffin and as \u201cGreen \n\nGold\u201d [17]. Similarly, it is used for making paper, \n\npulp, food, scaffolding, textiles, plywood, boards, \n\nraw materials for construction, fencing, clothes, \n\nreinforcing fibers,  etc, and bio-energy applications \n\n[10]. Also, it is used as an alternative source of \n\nenergy and helped to prevent soil erosion due to the \n\nclosely woven mat of intertwining roots and \n\nrhizomes [17, 18] with its high agro-climatic \n\nsuitability. \n\nmailto:gauchan@ku.edu.np\nhttp://orcid.org/0000-0002-3728-5076\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  55 \n\nIt is perennial plants with monocarpic nature having \n\nvegetative and reproductive cycle range from 3 to \n\n120 years [19], sporadic flowering with the \n\nrecalcitrance of seeds which made difficult for \n\nidentification and floral characterization of bamboo. \n\nMostly, the classification and identification of \n\nbamboo is done based on the vegetative pattern but \n\nit is not reliable due to influence of ecological factors \n\nan easily practicable because bamboo can produce a \n\nlimited number of seeds after a long period of its life \n\nspan which has short viability period only 3 to 6 \n\nmonths [17, 18, 19] and suffering from insects and \n\nrodents. Hence, the bamboo is regenerated through \n\nthe vegetative method by planting of rhizome off-\n\nset, branch & culm cuttings, nodal macro-cutting \n\nand layering [20, 21] but for this process, it needs a \n\nlarge mass of bamboo plant stocks. This leads to the \n\ndestruction of entire clumps and the gradual \n\ndepletion of bamboo resources. Again, it is not easily \n\navailable sufficiently due to seasonal dependency \n\nand low rooting capacity so it is highly expensive \n\n[17]. Similarly, it is difficult to transport and handle \ndue to heavyweight and long length [22, 23]. Also, \n\nthere is more chance of liable desiccation before \n\nrooting [24].  Moreover, yield production of bamboo \n\nis hardly possible [22-27] through conventional and \n\nnon-conventional methods. Therefore, it is necessary \n\nto apply other techniques of plant tissue culture \n\nbecause it is highly demanded the industry to \n\nagriculture. \n\nIn Vitro Propagation of Bamboo \nPlant tissue culture is the only advanced technique \n\nthat can be applied to solve the challenges of speedy \n\nand mass propagation of the bamboo species [27]. \n\nFor the conservation of bamboo and to fulfill the \n\ngrowing demand of the markets, micropropagation \n\nis the alternative method that provides rapid mass \n\nmultiplication of bamboo along with disease-free \n\nplants as well as the same clone [25-32]. \n\nMicropropagation is not only ensuring the supply of \n\nquality planting material regularly but it also helps \n\nin conserving of germplasm of bamboo [25]. \n\nDifferent types of explants viz. seed, seedlings, \n\ninflorescences, root, culm, mature clumps, nodal \n\nsegment, meristem domes or leaves, etc [33, 34, 35] \n\nare used for bamboo micropropagation. In bamboo, \n\nboth juvenile and mature plants can be considered \n\nas explants [36]. The nodal segments containing is \n\nconsidered as more effective explants for in vitro \n\nculture because of resumed food materials. Due to \n\nthe presence of highly active meristematic tissue in \n\nnodal segments, it develops into new plantlets [37]. \n\nThe response of explants depends on the physical \n\ncondition of plants, the health of mother plants, \n\ncollection of the season from field and size of \n\nexplants, and its position in mother plants [38]. \n\nUsing nodal explants for organogenesis reduces the \n\nchance of somaclonal variation [27].", "start_char_idx": 3793, "end_char_idx": 7673, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d02a859e-a1c8-4013-831f-5ab099cafe42": {"__data__": {"id_": "d02a859e-a1c8-4013-831f-5ab099cafe42", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e4a1547d-3963-498d-85a5-f14a8a4a5f10", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "6b95137e4fad6000fb6d05638d7a498408f6f5285b7c232f73045a3d105804e1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f2ba13b7-9943-45b2-8bc1-5cebbc590908", "node_type": "1", "metadata": {}, "hash": "fca0c6229b8a1109a1ff98cd6dadf4bc3c57a26bb9b42ea54ae8252e6ce8a34e", "class_name": "RelatedNodeInfo"}}, "text": "There is so much research conducted on \n\nmicropropagation of bamboo through nodal \n\nexplants [27-47] However, the developed protocols \n\nare either insufficient or not applicable because the \n\nprotocols are only limited to the research which is \n\nnot applicable for industrial mass production. It is \n\nintended to explore the suitable protocols on the \n\nmicropropagation technique for mass bamboo \n\npropagation. This review intense to give an \n\noverview of the recent literature reports to \n\nsummarizes the importance of micropropagation by \n\nusing nodal segments of bamboo species and factors \n\nthat influence it. \n\nCollection of explants \nNodal explants were collected from January to \n\nFebruary, March to April, May to June, July to \n\nAugust), September to October, and November to \n\nDecember [32, 41, 48]. The establishment of explants \n\nin culture was directly related to the collection of \n\nexplants seasons and plants species which \n\ninfluenced in pure culture. It was dependent on \n\nexternal factors i.e. contamination and concentration \n\nof the hormones in media [49]. Explants collected \n\nduring February-March and September-October \n\nshowed maximum bud break [48]. It was reported \n\nthat the rainy season was better for bud break in \n\nDendrocalamus strictus [49] Bambusa tulda [40] \n\nGigantochloa atroviolacea [50] with the high rate of \n\ncontamination [40, 51,  52]. The explants collected \n\nfrom November to January (winter months) \n\nproduced only 35-45% bud break response during \n\nthe culture of Bambusa vulagaris by [53]. Spring and \n\nsummer season (February to June) was a \n\ncomparatively better period for the collection of \n\nexplants [37]. However, Negi and Saxena [29, 30] \n\nhave obtained high aseptic cultures along with a 90% \n\nbud response from July to October in Bambusa \n\nbalcooa. According to Negi and Saxena (2011) and \n\nMehta et al. (2011), the best collection of explants \n\nwas in July for culture initiation in Bambusa nutans. \n\nBut Singh et al. (2012b) reported the collection of \n\nnodal explants at pre-monsoon induced maximum \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  56 \n\nbud break in Dendrocalamus asper. Ramanayake et al. \n\n(1995) observe that the influence of seasons on bud \n\nbreak was countable in D. giganteus and Berberis \n\nvulgaris. It was observed that seasonal effects on bud \n\ninitiation and found that February to March is a \n\ngood period for obtaining auxiliary buds for cultural \n\ndevelopment [54]. Moreover, Shivabalan et al. (2014) \n\nhave established a new culture form explants of B. \n\nbalcooa collected in December. In other seasons, the \n\nestablishment of pure culture is difficult due to the \n\nhigh scale of contamination suffering from the \n\nvariable pathogen in the summer and rainy season. \n\nBetter establishment of pure aseptic cultures and \n\nbud response of explants depends on the ratio of \n\ncontamination, handling, physiological state of \n\nexplants, species, type of explants, and the season of \n\ncollection [24, 43, 52, 55, 56, 57, 58, 59]. \n\nDifferent aged of the mother stock plants were also \n\nused for culture initiation. The new Culm (1 year) \n\nand lateral branched (frequently actively branched) \n\nof 2 to 5 years old bamboo can be used as explants \n\n[25, 43]. But Patel et al. (2015) have established in \n\nvitro culture from 2 to 30 years old explants and also \n\nSharma et al. (2012) used nodal explants from the \n\ncurrent year\u2019s growth mature and healthy clumps of \n\nBambusa nutans.", "start_char_idx": 7676, "end_char_idx": 11176, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f2ba13b7-9943-45b2-8bc1-5cebbc590908": {"__data__": {"id_": "f2ba13b7-9943-45b2-8bc1-5cebbc590908", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d02a859e-a1c8-4013-831f-5ab099cafe42", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "76707eda58b6871687336615f21156d9940faa0c192b450987c1c1bc488d599c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a6b11238-22ff-41fc-9ca9-674244e7e65d", "node_type": "1", "metadata": {}, "hash": "770999499d5b11c7d58e16d7ffa813f5849580a90b7d3c2836fd582846cebb88", "class_name": "RelatedNodeInfo"}}, "text": "Similarly, nodal explants of 40 years \n\nold B. nutans; 30 years old B. balcooa [60] and 10 years \n\nold B. tulda were used for multi proposed [59, 61]. \n\nThe aged of the explants could not effectually \n\ndetermine in the initiation of the shoot in vitro. \n\nFurthermore, there may be some result influenced \n\nby the age of the plants during the experiment but \n\nthere was no impact reason behind it. It depended \n\non the composition of media, concentration of \n\nhormones, contamination rate, and condition of the \n\nculture [31]. Also, various aged of the explants can \n\nbe established successfully in vitro culture through \n\nsingle nodal segments from bamboos. \n\nPosition of the Nodal Explants \nTo date, only limited researchers have mentioned \n\nthe position of nodal explants in mother plants. \n\nNodal segments from the healthy mature mother \n\nplant with disinfected lateral branches and were \n\nmore effective for the initiation of the culture [62]. \n\nChowdhury et al. (2004) recorded that the 1st and 2nd \n\nposition from the base of secondary branches of D. \n\nstrictus was the best for regeneration in \n\nmicropropagation. But, according to Mudoi et al. \n\n(2008, 2014), the 5th to 7th position of the B. balcooa, B. \n\nnutans and B.tulda explants from mother stock culm \n\nwas best for maximum regeneration in vitro culture \n\nrather than below 5th position because the base \n\nexplants can excaudate phenolic compound which \n\nresulted in browning problem on shoots. A similar \n\nresult was also illustrated in the report of Devi and \n\nSharma (2009) in which the top position of explants \n\nshowed a low frequency of bud break in comparison \n\nto the basal and mid Culm nodes in Arrundinaria \n\ncallosa Munro. Middle node explants of Culm were \n\nvery effective resulted in vitro propagation of the B. \n\nvulgaris, [63]. Another experiment revealed that the \n\nauxiliary branch of explants from healthy mother \n\nstock was found to be good for regeneration of the \n\nnew plants such as in D. hamiltonii [1, 64], D. asper \n\n[65], D. giganteus [56]. Similarly, Sharma and Sarma \n\n(2013) reported that young lateral buds also showed \n\nthe bud break in B. tulda. Therefore, it is stated that \n\nthe top and the base portion of the nodal segment in \n\nCulm bamboo can hardly regenerate in vitro \n\npropagation of bamboo. \n\nSurface Sterilization \nThe size of 2.5 mm explants was more effective than \n\na smaller size (5-7 mm) to initiate the culture within \n\na short period because of high endogenous \n\nhormonal effect [37, 41]. For the initiation of the \n\nculture, the explants were surface sterilized by \n\ntreating different kinds of chemicals for a certain \n\ntime to avoid the contamination. It was reported that \n\nexplants treated in 70% ethanol for the 30s to 1 min \n\n[25, 29, 30, 31, 33 39] followed running tap water \n\nafter washing in 4-6 drops of detergent (Tween \n\n20/Tween 80) for 30 mins. [29, 30, 33, 68] reduced the \n\nrate of contamination. It was reported that 0.1% \n\nMercuric chloride (HgCl2) was found more effective \n\nthan other surface sterilants (Sodium hypochlorite, \n\nPotassium Hypochlorite, Hydrogen Peroxide, etc.) \n\nfor various species of bamboo micropropagation [29-\n\n,31, 33, 35, 36, 38, 39, 65- 71]. So it is suggested that \n\n0.1% Mercuric Chloride was more effective for the \n\ndisinfection of the explants because the high \n\nconcentration of the HgCl2 retarded the growth of \n\nplants due to the impact of chemical in the internal \n\ntissues [72]. Wei et al.", "start_char_idx": 11177, "end_char_idx": 14637, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a6b11238-22ff-41fc-9ca9-674244e7e65d": {"__data__": {"id_": "a6b11238-22ff-41fc-9ca9-674244e7e65d", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f2ba13b7-9943-45b2-8bc1-5cebbc590908", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "580c497f6e5c4648610ff6edf97c77c8e65a38d26c9421e037cb319efae6a8e3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "50fddaa6-8e6a-43bd-8e9e-1016dbe29d72", "node_type": "1", "metadata": {}, "hash": "1edb81aebe644155beaeea017dbf94cf1ac13a04925d7a108f81883f5b932221", "class_name": "RelatedNodeInfo"}}, "text": "Wei et al. (2015) have reported that the \n\ntreatment of explants on 0.1% HgCl2 at lower \n\nduration enhanced the survival rate of explants and \n\nfrequency of bud break. When treatment time was \n\nincreased at the same concentration of HgCl2 in D. \n\nstrictus, the bacterial and fungal contamination was \n\ndecreased [36]. Similarly, for the establishment of a \n\npure culture of bamboo, different researchers have \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  57 \n\nused antiseptics like savlon, tempol, streptomycin \n\nsulphate, gentamicin, cetavelon, etc [29, 40, 42, 60] \n\nand fungicide like bavistin, benomyl, mancozeb, \n\ncarbendazim, tetracycline, etc [37, 41, 42. 49, 53, 56].   \n\nCulture Media \nAccording to Chang and Ho (1997), the nutritional \n\ncomposition is greatly varied in culture media which \n\ndepends on the types of tissues and plant species. So, \n\nfor the establishment of the culture, the proper \n\nmedia play a major role in the growth and \n\ndevelopment of the plants. It is difficult to consider \n\na unique media for all types of plants in tissue \n\nculture.  Not only for getting maximum auxiliary \n\nbud breaking, but MS [78] medium has also been \n\nwidely practiced for more superior responsive bud \n\nproliferation and further multiplication of bamboo \n\nin comparison to other media such as SH (Schenk \n\nand Hildebrandt 1972), B5 (Gamborg et al. 1968) and \n\nNN (Nitsch and Nitsch 1969), WP Medium (Lloyd \n\nand Crown 1980) [34-122]. But Kabade (2009) \n\nobserved that the WPM media is suitable for shoot \n\ninduction from nodal segments of B. bambos.  \n\nSimilarly, Shirgurkar et al. (1996), Singh et al. (2001), \n\nOgita et al. (2009), and Negi and Saxena (2011) \n\nreported that the half-strength rather than full \n\nstrength MS medium was better for successful in \n\nvitro culture in bamboo [123-124].  \n\nThe physical condition of the media is also a factor \n\nthat influences to grow plant tissue under in vitro \n\nculture. Several researchers reported that the \n\nproliferation and shoot multiplication of the bamboo \n\nwas successfully obtained under in vitro culture on \n\nsemi-solid/solid MS media [34, 35, 36, 39, 40-48]. \n\nMostly, 0.8% agar was widely used as a gelling agent \n\nto solidified/semi-solidified the media which \n\ninfluenced the plant metabolism [32-39, 76]. Some \n\nresearchers also used phytagel (0.2%) or Gelrite (0.2-\n\n0.35%) to agar which influenced high bud breaking \n\nin B. wamin [72] and shoot proliferation in B. oldhamii \n\n[77]. Similarly, it was noted that dwarf and a lower \n\nnumber of shoots per explant in MS solid media due \n\nto leaching and browning problems [79]. Similarly, \n\nSharma and Sarma (1998) have also observed \n\nleaching of phenolic exudates and poor growth of \n\nshoot in MS agar gelled medium. Several reporters \n\nmentioned that liquid MS media was also observed \n\nmore suitable than MS semi-solid/ solid media for \n\nproliferation and multiplication of the shoots in \n\nbamboo species [34, 43, 78]. The high rate of shoot \n\ninitiation has observed in the liquid medium \n\ncompared to agar gelled medium means attributed \n\nto easy availability and faster uptake of nutrients in \n\nliquid medium [81]. When culture initiated in liquid \n\nmedia generally shoots were grown faster and less \n\nrequired hardening time [82]. \n\nPlant Growth Regulators \nThe chemical substances which influenced either \n\npromote (positive) or inhibit (negative) the growth \n\nof the plant are plant growth regulators (PGR).", "start_char_idx": 14627, "end_char_idx": 18112, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "50fddaa6-8e6a-43bd-8e9e-1016dbe29d72": {"__data__": {"id_": "50fddaa6-8e6a-43bd-8e9e-1016dbe29d72", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a6b11238-22ff-41fc-9ca9-674244e7e65d", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "2fb7f5e83ca43e73cc48ce998113e949835ad6f9a8320659ea4539495160895b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "64aac2f2-2748-48ba-abaf-c3eb0fd6da04", "node_type": "1", "metadata": {}, "hash": "870b7066ac9ebfe9a36dac676aa7f47b6040b42c4b05fce327751a604e746bc8", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nlow quantity of PGR can change the morphological \n\nstructure of plants. Natural and synthetics \n\nphytohormones are widely used in tissue culture. \n\nMostly cytokinins and auxin are used for \n\ncallogenesis and histogenesis of bamboo. Different \n\nconcentration of the 6\u2013Benzyl aminopurine (BAP), \n\n6\u2013Benzyl adenine (BA), Napthalene Acetic Acid \n\n(NAA), Indole 3-Butyric Acid (IBA), Indole Acetic \n\nAcid (IAA),  Zeatin (ZN), kinetin (KN), Thidiazurn \n\n(TDZ) with the supplement of 3% sucrose and 100 \n\nmg/L Myo-Inositol was used on the \n\nmicropropagation of the bamboo. The growth \n\nregulators hormones used by researchers in Table 1. \n\nThere were various factors affect the initiation of an \n\naseptic culture of explants. The rate of percentage on \n\nbud break was varied with different concentrations \n\nof plant growth hormones, condition, physiological \n\nstatus of explants, size, the position of the explants, \n\nage of mother plants, the health of mother stock and \n\ncollection season of explants.  Mostly, BAP and BA \n\nwere widely used for micropropagation of bamboo \n\nbecause it might be cost-effective and autoclave \n\nnature [36], and ultimately BAP and BA showed \n\nsuccessful results when in cooperated within MS \n\nmedia for bud breaking, proliferation, and \n\nmultiplication of shoots of several bamboo species. \n\nBut Arya et al. (2003) could not obtain an axillary \n\nbud break in B. tulda in the presence of BAP only. \n\nVenkatachalam et al. (2015) have reported that 85% \n\nbud break was obtained separately or a combination \n\nof different concentrations of BAP, NAA, and KN \n\nwith a supplement of Additivesincooperation with  \n\nMS solid media. Moreover, similar combined effects \n\nof two cytokinins (BAP and KN) in different \n\nconcentrations have found successfully result in bud \n\nbreaking and shoot initiation of B. arundinancea Retz. \n\nWild [84]. However, In shoot initiation experiment, \n\ndifferent researchers have tried auxin (NAA, IAA) \n\nalong with different combinations of cytokinins \n\n(BAP, KN, and TDZ and also suggested that \n\nincreased levels of   BAP and KN retarded in bud \n\ninitiation [23, 57, 58, 62, 78]. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  58 \n\nTable 1. Micropropagation from Nodal segments/Explants \n\nBamboo Species \nPlant Growth regulators \n\nResults Ref \nBud breaking  Shoot Multiplication  Rooting  \n\nArundinaria callosa  \nMunro  \n\nLiquid MS + \nBAP(13.3 \u03bcM/L) \n\nLiquid MS  + \nBAP (13.3 \u03bcM/L) + IBA \n(1.0 \u03bcM/L)  \n\n1/2  MS Liquoid + IBA ( \n25 \u03bcM) + BAP ( 0.05 \n\u03bcM/L)  \n\nShoot \nmultiplication and \nRooting \n\n70 \n\nB. arundinacea BAP (5.0 mg/L) BAP (5.0 mg/L) NAA (3.0 mg/L) Mass multiplication 79 \n\nB. arundinacea Retz.", "start_char_idx": 18113, "end_char_idx": 20814, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "64aac2f2-2748-48ba-abaf-c3eb0fd6da04": {"__data__": {"id_": "64aac2f2-2748-48ba-abaf-c3eb0fd6da04", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "50fddaa6-8e6a-43bd-8e9e-1016dbe29d72", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "b70df2346111ca8e049b64e00bb172465d3bb27fbfa5542408ad07cc2ce5541d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7b5fc3aa-22cb-4277-bf2b-0ce9d5eb0655", "node_type": "1", "metadata": {}, "hash": "eaaf460d3917edbdcba7c99c53f65952f88b4155ce6471681ff0c08731be5637", "class_name": "RelatedNodeInfo"}}, "text": "Willd \n\nMS + \nBAP(3.0 mg/L ) \n+KN (0.5 mg/L)  \n\nMS +BAP (3.0 mg/L) + \nKN (0.5 mg/L)  \n\n\u00bd MS+ IBA ( 2.0 mg/L) +   \nKN (0.5 mg/L)  \n\nMass multiplication 94 \n\nB. balcooa - BAP (11.25\u03bcM/L) +KN  \n(4.5 \u03bcM/L) \n\n1/2 MS+ \nIBA (1.0 \u03bcM/L) \n\nIn Vitro \nregeneration \n\n67 \n\nB. balcooa BAP  \n(1.0 mg/L) \n\nBAP (1.0-5.0 mg/L) 1/2MS+NAA(1-3mg/L) \n+ IBA(1 -5. mg/L) \n\nMass multiplication 112 \n\nB. balcooa  MS +BAP (4.4 \n\u00b5M/L)+NAA \n(0.53\u00b5M/L) \n\nMS + BAP (4.4 \u00b5M/L)+ \nNAA (0.53\u00b5M/L) \n\nMS + NAA  (16.11 \n\u00b5M/L) \n\nMass multiplication \nand Rooting \n\n49 \n \n \n\nB. balcooa Roxb MS+ citirc acid \n(25mg/L) + \n\nascorbic (50 mg/L) \n+BAP (3.5 mg/L)   \n\nMS+ BAP (3 mg/L)+ \nNAA (0.5 mg/L)  \n\nMS +NAA ( 4 mg/L)  Mass multiplication \nand Rooting \n\n51 \n\nB. balcooa MS+TDZ (0.1 \n\nmg/L)+ Gelrite \n(2g/l)  \n\nMS + TDZ \n\n (0.1 mg/L)  \n\nMS + TDZ ( 0.01 mg/L) + \n\n2,4-D (0.5 mg/L).", "start_char_idx": 20816, "end_char_idx": 21652, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7b5fc3aa-22cb-4277-bf2b-0ce9d5eb0655": {"__data__": {"id_": "7b5fc3aa-22cb-4277-bf2b-0ce9d5eb0655", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "64aac2f2-2748-48ba-abaf-c3eb0fd6da04", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "7020fda90aa855b6cb9d4943a7ca6e41e4c4b646cc931a6279f4b780055997aa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1f22f885-73df-4db6-a8b8-54b8ff3fcb11", "node_type": "1", "metadata": {}, "hash": "e6187b8fbc2648ed9e050ea9554d78b3778f76a9c9ad0b328ac89c843721e57b", "class_name": "RelatedNodeInfo"}}, "text": "Mass multiplication \n\nand Rooting \n\n85 \n\nB.balcooa MS + BAP \n (1 mg/L)  \n\nMS+ BAP \n (1 mg/L) \n\nMS+  BAP  (1 mg/L) + \nNAA  (3mg/L) \n\nMass multiplication \nand Rooting \n\n61 \n\nB.balcooa MS+  BAP \n (4 mg/L)  \n\nLiquid  MS + BAP (4 \nmg/L)  \n\n MS Liquid+  IBA \n(1mg/L)  \n\nMass multiplication \nand Rooting \n\n68 \n\nB.balcooa Liquid MS + BAP (1 \nmg/L)  \n\nMS+BAP  (1.0-5.0 mg/L)  \u00bd MS+ NAA  \n(3 mg/L)/ IBA \n (5 mg/L)  \n\nMass multiplication \nand Rooting \n\n112 \n\nB.balcooa Liqiud MS + BAP \n(11.25 \u03bcM/L) + KN \n\n(4.5 \u03bcM/L)  \n\n Liquid MS  +  IBA (1 \n\u03bcM/L)   \n\n\u00bd MS Liquid) +  IBA  (1 \n\u03bcM/L)   \n\nMass multiplication \nand Rooting \n\n98 \n\nB.balcooa MS+ BAP (4.4 \n\n\u03bcM/L) + KN (2.32 \n\u03bcM/L)+ Gelrite \n(0.2% w/v)  \n\n Liquid  MS + BAP  \n\n(6.6 \u03bcm/L)+KN ( 2.32 \n\u03bcM/L)+ Coconut water \n(2.5% (v/v)  \n\n1/2 MS +IAA (5.71 \n\n\u03bcM/L)+ IBA \n (4.9 \u03bcM/L)+NAA  (5.37 \n\u03bcM/L)   \n\nMass multiplication \n\nand Rooting \n\n34, 35 \n\nB.balcooa MS + BAP (3 mg/L) MS + BAP (5 mg/L) MS +NAA (4.5 mg/L) Mass multiplication \nand Rooting \n\n124 \n\nB. bambos  MS + BAP (4.4 \n\u03bcM/L)  \n\nMS+  BAP (4.4 \u03bcM/L) + \nKN (1.16 \u03bcM/L)  \n\nMS+  IBA (9.80 \u03bcM/L)  Mass multiplication \nand Rooting \n\n49 \n\nB.bambos MS + BAP (4.4 \n\n\u00b5M/L)+KN ( \n1.16\u00b5M/L) \n\nMS + BAP (4.4 \n\n\u00b5M/L)+KN ( 1.16 \u00b5M/L) \n\nMS + IBA (9.80\u00b5M/L) Mass multiplication \n\nand Rooting \n\n49 \n\nB.bambos \u2026 BAP (5.0 mg/L)  NAA (3.0 mg/L) Micropropagat-ion 48 \nB. edulis BAP (1mg/L)/ \n\nBAP(1mg/L)+ \n\nNAA(1mg/L)  \n\nTDZ (0.01 mg/L) TDZ (0.01 mg/L) Micropropag-ation \nand In- vitro \n\nflowering \n\n85 \n\nB.glaucescensWilld MS + BA (5 \u03bcM/L) Liquid MS+ BA(5 \u03bcM/L) \n\n+ KN 15 \u03bcM/L)  \n\nMS+ IBA (25 \u03bcM/L)  Mass multiplication \n\nand Rooting \n\n99 \n\nB. nutans Wall ex. \nMunro  \n\nMS+ BAP (1.0 \nmg/L)  \n\nMS +BAP ( 0.5 mg/L) + \nNAA  (0.1 mg/L).  \n\nMS+ NAA (2.0 mg/L)  Mass multiplication \nand Rooting \n\n29 \n\nB. nutans Wall ex. \n\nMunro  \n\nLiquid MS+  \n\nBAP (1 mg/L)  \n\nMS+ BAP (1.0-5.0 mg/L)   \u00bd MS +NAA(3.0 mg/L)/  \n\nIBA (5.0 mg/L)  \n\nMass multiplication \n\nand Rooting \n\n112 \n\nB. nutans Wall ex.", "start_char_idx": 21656, "end_char_idx": 23611, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1f22f885-73df-4db6-a8b8-54b8ff3fcb11": {"__data__": {"id_": "1f22f885-73df-4db6-a8b8-54b8ff3fcb11", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7b5fc3aa-22cb-4277-bf2b-0ce9d5eb0655", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "1a19188cbdb7c57903d269f99c71941a0164a8771ad48dfe85f3935c0f188b83", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "794d9724-a3bf-43d7-96a2-016bd5d8d903", "node_type": "1", "metadata": {}, "hash": "0865ec1f96d0ccc431435a26ffdedfcdccf427eb8a00399e121cdbf2d959976e", "class_name": "RelatedNodeInfo"}}, "text": "Munro  \n\nMS+   \nBA (2.22 \u03bcM/L)  \n\nLiquid MS+  \nBA  (2.22 \u03bcM/L) \n\nMS+ IBA (49.0 \u03bcM/L)  Mass multiplication \nand Rooting \n\n97 \n\nB.nutans MS +  \nBAP (4.44 \u00b5M/L)+ \n\n2,4-D (4.2 \u00b5M/L) + \n3% Sucrose \n\nMS + TDZ (6.49 \u00b5M/L)+ \nNAA ( 0.74 \u00b5M/L) \n\nMs +NAA (16.11 \u00b5M/L) \n+2% sucrose \n\nMass multiplication \nand Rooting \n\n58 \n\nB. nutans Wall ex. \nMunro  \n\nMS+ BA (4.4 \u03bcM/L \n+ KN (2.32) \n\nLiquid MS +  \nBA  (13.2 \u03bcM/L) +  \nKN (2.32 \u03bcM/L) +  \n\nIBA (0.98 \u03bcM/L)  \n\n\u00bd MS+IBA (9.8 \u03bcM/L) + \nIAA (2.85 \u03bcM/L)+  \nAA  (2.68 \u03bcM/L)  \n\nMass multiplication \nand Rooting \n\n34, 35 \n\nB. nutans Wall ex. \n\nMunro  \n\nMS +  \n\nBAP (5.0 \u00b5M/L)  \n\nMS + BAP (5.0 \u00b5M/L)  MS + IBA (10.0 \u00b5M/L) Shoot \n\nmultiplication and \nRooting \n\n119 \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  59 \n\nB. oldhamii Munro  MS+  \nTDZ (0.45 \u03bcM/L)+  \n\nGelrite (2.2 g/L) \n\nLiquid MS +  \nTDZ (2.27 \u03bcM/L)  \n\nMS basal +   \nNAA (10.74-26.85 \u03bcM/L)   \n\nShoot \nmultiplication and \n\nRooting \n\n85 \n\nB. pallida  Liquid MS+ \n\nascorbic acid (50 \nmg/L) + citric acid \n\n(25 mg/L) + \ncysteine (25 \nmg/L)+  \n\nNAA 1.34 \u03bcM/L+ \nTDZ (1.125 \u03bcM/L)  \n\nLiquid MS+ \n\nNAA  (1.34 \u03bcM/L)+  \nBAP ( 4.44 \u03bcM/L)  \n\n\u00bd MS+ 2% sucrose +1% \n\nglucose + 0.6% agar after \ntreatment of IBA (0.5 \n\n\u03bcM/L) for 30 min  \n\nMass multiplication \n\nand Rooting \n\n111 \n\nB. pallida  MS+ BA (1 mg/L) + \nGelrite (2.5% )  \n\nMS+ BA (3 mg/L)  MS+ NAA ( 2.0 mg/L)  Mass multiplication \nand Rooting \n\n88 \n\nB. salarkhanii Liquid MS+  \n\nBAP (1 mg/L)  \n\nMS+ BAP (1.0-5.0 mg/L)  \u00bd MS+ NAA (3 \n\nmg/L)/IBA(5 mg/L)  \n\nMass multiplication \n\nand Rooting \n\n112 \n\nB. tulda MS+ BA (1.0 mg/L)  Semi-solid MS+ BA (1.0 \n\nmg/L)  \n\n MS+ NAA(5 mg/L)  Mass multiplication \n\nand Rooting \n\n88 \n\nB. tulda semi-solid MS+  \n\nBA (10 \u03bcM/L) + \nIAA ( 0.1 \u03bcM/L) \n\n MS (L) + glutamine (100 \n\n\u03bcM/L ) +  \nIAA  (0.1 \u03bcM/L) +   \nBAP (12 \u03bcM/L)   \n\nMS liquid medium + 40 \n\n\u03bcM/L Coumarin \n\nMass multiplication \n\nand Rooting \n\n47 \n\nB. tulda MS+ BAP (3mg/L)  Liquid MS+ KN (2mg/L) \n+ BAP (3mg/L)  \n\n\u00bd MS+ IBA (3mg/L)+   \ncoumarin 10 mg/L + 3% \n\nsucrose \n\nMass multiplication \nand Rooting \n\n46 \n\nB. tulda MS (Liquid) +  \n\nBAP (8.8 \u00b5M/L)+ \nKN(4.", "start_char_idx": 23613, "end_char_idx": 25734, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "794d9724-a3bf-43d7-96a2-016bd5d8d903": {"__data__": {"id_": "794d9724-a3bf-43d7-96a2-016bd5d8d903", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1f22f885-73df-4db6-a8b8-54b8ff3fcb11", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "17d534bfa32c55970b70f1fc57f1eb80289f790313deaa61ba134586e71ee66b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63bc2808-1fdf-42a8-a3f4-3adc2093feea", "node_type": "1", "metadata": {}, "hash": "3addf2ee4e2a6fde91e932b48c6e49fa271cfaa1cc6f29900831dc556d59a637", "class_name": "RelatedNodeInfo"}}, "text": "8 \u00b5M/L)+ \nKN(4.46 \u00b5M/L) + \n2% Sucrose \n\nMS (Liquid) + \n\nBAP(8.8\u00b5M/L)+KN ( 4.46 \n\u00b5M/L) + 2% Sucrose \n\nMS (Liquid) + IBA (18.8 \n\n\u00b5M/L) + 2% Sucrose \n\nMass multiplication \n\nand Rooting \n\n122 \n\nB. tulda Liquid MS +  \nBAP (2.5 mg/L) + \n\nKN (1mg/L) +  \n8% coconut water  \n\nLiquid MS+  BAP (2.0 mg \n/l) + KN  (1.0 mg/L)+ 8% \n\ncoconut water  \n\n\u00bd MS + IBA (0.2 mg/L)   Mass multiplication \nand Rooting \n\n98 \n\nB. ventricosa BAP (4.44 \u03bcM/L) BAP (4.44\u03bcM/L) NAA (5.4 \u03bcM/L) + BAP  \n(0.44 \u03bcM/L) \n\nIn Vitro \nregeneration \n\n53 \n\nB. ventricosa MS+  \n\nBA (22.2 \u03bcM/L)   \n\nMS+  BA (22.2 \u03bcM/L) \n\n+ TDZ (0.23 \u03bcM/L)+  \nNAA (0.27 \u03bcM/L)  \n\nMS + NAA(2.7 \u00b5M/l) \n\n+IBA( 4.9 \u03bcM/L)+ \n BA (4.4 \u00b5\u039c/l)  \n\nMass multiplication \n\nand Rooting \n\n78 \n\nB.vulgaris Modified MS+   \nBAP(2 mg/L)  \n\nModified MS+   \nBAP (2 mg/L)  \n\nModified MS+   \nIBA  (20 mg/L)  \n\nMass multiplication \nand Rooting \n\n113 \n\nB. vulgaris  Liquid MS+   \nBAP (1 mg/L)  \n\nMS+ BAP (1.0-5.0 mg/L)  \u00bd MS +NAA  \n(3.0 mg/L)/IBA  \n\n( 5.0 mg/L)  \n\nMass multiplication \nand Rooting \n\n112 \n\nB. vulgaris  Liquid MS+  \nBAP (1 mg/L)  \n\nMS+ BAP (1.0-5.0 mg/L)   \u00bd MS+ NAA (3 mg/L) / \nIBA(5 mg/L)  \n\nMass multiplication \nand Rooting \n\n112 \n\nB. vulgaris MS+  \nBAP (2.0 mg/ l)   \n\nMS +BAP (4.0 mg/L)  \u00bd MS+ IBA \n (3.0 mg/L)  \n\nMass multiplication \nand Rooting \n\n64 \n\nB. wamin  MS (L) + BAP \n (5.0 mg/L)  \n\nSemisolid MS+ BAP (2.0 \nmg/L) +KN \n\n (0.8 mg/ l)  \n\n\u00bd MS+ IBA  \n(7.5 mg/L)  \n\n8 80 \n\nD. asper BAP ( 0-2.0 mg/L + \nCW (0-20.0 mg/L) \n\nBAP (5.0 Mg/L)  NAA (0.5 mg/L) In Vitro culture 2,96 \n\nD. asper  MS+ BAP  \n( 5 mg/L)  \n\nMS (L)  BAP  ( 5 mg/L) \n+Ads ( 40 mg/L) \n\n MS (liquid) + IBA (1 \nmg/L)  \n\nShoot \nmultiplication and \n\nRooting \n\n103 \n\nD.asper MS+ BA  \n\n(0.1-15 mg/L)  \n\nMS (L) + IBA (3 mg/L)  MS + IBA  \n\n(10 mg/L)  \n\nShoot \n\nmultiplication and \nRooting \n\n25 \n\nD. asper BAP  \n\n(3.0 mg/L)   \n\n BAP  \n\n(1.0-4.0 mg/L \n\nNAA (2.0 mg/L) or IBA \n\n(10.0 mg/L) \n\nShoot \n\nmultiplication and \nRooting \n\n48 \n\nD.asper MS + BAP (15 \n\u03bcM/L)  \n\nMS + BAP (10 \u03bcM/L) + \nAds (75 \u03bcM/L)+  \nTable Sugar 3%   \n\n\u00bd MS +IBA  \n( 5 \u03bcM/L) + NAA  \n(5 \u00b5\u039c/l) \n\nShoot \nmultiplication and \nRooting \n\n21 \n\nD.", "start_char_idx": 25719, "end_char_idx": 27816, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63bc2808-1fdf-42a8-a3f4-3adc2093feea": {"__data__": {"id_": "63bc2808-1fdf-42a8-a3f4-3adc2093feea", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "794d9724-a3bf-43d7-96a2-016bd5d8d903", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "aa84032049d8a7518bf9f1b6134f8c097bb46b98556c1e7c740220e31344ed64", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "86610f34-5218-47f9-a2c9-2d00ef97c894", "node_type": "1", "metadata": {}, "hash": "70828fa16ba7af9fd81c260e4b52b982e4a05d6965a26d874dfffb3071cb4f66", "class_name": "RelatedNodeInfo"}}, "text": "asper  MS+BAP (8.86 \n\u03bcM/L)+ \n\nAds (13.5 \u03bcM/L)  \n\nMS+BAP  \n(8.86 \u03bcM/L) + Ads (13.5 \n\n\u03bcM/L)  \n\nMS+ IBA  \n(14.76 \u03bcM/L)+ \n\n NAA (3.67 \u03bcM/L)  \n\nShoot \nmultiplication and \n\nRooting \n\n59 \n\nD.asper {Schult. & \n\nSchult.f.} Backer ex \nk. Heyne)  \n\nMS+ BAP \n\n (15 \u03bcM/L)  \n\nMS + BAP (10 \u03bcM/L)+ \n\nAds (75 \u03bcM/L)  \n\n\u00bd MS +  IBA (5 \u00b5\u039c/l) +  \n\nNAA (5 \u03bcM/L)  \n\nShoot \n\nmultiplication and \nRooting \n\n66 \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  60 \n\nD.asper MS+ BA (3 mg/L)  Liquid  MS  + BA  (3 \nmg/L) + Ads  (50 mg/L)  \n\nMS (Liquid) + IBA (1.0 \nmg/L)  \n\nShoot \nmultiplication and \n\nRooting \n\n2 \n\nD.BrandisiiKurz.   MS (L) + Ascorbic \n\nacid (25mg/L) \n+Citric acid \n\n(12.5mg/L)+Cystei\nne (12.5mg/L) + \nGlutamic acid (50 \n\nmg/L) +  \nTDZ (0.25 mg/L)+  \n\nNAA (0.25 mg/L)  \n\nMS (Liquid) +  \n\nNAA (0.25 mg/L) +  \nBAP (2.5 mg/L)  \n\n \u00bd MS (L)+  \n\nNAA (1mg/L)  \n\nShoot \n\nmultiplication and \nRooting \n\n86 \n\nD. giganteus BAP  \n(2.0 -5.0 mg/L) \n\nKN (10.0 \u00b5M/L) +  \nBAP (0.5 \u00b5M/L) \n\n\u2026 Mass multiplication 73 \n\nD.giganteus BAP (30.0 \u00b5M/L) BAP (20.0 \u00b5M/L) IBA (25 \u00b5M/L) + BAP \n(0.05 \u00b5M/L) \n\nRapid \nmultiplication \n\n48 \n\nD.giganteus ..   IBA+TDZ+ Coumarin Root induction 105 \nD. giganteus Munro  Semi-solid MS+ \n\nBAP (2 mg/L) +  \nKN (0.1 mg/L)+  \nBenlate (1g/L)  \n\n MS (Liquid) +  \n\nBAP(6 mg/L)+   \nKN (1 mg/L) + \n 8% (v/v) coconut water  \n\n\u00bd MS+  IBA (3 mg/L) +  \n\n10 mg/L Coumarin \n\nShoot \n\nmultiplication and \nRooting \n\n106 \n\nD.hamiltoniiNees et \nArn.  \n\nEx Munro  \n\nMS+ 2% sugar \nfollowed by MS+  \n\nBAP (8 \u03bcM/L)+ \nNAA (1 \u03bcM/L)  \n\nMS+  BAP (8 \u03bcM/L)+   \nNAA (1 \u03bcM/L)  \n\nMS+IBA  \n(100 \u03bcM/L) followed by \n\ngrowth regulator free \nmedia  \n\nShoot \nmultiplication and \n\nRooting \n\n102 \n\nD.hamiltonii ARN. \nEx MUNRO  \n\nMS+  TDZ (3.0 \n\u03bcM/L)  \n\nMS+ TDZ  \n(1.5 \u03bcM/L) + ascorbic acid  \n(56.0 \u03bcM/L) \n\n\u00bd MS+ IBA  \n(25.0 \u03bcM) + Choline \nChloride (36.0 \u03bcM/L) \n\nShoot \nmultiplication and \nRooting \n\n62 \n\nD. Longispathus \nKURZ.  \n\nMS+ BAP (12 \n\u03bcM/L)+  KN (3 \n\n\u03bcM/L)  \n\n MS (L) + BAP (15 \u03bcM/L)+ \nIBA (1 \u03bcM/L) + Coconut \n\nWater (10%)  \n\n\u00bd MS+ IBA (1\u03bcM/L)+ \nIAA (1\u03bcM/L) \n\n+Coumarin(68 \u03bcM/L).", "start_char_idx": 27817, "end_char_idx": 29902, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "86610f34-5218-47f9-a2c9-2d00ef97c894": {"__data__": {"id_": "86610f34-5218-47f9-a2c9-2d00ef97c894", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63bc2808-1fdf-42a8-a3f4-3adc2093feea", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "31062b549113b8f2982c555925716fc841e6653c6b8d8bd58c907a4fe23968db", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f96bf959-071c-4974-97f3-c41d835b3ba1", "node_type": "1", "metadata": {}, "hash": "3e077b69003df2ba908a8a23a49547f699df8719826a39fe1105000979e120ba", "class_name": "RelatedNodeInfo"}}, "text": "Shoot \nmultiplication and \n\nRooting \n\n28 \n\nD. membranaceus MS +BAP (1-5mg/L \n\nNAA (0.5 g/L) \n\nBAP (1-5mg/L) + NAA ( \n\n0.5 mg/L) \n\nNAA ( 3.0 mg/L) / IBA \n\n(10.0 mg/L) \n\nMass multiplication 73, 79 \n\nD. membranaceus MS +  \n\nBAP (4.4 \u00b5M/L) +  \nKN ( 1.16 \u00b5M/L) \n\nMS + BAP (4.4 \u00b5M)+ \n\nKN ( 1.16 \u00b5M/L) \n\n1/2 MS +  \n\nNAA (5.37 \u00b5M/L) + \nBAP( 4.4 \u00b5M/L) \n\nMass multiplication \n\nand Rooting \n\n49 \n\nD. strictus MS +  \n\nBAP (2.0-5.0 mg/L)  \n\nBAP (2.0-5.0 mg/L)   Mass multiplication 73 \n\nD. strictus Nees White medium  Liquid MS+ BA (0.5 \n\nmg/L)+ KN (0.5 mg/L)+ \nCoconut Water (200 ml/L)   \n\nSolid MS+  \n\nIBA (0.25 mg/L)  \n\nShoot \n\nmultiplication and \nRooting \n\n1 \n\nD.strictus Nees MS (L)+  \nBA (0.5 mg/L)+  \nAds (15 mg/L)  \n\nMS (L)+ IBA (0.5 mg/L)+ \nAds (15 mg/L)  \n\n 1/2MS Liquid +  \nIBA ( 0.25 mg/L)  \n\nShoot \nmultiplication and \nRooting \n\n45 \n\nD.strictus Nees MS + BAP \n (2 mg/L)  \n\nMS + BAP  \n(4 mg/L) + Ads (15 mg/L)  \n\nMS+IBA  (5mg/L)  Shoot \nmultiplication and \n\nRooting \n\n75 \n\nD.strictus Nees MS+ IAA \n (0.5 mg/L)+ Ads \n\n(15 mg/L )  \n\n  \u00bd MS+ IBA  (1 \nmg/L)+NAA(1mg/L)+ \n\n2,4-D (0.5mg/L)+ \nPhloroglucinol (1mg/L) \n\nShoot \nmultiplication and \n\nRooting \n\n56 \n\nD.strictus Nees MS+  BAP  \n(4 mg/)+ TDZ  (0.25 \n\nmg/L)  \n\nMS+ BAP (4mg/ L)+ TDZ \n(0.25 mg/ L)  \n\nLiquid MS+ BAP (2.5 \nmg/ L)+ IAA ( 5 mg/ L)  \n\nShoot \nmultiplication and \n\nRooting \n\n31 \n\nD.strictus Nees MS+ BAP  \n(4 mg/L)  \n\nMS + BAP   \n(4 mg/L)  \n\nMS+ NAA  \n(3 mg/L)  \n\nShoot \nmultiplication and \n\nRooting \n\n44 \n\nD.strictus (Roxb.) \n\nNees \n\nMS+ 2,4-D  \n\n(5 mg/L)  \n\n ------------ MS + 2,4-D  \n\n(0.5 mg/L) \n\nCallus initiation \n\nShoot \nmultiplication and \n\nRooting \n\n31 \n\nMelocanna baccifera MS+ BAP (3 mg/L)  Liquid MS+ KN (2mg/L) \n+ BAP (3mg/L)  \n\n\u00bd MS+ IBA (3 mg/L)+ \nCoumarin (10 mg/L)+ \n\n 3% sucrose,  \n\nShoot \nmultiplication and \n\nRooting \n\n46 \n\nThamnocalamusspat\nhiflorus (Trin.) \n\nMunro  \n\n\u00bd strength MS  MS medium + BAP  \n\n( 5.0 \u03bcM/L) + IBA (1.0 \n\u03bcM/L)  \n\n\u00bd MS+ IBA \n\n (150 \u03bcM/L) \n\nShoot \n\nmultiplication and \nRooting \n\n91 \n\nThyrsostachysoliveri Liquid MS+BAP  \n(1 mg/L)  \n\nMS+ BAP (1.0-5.0 mg/L)   \u00bd MS + NAA (3.0 \nmg/L)/ IBA  \n\n(5.0 mg/L)   \n\nShoot \nmultiplication and \n\nRooting \n\n112 \n\n\n\nNepal J Biotechnol.", "start_char_idx": 29906, "end_char_idx": 32077, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f96bf959-071c-4974-97f3-c41d835b3ba1": {"__data__": {"id_": "f96bf959-071c-4974-97f3-c41d835b3ba1", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "86610f34-5218-47f9-a2c9-2d00ef97c894", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "bc29ab2cbe7c693a887b4a069c594f23905cba961f74276ca8c8482c10103f66", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3aad6e15-72e6-492e-9bec-45b3093121d9", "node_type": "1", "metadata": {}, "hash": "bca7d17ef7157485a83144829fed098eb4d910245abc437e9043eadee3b96f50", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  61 \n\nContamination: Bottleneck for culture \nestablishment \nSurface and systemic contamination is the major \n\nincidence in tissue culture of bamboo because it has \n\nlarge intercellular spaces and vessel cavities at the \n\ncut end which accumulated contaminating agents \n\ndeeply. Therefore, various bacterial and fungal \n\ncontaminations have been serious problems \n\nobtained in bamboo in vitro culture. According to \n\nRamirez et al. (2009), several bacteria like \n\nXanthomonas, Pseudomonas, Agrobacterium and \n\nErwinia and Bacillus sp. by Cruz- Martin et al. (2007) \n\nare contaminating agents in some species of bamboo \n\ntissue culture while Pantoea agglomerans and P. \n\nananatis by Nadha et al. (2012) are the main bacterial \n\ncontaminates in the nodal segments of G. angistifolia.  \n\nTo overcome this problem, there is either \n\nincorporation of antibiotics in media or explants \n\ntreatment in antifungal. Surface sterilization of all \n\npieces of equipment including explants has been \n\ndone with care to escape contamination [35-40]. \n\nYasodaha et al. (2008) have used Streptomycin and \n\nKanamycin during the culture initiation stages. The \n\ncombinations of Bavistin with streptomycin [55], \n\nstreptomycin sulphate and tetracycline \n\nhydrochloride [68], bacteriocin [88] gentamycin [89] \n\nare used successfully to reduce contamination. \n\nWhen there is not directly used of tap water for \n\nwashing explants during surface sterilization, it \n\nhelps to minimize the contamination [38]. Bavistine \n\n(Cardazamine) and Mancozebas antifungal and \n\nGentamycin as antibacterial [25, 41-45] are used as \n\nanti infections. Also, Ali et al. (2009) have been found \n\nthat the combination of the antibiotics streptomycin, \n\nrifampicin and ciprofloxacin with the fungicide \n\nBavistine is successfully disinfecting in the \n\nmicropropagation of nodal explants of B. tulda, B. \n\nbalcooa, B. bamboos, and D. asper.  Oprin et al. (2004) \n\nfound that rinsing explants with acetone for 3-4 \n\ntimes with a bleaching solution help to disinfect the \n\nexplants. During surface sterilization of the explants, \n\nit is useful to apply the pre-treatment technique with \n\nthe combination of the standard disinfected \n\ncompounds. It is proved by Jimenez et al. (2006) \n\nwhen they pretreated the explants in Extran, Agri-\n\nmycine, and benomyl before surface sterilization in \n\nsodium hypochlorite and plant preservative \n\nmixture. The contamination was reduced from 2% to \n\n11% in Gaudua angustifolia.  Ramanayake et al. (1995) \n\nhave suggested that it can be controlled systemic \n\nfungal contamination in B. vulgaris by supplement of \n\nbenomyl in the culture medium.  \n\nSimilarly, the other major problem for the bamboo in \n\nvitro propagation is browning and necrosis of the \n\nshoots in the initiation of culture, shooting stage and \n\nrooting stage due to phenolic compounds exudation \n\n[25] and increase the production of polyphenol \n\noxidase by wounding of the tissue. It converts \n\nbrowning into blackish, and later on, it dies because \n\nthe increased production of polyphenol is \n\nphytotoxic to the explants [45]. According to Huang \n\net al. (2002) and Oprins et al. (2004), the browning of \n\nthe plants depends on the species, age and position \n\nof the tissue, age of mother plants, the season of \n\nexplants collection, used nutrient media and used \n\nsterilizing agents. Because of the exudation phenolic \n\ncompound by the plant itself, there was encounter \n\nbrowning problem during shoots multiplication \n\nwhich decreased the multiplication rate. But Huang \n\net al.", "start_char_idx": 32058, "end_char_idx": 35682, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3aad6e15-72e6-492e-9bec-45b3093121d9": {"__data__": {"id_": "3aad6e15-72e6-492e-9bec-45b3093121d9", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f96bf959-071c-4974-97f3-c41d835b3ba1", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "27c7277402a530f50a8bd4a8f31c0eda58e3d1603edd89ece65bdcae6ae5691b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "946b2fb3-9a92-4ceb-8982-c0eae52fd77f", "node_type": "1", "metadata": {}, "hash": "61cf4b6aca0fcf91e7e3a08483c61a21efa6c0734cec4e8c7d0fed333a61d869", "class_name": "RelatedNodeInfo"}}, "text": "But Huang \n\net al. (2002) observed that the browning of bamboo \n\nis in higher pH values of 7 and 8 while in the acidic \n\nnutrient media with standard pH 5.7 has a relatively \n\nlow browning rate.  Such a problem is solved by the \n\nsupplement of some additives along with plant \n\ngrowth regulators in the media [46]. The browning \n\nis occurred according to species, tissue or organ, and \n\nnutrient medium in vitro [47].  Different types of \n\nantioxidants (Ascorbic acid, Cysteine, Activated \n\nCharcoal, Citric acid, Adenine sulphate, \n\nPolyvinylpyrrolidone (PVP) ) with various \n\nconcentrations are either substituted into the media \n\nreduced the browning problem or soaking the \n\nexplants in a liquid solution of those mentioned \n\n[73,74] to reduce the browning percentage in \n\nmultiple shoots. Waikhom and Louis (2014) have \n\nshown that the addition of NaCl and Silicon in MS \n\nmedia significantly enhances the activities of \n\nantioxidant enzymes. But some researchers have \n\nsucceeded to overcome serious browning and \n\nleaching problem by frequently transfer the clumps \n\nto the fresh medium without the incorporation of \n\nany antioxidants with media and treatments [74]. \n\nHuang et al. (2002) found in B. oldhamii, D. latiflorus, \n\nand P. nigra browning control when they used PVP, \n\nactivated charcoal, ascorbic acid, cysteine, ferulic \n\nacid, and thiourea. \n\nShoot Multiplication \nThe size and number of propagules have a vital role \n\nin the shoot multiplication. Three to four propagules \n\nfor each culture to multiply was observed effective \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  62 \n\n[29-31] than individual propagule cultured [38]. \n\nFurthermore, BAP was extensively used on in vitro \n\nmultiplication of different bamboos shoot [86, 87, \n\n124]. A higher concentration of BAP was an impact \n\non decreasing numbers and length of shoots. KN \n\nalone is not a significant response in shoot \n\nmultiplication in B. balcooa, which results in the \n\nclumps dried and browning. Incorporation between \n\nKN and BAP was found effective in shoot \n\nmultiplication [88]. Similar to the synergistic effect of \n\nKN and BA on the shoot multiplication rate was \n\nreported in B. balcooa [59]; B. glaucescens [89]; D. \n\ngiganteus [40];  B .tulda and M. baccifera [38]. \n\nSimilarly, TDZ has been reported an effective \n\ncytokinin for shoot proliferation [82, 90]. The effect \n\nof coconut water on bamboo shoot multiplication \n\nhas been reported by different researchers. Saxena \n\nand Bhojwani (1993) have found that the addition of \n\n10% coconut milk (CM) as an additive in the media \n\nis better for shoot proliferation and multiplication in \n\nD. giganteus. Ramanayake et al. (2001) reported that \n\na high level of sucrose (4%) adversely affected the \n\nshoot multiplication in D. giganteus. There are 3% \n\nsucrose is widely used in tissue culture. \n\nDevi and Sharma (2009) have found IBA was \n\nsuperior over NAA for shoot multiplication in \n\nArundinaria callosa Munro. The use of IAA and NAA \n\nin conjunction with BA and KN was found to \n\nincrease the length of shoots but lowered the \n\nmultiplication rate [29]. Further, Gibberellic acid \n\n(GA) was effective and enhances for multiplication \n\nof shoot in B. vulgaris [63]. Rathore et al. (2009) only \n\none researcher who has accounted for that the \n\ncombined effect of NAA and BAP was effective for \n\nshoot multiplication of B. balcooa and B. bambos.  \n\nRooting \nIn vitro rooting is the bottleneck for the researchers \n\nin bamboo. Generally, NAA, IAA, and IBA are used \n\nindividuals or combined for root initiation.", "start_char_idx": 35664, "end_char_idx": 39266, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "946b2fb3-9a92-4ceb-8982-c0eae52fd77f": {"__data__": {"id_": "946b2fb3-9a92-4ceb-8982-c0eae52fd77f", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3aad6e15-72e6-492e-9bec-45b3093121d9", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "dc0b7cafb1db23102810cc03f247cc4e92c44599ff30b5ec12b691a87ca822f4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "667465f2-2251-4be3-ae0a-522d0de3d332", "node_type": "1", "metadata": {}, "hash": "1ec7eac0d56126a192a1348bf009d7806ce5136a9f887cd59e24955a983ef5e7", "class_name": "RelatedNodeInfo"}}, "text": "These \n\nthree hormones were more suitable for rooting in D. \n\nasper [84, 91,92,93,94, 97, and 98]. Clusters of 3-5 \n\nshoots were effective for transferring into rooting \n\nmedium [29, 51, 58, 96, 99, 123, 124]. Full strength MS \n\nand half-strength MS media with the supplement of \n\nrooting hormones were frequently practiced for In \n\nvitro rooting. Arya et al. (2008) reported 80-90% of \n\nroots obtained in MS medium with supplementing \n\nof NAA or IBA within 5 weeks of transferring while \n\nworking in D. asper and D. falcatum. But Singh et al. \n\n(2012b) have observed that 100% rooting in D. asper \n\nby using a combination of IBA and NAA. Whereas \n\nfor B. tulda and B. balcooa a two-step treatment of 7 \n\ndays on liquid MS medium supplemented with  IBA \n\nand then transferred in vitro shoots to basal MS \n\nmedium (pulse treatment) without any rooting \n\nhormones, was followed for in vitro rooting [43]. The \n\ncombination of IBA and NAA for rooting has been \n\nalso reported by Islam and Rahman (2005); Arya et \n\nal. (2006) and Rathore et al. (2009) in many important \n\nbamboo species. Some studies proved that IBA was \n\nfound to be the most favorable root inducer \n\ncompared to NAA and IAA on several bamboos \n\nsuch as Drepanostachym falcatum [40], Oxytenanthera \n\nabyssinica [94], D. hookeri [95], D. hamiltonii [92]); \n\nMelocanna baccifera [100]. On rooting medium shoots \n\nalso elongated and good root and shoot system \n\ndeveloped in 5-7 weeks. Saxena (1990) working on B. \n\ntulda has reported supplementing of Coumarin in \n\nrooting media resulted in better root induction and \n\nelongation. Similarly, Ramanayake and Yakandwala \n\n(1997) working on D. giganteus and Sood et al. (2009) \n\non D. hamiltonii  observed a high frequency of \n\nrooting when IBA was used in combination with \n\nCoumarin. In the case of D. strictus, up to 90%, \n\nrooting was found in medium containing IBA [38]. \n\nHowever, well-developed roots with healthy shoots \n\nwere observed in half-strength MS medium \n\nsupplemented with NAA [36]. Negi and Saxena \n\n(2011) have a document that the highest rooting \n\nfrequency was obtained on \u00bd MS media with \n\nsupplemented of IAA, IBA and NAA in B. nutans \n\nand also similar result obtained by Kapoor and Rao \n\n(2006)  who reported that 100% rooting in \u00bd MS \n\nmedia containing the optimal concentration of BA \n\nand NAA in B. bambos. Sanjaya et al. (2005) have \n\nachieved In vitro rhizome in P. stocksii with \n\ncontinued subculturing of rooted plantlets on \n\nmedium containing \u00bd strength of Major salts within \n\nthe addition of  IBA, BA, ascorbic acid, citric acid, \n\ncysteine and glutamine in different concentration. \n\nChowdhury et al. (2004) obtained in vitro rhizome in \n\nD. strictus culturing in rooting media that have \u00bd \n\nstrength major salts and IBA.  Again, several \n\nresearchers observed half-strength MS \n\nsupplemented with NAA and IBA was better than \n\nthat of the full strength of MS media [21, 25, 29, 78] \n\nin various bamboo species. Islam and Rehman (2005) \n\nhave accounted that the couple of NAA and IBA \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  63 \n\nwere suitable for the rooting of bamboo.", "start_char_idx": 39267, "end_char_idx": 42434, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "667465f2-2251-4be3-ae0a-522d0de3d332": {"__data__": {"id_": "667465f2-2251-4be3-ae0a-522d0de3d332", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "946b2fb3-9a92-4ceb-8982-c0eae52fd77f", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "57d811b1e8e6c92ea3993123119b60370726830b51892ebefc08b2441ea4ea62", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "43cf0950-b65e-4b7b-bbb9-3b55892bcb33", "node_type": "1", "metadata": {}, "hash": "aaeacdeebef9845ca7291eadcc9538ac35abe6312a5362b8910994130557121a", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  63 \n\nwere suitable for the rooting of bamboo. But IBA was \n\nfound better than NAA in B. arundinacea and D. \n\ngiganteus for rooting by. Again, the IBA \n\nsupplemented medium showed only poorly \n\ndeveloped roots in B.nutans and B. balcooa [29]. But \n\nRavikumar et al. (1998) reported that IBA \n\nsupplement in MS media was more effective to root \n\ninduction in D. strictus and D. asper respectively \n\nwhich was also supported by Bag et al. (2000) in the \n\nresult of Thamnocalamus spathiflorus. Singh et al. \n\n(2012b) reported that the combination of IBA and \n\nNAA was a synergistic effect for rooting in D. asper \n\nin place of single use. But Mudoi and Borthakur \n\n(2009) observed a combination of NAA and BAP was \n\nfound most effective in rooting of B. balcooa which \n\nwas also confirmed by Goyal et al. (2015) in D. \n\nstrictus. The limited number of the report was \n\navailable for rooting in IAA. Kapruwan et al. (2014) \n\nreported IAA for in vitro rooting in D. strictus. The \n\neffect of growth regulators on rooting was varied \n\nspecies to species [95]. Negi and Saxena (2011) have \n\npracticed successfully rooting in full strength MS \n\nliquid media with the supplement of IAA, IBA, and \n\nNAA. And the high concentration of cytokine \n\nintroduced for rooting might have resulted in cell \n\ndeath and cell cultures became yellowing leaves and \n\nreduced root mass in intact plants [97-100]. \n\nHardening \nThe transfer of in vitro propagated plantlets form Lab \n\nto land is another big nutshell of micro-propagation \n\n[22]. The plant developed in vitro is unable to survive \n\nin vivo directly due to lack of adaptation and proper \n\nhardening [43] however it has well-developed roots. \n\nTo overcome the bottleneck of hardening, \n\nresearchers have followed various hardening \n\nprocedures. In general, the healthy and well-rooted \n\nplantlets are washed to free from the rooting \n\nmedium and transferred to the pot containing \n\ngrowth supporting composition such as soil, sand, \n\nsoil rite, perlite, cocopeat, agro peat, vermiculite, \n\ncompost, farmyard manure, etc either alone or in \n\nvarious ratios [22]. Most researchers have used \n\nmention substrate in 1:1:1 ratio or modified. Some \n\nresearchers have described the primary hardening \n\nand secondary hardening to obtained maximum \n\nnumbers of plantlets. Like, In vitro plantlets were \n\ntransferred to \u00bd strength MS liquid medium without \n\nplant growth regulators and vitamins for hardening \n\nin D. asper [65], B. nutans [109], and D. hamiltonii [54]. \n\nThen when plantlets transferred to polybags 1:1:1 \n\ncomposition of Sand: Farmyard manure: Soil they \n\nobtained high rate plantlets. The mortality \n\npercentage of plants is increased when the direct \n\ntransfer of in vitro propagated plantlets to the \n\nexternal environment because of their inability to \n\nsurvive against biotic and abiotic stresses [91]. But \n\nNegi and Saxena (2011) obtained a 95.83% success \n\nrate by directly hardening in 2:1 mixture of soil: agro \n\npeat in B. nutans.", "start_char_idx": 42378, "end_char_idx": 45373, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "43cf0950-b65e-4b7b-bbb9-3b55892bcb33": {"__data__": {"id_": "43cf0950-b65e-4b7b-bbb9-3b55892bcb33", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "667465f2-2251-4be3-ae0a-522d0de3d332", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "4d724bc6dadf8794c942cc662515826b9dd2fe120f9c2cf1cb0c413e0543ce1a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2b4301a4-1161-4d07-97d7-6f373f0890f8", "node_type": "1", "metadata": {}, "hash": "75f92537675ce34928d01388054a8925be109ad7776639f7719972a642d8e2a7", "class_name": "RelatedNodeInfo"}}, "text": "Similarly, several workers reported \n\non hardening of in vitro plants in the mixture of soil: \n\nsand: compost cocopeat (1:1:1) in B.nutans [87, 110]; \n\nsoil: sand: cow dung(1:1:2) in B. balcooa [25] and B. \n\nnutans (Sharma and Sarma2014); soil mixture of peat, \n\nperlite, and vermiculite (1:1:1) in B. oldhamii [77], \n\nperlite, soil, and farmyard manure (1:1:1) in D. \n\nstrictus [36]; 3:1 ratio of coco peat and vermicompost \n\n(3:1) in B. balcooa [43]. Without other substrates \n\ncomposition with soil, In vitro plantlet was \n\nacclimatized successfully in B. bambos [22, 29, 33, 41, \n\n42, 62, 65] \n\nBA- 6- Benzyl Adenine, BAP- 6- Benzyl \n\naminopurine, TDZ- Thidiazuron, IBA- Indole -3-\n\nbutyric acid, IAA- Indole -3-acetic acid, NAA- \u03b1-\n\nNaphthalene acetic Acid, Ads- Adenine sulphate, \n\nKN- Kinetin, MS-Murrashige and Skoog media, CW-\n\nCoconut water. \n\nConclusion \nAs a fast-growing and high potential economic \n\ndevelopment of the country, the demanding bamboo \n\nhas enhanced the depleting rootstock of bamboo \n\nrapidly. Bamboo has a high capacity to carbon \n\nsequester and it is the mitigation of climate change \n\nand environments. Similarly, it is an alternative \n\nsource of the forest. So that it has a great role in \n\nconservation biology and is become a priority \n\nconcern.  With knowledge of the awareness of the \n\nconservation biology and environment however \n\npeople have to fulfill the enormous demands of the \n\nmarkets, they have to exploit the limited resources.  \n\nHarvesting from the resources means that a large \n\nscale of bamboo plantlets are necessary highly \n\nthrough micropropagation to fill up the gap of plant \n\nstocks. Mass yield production protocols along with \n\nfactors influence on it are discussed in the review. \n\nSeveral protocols are reported by several \n\nresearchers. Nodal explants are better explants for \n\nmicropropagation technique with the supplement of \n\nproper plant growth regulators in MS media at \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  64 \n\ndecontamination condition, the proper season of \n\nexplants collection, and appropriate position of the \n\nnodal segment in mother plant stocks. BA/ BAP is \n\nthe best cytokine for bud initiation and shoots \n\nmultiplication bamboo species. In vitro rooting is \n\nspecific for bamboo species. IBA is a more effective \n\nrooting hormone for bamboo in comparison to other \n\nNAA and IAA. But several studies indicated that a \n\ncouple of IBA and NAA is also effective for rooting \n\nin in vitro. It is also reported that the incorporation of \n\nNAA, IBA, and IAA is also suitable for rooting in \n\nsome bamboo species. Sand alone is restricted for \n\nhardening but the combination of different \n\nsubstrates varies, the ratio is appreciable for \n\nhardening the in vitro rooted plants. The more \n\nrelevant protocols have to develop by addressing \n\nthose issues properly. Future research must be \n\nfocused to generate a large scale of bamboo plants. \n\nAuthor\u2019s Contribution \nMMS, JL and DPG were equally contributed to \n\npreparing framework of literature, \n\nconceptualization, Review and editing the final \n\ndraft. MMS was involved in writing original draft of \n\nthis review article. JL was Project coordinator and \n\nDPG was Project in charge. All authors read and \n\napproved the final manuscript. \n\nCompeting Interest \nThe authors declare that they have no competing \n\ninterest, which includes personal, financial, or any \n\nother kind of relationship with people or \n\norganizations that could inappropriately affect this \n\nreview.", "start_char_idx": 45374, "end_char_idx": 48921, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2b4301a4-1161-4d07-97d7-6f373f0890f8": {"__data__": {"id_": "2b4301a4-1161-4d07-97d7-6f373f0890f8", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "43cf0950-b65e-4b7b-bbb9-3b55892bcb33", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "2493820ba6ac8ff75c1c4b5042d3540708a6f3d03e76fd70d157eb7688096a0f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1c8282e6-ce0f-4ebf-8ad8-b7781c52d0be", "node_type": "1", "metadata": {}, "hash": "8c95c0347d70aced9176b5c89d4b7755618d03a9a7bb90fb6760e2057214c560", "class_name": "RelatedNodeInfo"}}, "text": "Acknowledgments \nIt is our radiant sentiment to place on record our best \n\nregards as well as the deepest sense of gratitude to \n\nthe Department of Biotechnology, School of Science, \n\nKathmandu University, and President of Tarai \n\nMadhesh Chure Conservation Broad for providing \n\nus financial assistance.  \n\nAbbreviations \nBenzyl amino purine (BAP), 6 \u2013Benzyl adenine (BA), \n\nNaphthalene Acetic acid (NAA), Indole 3-Butyric \n\nacid (IBA), Indole Acetic acid (IAA), Zeatin (ZN), \n\nKinetin (KN), Thidiazurn (TDZ), MS (Murashige \n\nand Skoog), Plant Growth Regulators PGRs, \n\nCoconut Water (CW), Adenine Sulphate (Ads) etc. \n\nReferences \n1. Godbole S, Sood A, Thakur R, Sharma M, Ahuja PS. Somatic \n\nembryogenesis and its conversion into plantlets in a \nmultipurpose bamboo, Dendrocalamus hamiltonii Nees et Arn. \n\nEx Munro. Current science. 2002 Oct 10:885-9. \n2. Kumar V, Banerjee M. Albino regenerants proliferation of \n\nDendrocalamus asper in vitro. World Journal of Agricultural \nSciences. 2014;10(1):09-13. \n\n3. Grass Phylogeny Working Group, Barker NP, Clark LG, \n\nDavis JI, Duvall MR, Guala GF, Hsiao C, Kellogg EA, Linder \nHP, Mason-Gamer RJ, Mathews SY. Phylogeny and \n\nsubfamilial classification of the grasses (Poaceae). Annals of \nthe Missouri Botanical Garden. 2001 Jul 1:373-457. \nhttps://doi.org/10.2307/3298585 \n\n4. Clark LG, Londo\u00f1o X, Ruiz-Sanchez E. Bamboo taxonomy \nand habitat. InBamboo 2015 (pp. 1-30). Springer, Cham. \n\nhttps://doi.org/10.1007/978-3-319-14133-6_1 \n5. Kigomo BN. Guidelines for Growing Bamboo. KEFRI \n\nGuideline Series: No. 4. Kenya Forestry Research Institute \n(KEFRI). Downtown Printing Works Ltd. Nairobi, Kenya. \n59pp. 2007. \n\n6. Mudoi KD, Saikia SP, Goswami A, Gogoi A, Bora D, \nBorthakur M. Micropropagation of important bamboos: a \n\nreview. African Journal of Biotechnology. 2013;12(20). \n7. Banik RL. Review of conventional propagation research in \n\nbamboos and future strategy. INBAR. 1994;5:115-42. \n8. Banik RL. Selection criteria and population enhancement of \n\npriority bamboos. INBAR. 1995 May;7:99-110. \n\n9. Banik RL. Bamboo silviculture. InBamboo 2015 (pp. 113-174). \nSpringer, Cham. https://doi.org/10.1007/978-3-319-14133-\n\n6_5 \n10. FAO F. Agriculture Organization: Global Forest Resources \n\nAssessment. FAO, Rome, Italy. 2010. \n\n11. BPG (Bamboo Phylogeny Group). An updated tribal and \nsubtribal classification of the bamboos (Poaceae: \n\nBambusoideae). In: Proceedings of the 9th World Bamboo \nCongress. Antwerp, Belgium: World Bamboo Organization. \n\n2012;3-27. \n12. Soreng RJ, Peterson PM, Romaschenko K, Davidse G, Zuloaga \n\nFO, Judziewicz EJ, Filgueiras TS, Davis JI, Morrone O. A \n\nworldwide phylogenetic classification of the Poaceae \n(Gramineae). Journal of Systematics and Evolution.", "start_char_idx": 48924, "end_char_idx": 51668, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1c8282e6-ce0f-4ebf-8ad8-b7781c52d0be": {"__data__": {"id_": "1c8282e6-ce0f-4ebf-8ad8-b7781c52d0be", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2b4301a4-1161-4d07-97d7-6f373f0890f8", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "d6b20d040aefbba849bba626d5ebca5c2ef3908a2408943b8362eabcffbaab41", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "775bdc2d-34df-457b-9cdc-778418e71c7e", "node_type": "1", "metadata": {}, "hash": "536d27b1a76f0fa066bef0f71794720604b6e9a8d29baa2328d342ca80971e6d", "class_name": "RelatedNodeInfo"}}, "text": "Journal of Systematics and Evolution. 2015 \n\nMar;53(2):117-37. https://doi.org/10.1111/jse.12150 \n13. Bystriakova N, Kapos V, Lysenko I, Stapleton CM. \n\nDistribution and conservation status of forest bamboo \nbiodiversity in the Asia-Pacific Region. Biodiversity & \nConservation. 2003 Sep 1;12(9):1833-41. \n\nhttps://doi.org/10.1023/A:1024139813651 \n14. Shrestha K. Distribution and status of bamboos in Nepal. \n\nNatural History Museum, Plant Department, Nepal. 2001. \n15. Bhattacharya S, Ghosh JS, Sahoo DK, Dey N, Pal A. Screening \n\nof superior fiber-quality-traits among wild accessions of \nBambusa balcooa: efficient and non-invasive evaluation of fiber \ndevelopmental stages. Annals of Forest Science. 2010 \n\nJan;67(6):611 https://doi.org/10.1051/forest/2010024 \n16. Hsiung W. Prospects for bamboo development in the world. \n\nJournal de La American Bamboo Society. 1988;8(1-2):168. \n17. Benton A, Thomson L, Berg P, Ruskin S. Bamboo (various \n\nspecies). Farm And Forestry Production And Marketing. 2011. \n\n18. Gantait S, Pramanik BR, Banerjee M. Optimization of planting \nmaterials for large scale plantation of Bambusa balcooa Roxb.: \n\nInfluence of propagation methods. Journal of the Saudi \nSociety of Agricultural Sciences. 2018 Jan 1;17(1):79-87. \n\nhttps://doi.org/10.1016/j.jssas.2015.11.008 \n19. Tewari DN. Silviculture and management of bamboos in \n\nIndia. A monograph on bamboo. International Book \n\nDistributors, Dehra Dun. 1992:169-86. \n20. Austin AT, Marchesini VA. Gregarious flowering and death \n\nof understorey bamboo slow litter decomposition and \nnitrogen turnover in a southern temperate forest in Patagonia, \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  65 \n\nArgentina. Functional Ecology. 2012 Feb;26(1):265-73. \nhttps://doi.org/10.1111/j.1365-2435.2011.01910.x \n\n21. Singh SR, Singh R, Kalia S, Dalal S, Dhawan AK, Kalia RK. \nLimitations, progress and prospects of application of \n\nbiotechnological tools in improvement of bamboo-a plant \nwith extraordinary qualities. Physiology and Molecular \n\nBiology of Plants. 2013 Jan 1;19(1):21-41. \nhttps://doi.org/10.1007/s12298-012-0147-1 \n\n22. Embaye K. Ecological aspects and resource management of \n\nbamboo forests in Ethiopia. 2003. \n23. Haile B. Study on establishment of bamboo processing plants \n\nin Amhara Regional State. Addis Ababa University. 2008. \n24. Singh J, Sharma R, Dhakad AK, Chauhan SK. Defining \n\ngrowth, quality and biomass production of different bamboo \n\nspecies in central plains of Punjab. Journal of Pharmacognosy \nand Phytochemistry. 2018;7(5):1328-32. \n\n25. Arya S, Satsangi R, Arya ID. Large scale plant production of \nedible bamboo Dendrocalamus asper through somatic \n\nembryogenesis. Bamboo Science & Culture. 2008 Jun 1;21(1). \n26. Ray SS, Ali MN. Factors influencing micropropagation of \n\nbamboo species using nodal explants: A review. Research \n\nJournal of Pharmaceutical Biological and Chemical Sciences. \n2016 Sep 1;7(5):2877-89. \n\n27.", "start_char_idx": 51631, "end_char_idx": 54613, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "775bdc2d-34df-457b-9cdc-778418e71c7e": {"__data__": {"id_": "775bdc2d-34df-457b-9cdc-778418e71c7e", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1c8282e6-ce0f-4ebf-8ad8-b7781c52d0be", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "e00c4a38debae0bae8a4eea32b3ee1aef17b80918efa69b97106ec5de52af7d4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3b5c7ca5-7089-4f4d-b5da-f0fbc945586b", "node_type": "1", "metadata": {}, "hash": "96a841be72899737dd717eac60e68014888656fad6487c82df8f233d60381eb4", "class_name": "RelatedNodeInfo"}}, "text": "2016 Sep 1;7(5):2877-89. \n\n27. Viswanath S. Dendrocalamus stocksii (Munro.): A potential \nmultipurpose bamboo species for Peninsular India. Institute \n\nof Wood Science and Technology; 2013. \n28. Saxena S, Bhojwani SS. In vitro clonal multiplication of 4-year-\n\nold plants of the bamboo, Dendrocalamus longispathus Kurz. In \n\nVitro Cellular & Developmental Biology-Plant. 1993 Jul \n1;29(3):135-42. https://doi.org/10.1007/BF02632285 \n\n29. Mudoi KD, Saikia SP, Borthakur M. Effect of nodal positions, \nseasonal variations, shoot clump and growth regulators on \n\nmicropropagation of commercially important bamboo, \nBambusa nutans Wall. ex. Munro. African Journal of \nBiotechnology. 2014;13(19):1961-72. \n\nhttps://doi.org/10.5897/AJB2014.13659 \n30. Singh SR, Singh R, Kalia S, Dalal S, Dhawan AK, Kalia RK. \n\nLimitations, progress and prospects of application of \nbiotechnological tools in improvement of bamboo-a plant \nwith extraordinary qualities. Physiology and Molecular \n\nBiology of Plants. 2013 Jan 1;19(1):21-41. \nhttps://doi.org/10.1007/s12298-012-0147-1 \n\n31. Kapruwan S, Bakshi M, Kaur M. Rapid in vitro propagation of \nthe solid bamboo, Dendrocalamus strictus nees, through \n\naxillary shoot proliferation. Biotechnology International. \n2014;7(3):58-68. \n\n32. Gillis K, Gielis J, Peeters H, Dhooghe E, Oprins J. Somatic \n\nembryogenesis from mature Bambusa balcooa Roxburgh as \nbasis for mass production of elite forestry bamboos. Plant \n\nCell, Tissue and Organ Culture. 2007 Nov 1;91(2):115-23. \nhttps://doi.org/10.1007/s11240-007-9236-1 \n\n33. Jim\u00e9nez VM, Castillo J, Tavares E, Guevara E, Montiel M. In \nvitro propagation of the neotropical giant bamboo, Guadua \nangustifolia Kunth, through axillary shoot proliferation. Plant \n\nCell, Tissue and Organ Culture. 2006 Sep 1;86(3):389-95. \nhttps://doi.org/10.1007/s11240-006-9120-4 \n\n34. Negi D, Saxena S. In vitro propagation of Bambusa nutans Wall. \nex Munro through axillary shoot proliferation. Plant \n\nBiotechnology Reports. 2011 Jan 1;5(1):35-43. \nhttps://doi.org/10.1007/s11816-010-0154-z \n\n35. Negi D, Saxena S. Micropropagation of Bambusa balcooa Roxb. \n\nthrough axillary shoot proliferation. In Vitro Cellular & \nDevelopmental Biology-Plant. 2011 Oct;47(5):604-10. \n\nhttps://doi.org/10.1007/s11627-011-9403-2 \n36. Devi WS, Bengyella L, Sharma GJ. In vitro seed germination \n\nand micropropagation of edible bamboo Dendrocalamus \n\ngiganteus Munro using seeds. Biotechnology. 2012 Mar \n1;11(2):74-80. https://doi.org/10.3923/biotech.2012.74.80 \n\n37. Oprins J, Grunewald W, Gillis K, Delaere P, Peeters H, Gielis \nJ. Micropropagation: a general method for commercial \n\nbamboo production. InWorld Bamboo Congress 2004 (Vol. 7, \npp. 1-11). \n\n38. Thakur R, Sood A. An efficient method for explant \nsterilization for reduced contamination.", "start_char_idx": 54583, "end_char_idx": 57379, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3b5c7ca5-7089-4f4d-b5da-f0fbc945586b": {"__data__": {"id_": "3b5c7ca5-7089-4f4d-b5da-f0fbc945586b", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "775bdc2d-34df-457b-9cdc-778418e71c7e", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "20405016af279213dd88db004234836381bcc512ac5b70fdda161e6407ee93a5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c00351aa-40a7-4c92-bc1b-4683a0376ea1", "node_type": "1", "metadata": {}, "hash": "5d81c69367820b671022f7316c4ab7b669a68be2cd96d9b52868073b3aca780c", "class_name": "RelatedNodeInfo"}}, "text": "Plant Cell, Tissue and \n\nOrgan Culture. 2006 Mar;84(3):369-71. \nhttps://doi.org/10.1007/s11240-005-9034-6 \n\n39. Yuan JL, Yue JJ, Wu XL, Gu XP. Protocol for callus induction \nand somatic embryogenesis in Moso bamboo. PloS one. 2013 \nDec 11;8(12):e81954. \n\nhttps://doi.org/10.1371/journal.pone.0081954 \n40. Sood A, Nadha HK, Sood S, Walia S, Parkash O. Large scale \n\npropagation of an exotic edible bamboo, Phyllostachys \npubescens Mazel ex H. De Lehale (Moso Bamboo) using seeds. \nIndian J Exp Biol. 2014;52:755-758. \n\n41. Prutpongse P, Gavinlertvatana P. In vitro micropropagation of \n54 species from 15 genera of bamboo. Hort Science. 1992 May \n\n1;27(5):453-4. https://doi.org/10.21273/HORTSCI.27.5.453 \n42. Malini N, Anandakumar CR. Micropropagation of bamboo \n\n(Bambusa vulgaris) through nodal segment. Int J Forest Crop \nImprov. 2013;4:36-9. \n\n43. Sandhu M, Wani SH, Jim\u00e9nez VM. In vitro propagation of \n\nbamboo species through axillary shoot proliferation: a review. \nPlant Cell, Tissue and Organ Culture (PCTOC). 2018 Jan \n\n1;132(1):27-53. https://doi.org/10.1007/s11240-017-1325-1 \n44. Goyal AK, Pradhan S, Basistha BC, Sen A. Micropropagation \n\nand assessment of genetic fidelity of Dendrocalamus strictus \n(Roxb.) nees using RAPD and ISSR markers. 3 Biotech. 2015 \nAug 1;5(4):473-82. https://doi.org/10.1007/s13205-014-0244-\n\n7 \n45. Chowdhury P, Das M, Sikdar SR, Pal A. Influence of the \n\nphysiological age and position of the nodal explants on \nmicropropagation of field-grown Dendrocalamus strictus Nees. \n\nPlant Cell Biotechnology and Molecular Biology. 2004 Mar \n31;5(1-2):45-50. \n\n46. Waikhom SD, Louis B. An effective protocol for \n\nmicropropagation of edible bamboo species (Bambusa tulda \nand Melocanna baccifera) through nodal culture. The Scientific \n\nWorld Journal. 2014 May;2014. \nhttps://doi.org/10.1155/2014/345794 \n\n47. Mishra Y, Patel PK, Yadav S, Shirin F, Ansari SA. A \n\nmicropropagation system for cloning of Bambusa tulda Roxb. \nScientia Horticulturae. 2008 Feb 1;115(3):315-8. \n\nhttps://doi.org/10.1016/j.scienta.2007.10.002 \n48. Arya S, Kant A, Sharma D, Arya ID. Micropropagation of Two \n\nEconomically Important Bamboos: Drepanostachyum falcatum \n(NEES) Keng and Bambusa balcooa Roxb. Indian Forester. 2008 \nSep 1;134(9):1211-21. \n\n49. Anand M, Brar J, Sood A. In vitro propagation of an edible \nbamboo Bam-busa Bambos and assessment of clonal Fidelity \n\nthrough molecular markers. Journal of Medical and \nBioengineering Vol. 2013 Dec;2(4). \n\nhttps://doi.org/10.12720/jomb.2.4.257-261 \n50. Oka GM, Triwiyono A, Awaludin A, Siswosukarto S. Effects \n\nof node, internode and height position on the mechanical \n\nproperties of Gigantochloa atroviolacea bamboo.", "start_char_idx": 57380, "end_char_idx": 60068, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c00351aa-40a7-4c92-bc1b-4683a0376ea1": {"__data__": {"id_": "c00351aa-40a7-4c92-bc1b-4683a0376ea1", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3b5c7ca5-7089-4f4d-b5da-f0fbc945586b", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "49ee9382fc780c310fc380a830500c8fc3fb03cc458ad27ad9c4c7aeaa40b586", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d854ee45-c5e1-49e8-b940-467459158088", "node_type": "1", "metadata": {}, "hash": "b60a09da1e7778ef1f595f5728c75beae092cbcea25477118ecac418431bf10d", "class_name": "RelatedNodeInfo"}}, "text": "Procedia \nEngineering. 2014 Jan 1;95:31-7. \n\nhttps://doi.org/10.1016/j.proeng.2014.12.162 \n51. Patel B, Gami B, Patel N, Bariya V. One step pre-hardening \n\nmicropropagation of Bambusa balcooa Roxb. Journal of \nPhytology. 2015 Nov 10:1-9. \nhttps://doi.org/10.5455/jp.2015-06-02 \n\n52. Ganesan M, Jayabalan N. Carbon source dependent somatic \nembryogenesis and plant regeneration in cotton, Gossypium \n\nhirsutum L. cv. SVPR2 through suspension cultures. I J Exp \nBiol. 2005;43:921-925. \n\n53. Huang LC, Lee YL, Huang BL, Kuo CI, Shaw JF. High \n\npolyphenol oxidase activity and low titratable acidity in  \nbrowning bamboo tissue culture. In Vitro Cellular & \n\nDevelopmental Biology-Plant. 2002 Jul 1;38(4):358. \nhttps://doi.org/10.1079/IVP2002298 \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  66 \n\n54. Kant A, Arya S, Arya ID. Micropropagation protocol for \nMelocanna baccifera using nodal explants from mature clump. \n\nIn8th World Bamboo Congress, Thailand 2009 (pp. 2-12). \n55. George EF, Hall MA, De Klerk GJ. Plant propagation by tissue \n\nculture 3rd Edition. The Netherland, The Back Ground \nSpringer. 2008. \n\n56. Chaturvedi HC, Sharma M, Sharma AK. In vitro regeneration \nof Dendrocalamus strictus Nees through nodal segments taken \nfrom field-grown culms. Plant science. 1993 Jan 1;91(1):97-101. \n\nhttps://doi.org/10.1016/0168-9452(93)90192-3 \n57. Bisht P, Pant M, Kant A. In vitro propagation of Gigantochloa \n\natroviolaceae Widjaja through nodal explants. J Am Sci. \n2010;6:1019-26. \n\n58. Mehta R, Sharma V, Sood A, Sharma M, Sharma RK. \n\nInduction of somatic embryogenesis and analysis of genetic \nfidelity of in vitro-derived plantlets of Bambusa nutans Wall., \n\nusing AFLP markers. European Journal of Forest Research. \n2011 Sep 1;130(5):729-36. https://doi.org/10.1007/s10342-\n\n010-0462-4 \n59. Nadha HK, Rahul K, Sharma RK, Anand M, Sood A. In vitro \n\npropagation of Dendrocalamus asper and testing the clonal \n\nfidelity using RAPD and ISSR markers. International Journal \nof Current Research. 2013;5(8):2060-7. \n\n60. Kiran A, Ansari SA. Adventitious rhizogenesis in relation to \nseasonal variation, size of culm branch cuttings and IAA \n\ntreatment in bamboos. Indian forester. 2000;126(9):971-84. \n61. Mudoi KD, Borthakur M. In vitro micropropagation of \n\nBambusa balcooa Roxb. through nodal explants from field-\n\ngrown culms and scope for upscaling. Current science. 2009 \nApr 10:962-6. \n\n62. Singh SR, Dalal S, Singh R, Dhawan AK, Kalia RK. \nMicropropagation of Dendrocalamus asper {Schult. & Schult. F.} \n\nBacker ex k. Heyne): an exotic edible bamboo. Journal of Plant \nBiochemistry and Biotechnology.", "start_char_idx": 60069, "end_char_idx": 62726, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d854ee45-c5e1-49e8-b940-467459158088": {"__data__": {"id_": "d854ee45-c5e1-49e8-b940-467459158088", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c00351aa-40a7-4c92-bc1b-4683a0376ea1", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "99c2042093ebe2290b91e23e411b0ec480f9a40155c7e4b0fc106bdcd5a66335", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "36c2289d-50c0-4247-8c6c-e9d5c3f8c13d", "node_type": "1", "metadata": {}, "hash": "3775ec12235ce1e0e5b3c6dc101aa095602fe9ce9c9b2c14939a9f2d91e6e222", "class_name": "RelatedNodeInfo"}}, "text": "Journal of Plant \nBiochemistry and Biotechnology. 2012 Jul 1;21(2):220-8. \nhttps://doi.org/10.1007/s13562-011-0095-9 \n\n63. Sivabalan S, Ramkumar P. Detection of albino in \nmicropropagated shoots of Bambusa balcooa Roxb, using PCR \n\nbased techniques. International Journal of Engineering \nResearch. 2014 Mar;3(3). \n\n64. Ramanayake SM, Yakandawala K. Micropropagation of the \n\ngiant bamboo (Dendrocalamus giganteus Munro) from nodal \nexplants of field grown culms. Plant Science. 1997 Nov \n\n21;129(2):213-23. https://doi.org/10.1016/S0168-\n9452(97)00185-4 \n\n65. Rathore TS, Rai VR. Micropropagation of Pseudoxytenanthera \nstocksii Munro. In Vitro Cellular & Developmental Biology-\nPlant. 2005 May 1;41(3):333-7. \n\nhttps://doi.org/10.1079/IVP2004625 \n66. Singh SR, Dalal SU, Singh RO, Dhawan AK, Kalia RK. \n\nSeasonal influences on in vitro bud break in Dendrocalamus \nhamiltonii Arn. ex Munro nodal explants and effect of culture \n\nmicroenvironment on large scale shoot multiplication and \nplantlet regeneration. Indian J Plant Physiol. 2012;17:9-21. \n\n67. Das M, Pal A. In vitro regeneration of Bambusa balcooa Roxb.: \n\nfactors affecting changes of morphogenetic competence in the \naxillary buds. Plant cell, tissue and organ culture. 2005 Apr \n\n1;81(1):109-12. https://doi.org/10.1007/s11240-004-3017-x \n68. Gantait S, Mandal N, Nandy S. Advances in \n\nmicropropagation of selected aromatic plants: a review on \nvanilla and strawberry. Am J Biochem Mol Biol. 2011;1(1):1-9. \nhttps://doi.org/10.3923/ajbmb.2011.1.19 \n\n69. Nayak S, Hatwar B, Jain A. Effect of cytokinin and auxins on \nmeristem culture of Bambusa arundinacea. Der Pharmacia \n\nLetter. 2010;2(1):408-14. \n70. Devi WS, Sharma GJ. In vitro propagation of Arundinaria \n\ncallosa Munro-an edible bamboo from nodal explants of \n\nmature plants. The Open Plant Science Journal. 2009 Jul 2;3(1). \nhttps://doi.org/10.2174/1874294700903010035 \n\n71. Hirimburegama K, Gamage N. Propagation of Bambusa \nvulgaris (yellow bamboo) through nodal bud culture. Journal \n\nof Horticultural Science. 1995 Jan 1;70(3):469-75. \nhttps://doi.org/10.1080/14620316.1995.11515317 \n\n72. Sood A, Ahuja PS, Sharma M, Sharma OP, Godbole S. In vitro \nprotocols and field performance of elites of an important \n\nbamboo Dendrocalamus hamiltonii Nees et Arn. Ex Munro. \nPlant Cell, Tissue and Organ Culture. 2002 Oct 1;71(1):55-63. \n\nhttps://doi.org/10.1023/A:1016582732531 \n73. Arya ID, Satsangi R, Arya S. Rapid micropropagation of \n\nedible bamboo Dendrocalamus asper. Journal of Sustainable \n\nForestry. 2001 Aug 28;14(2-3):103-14. \nhttps://doi.org/10.1300/J091v14n02_06 \n\n74. Jha A, Das S, Kumar B. Micropropagation of Dendrocalamus \nhamiltonii through nodal explants. Global Journal of Bio-\nScience and Biotechonolgy.", "start_char_idx": 62677, "end_char_idx": 65420, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "36c2289d-50c0-4247-8c6c-e9d5c3f8c13d": {"__data__": {"id_": "36c2289d-50c0-4247-8c6c-e9d5c3f8c13d", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d854ee45-c5e1-49e8-b940-467459158088", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "8ad59e30f762cf549dbe5bcb361c32b1dc4a8fe611e75b0ca3f52029c99e057a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b9db412a-4e50-4b9a-a472-9dd0c80e6f77", "node_type": "1", "metadata": {}, "hash": "27d360ab2ec2d140b48b8283c1206c3894a7ff9ec2211c7cf602a23751e98b08", "class_name": "RelatedNodeInfo"}}, "text": "Global Journal of Bio-\nScience and Biotechonolgy. 2013;2(4):580-2. \n\n75. Pandey BN, Singh NB. Micropropagation of Dendrocalamus \nstrictus nees from mature nodal explants. Journal of Applied \n\nand Natural Science. 2012 Jun 1;4(1):5-9. \nhttps://doi.org/10.31018/jans.v4i1.213 \n\n76. Raju RI, Roy SK. Mass propagation of Bambusa bambos (L.) \nVoss through in vitro culture. Jahangirnagar University \nJournal of Biological Sciences. 2016;5(2):15-26. \n\nhttps://doi.org/10.3329/jujbs.v5i2.32514 \n77. Cheah KT, Chaille LC. Somatic embryogenesis from mature \n\nBambusa ventricosa. College of Tropical Agriculture and \nHuman Resources, University of Hawaii. 2011. \n\n78. Wei Q, Cao J, Qian W, Xu M, Li Z, Ding Y. Establishment of \nan efficient micropropagation and callus regeneration system \nfrom the axillary buds of Bambusa ventricosa. Plant Cell, Tissue \n\nand Organ Culture (PCTOC). 2015 Jul 1;122(1):1-8. \nhttps://doi.org/10.1007/s11240-015-0743-1 \n\n79. Arya ID, Arya S. In vitro shoot proliferation and somatic \nembryogenesis: means of rapid bamboo multiplication. In10th \n\nWorld Bamboo Congress, Propagation, Plantation and \nManagement, Korea 2015. \n\n80. Arshad SM, Kumar A, Bhatnagar SK. Micropropagation of \n\nBambusa wamin through shoot proliferation of mature nodal \nexplants. Journal of Biological Research. 2005;3:59-66. \n\n81. Saxena S. In vitro propagation of the bamboo (Bambusa tulda \nRoxb.) through shoot proliferation. Plant cell reports. 1990 \nDec 1;9(8):431-4. https://doi.org/10.1007/BF00232266 \n\n82. Das M, Pal A. Clonal propagation and production of \ngenetically uniform regenerants from axillary meristems of \n\nadult bamboo. Journal of Plant biochemistry and \nbiotechnology. 2005 Jul 1;14(2):185-8. \n\nhttps://doi.org/10.1007/BF03355956 \n83. Murashige T, Skoog F. A revised medium for rapid growth \n\nand bio assays with tobacco tissue cultures. Physiologia \n\nplantarum. 1962 Jul;15(3):473-97. \nhttps://doi.org/10.1111/j.1399-3054.1962.tb08052.x \n\n84. Garc\u00eda-Ram\u00edrez Y, Gonz\u00e1les MG, Mendoza EQ, Seijo MF, \nC\u00e1rdenas ML, Moreno-Berm\u00fadez LJ, Ribalta OH. Effect of BA \n\ntreatments on morphology and physiology of proliferated \nshoots of Bambusa vulgaris Schrad. Ex Wendl in temporary \nimmersion. American Journal of Plant Sciences. 2014 Jan \n\n24;2014. https://doi.org/10.4236/ajps.2014.52027 \n85. Lin CS, Kalpana K, Chang WC, Lin NS. Improving multiple \n\nshoot proliferation in bamboo mosaic virus-free Bambusa \noldhamii Munro propagation by liquid culture. Hortscience. \n\n2007 Aug 1;42(5):1243-6. \nhttps://doi.org/10.21273/HORTSCI.42.5.1243 \n\n86. Kavitha B, Kiran S. An Efficient Technique for in vitro \n\npropagation of Dendrocalamus Brandisii Kurz using Nodal \nSegments. Global Journal of Microbiology and Biotechnology. \n\n2014;2(1):1-0. \n87. Somashekar PV, Rathore TS, Shashidhar KS.", "start_char_idx": 65371, "end_char_idx": 68161, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b9db412a-4e50-4b9a-a472-9dd0c80e6f77": {"__data__": {"id_": "b9db412a-4e50-4b9a-a472-9dd0c80e6f77", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "36c2289d-50c0-4247-8c6c-e9d5c3f8c13d", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "6f187175cf36b185585805f5ec57152fcda684835ebcdc22358b103b5f48d1d6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e0532fe4-d691-4590-8b69-ceb2a8d3ad70", "node_type": "1", "metadata": {}, "hash": "337c0e9113e2f3948f89133eaf848ff668d9d437c87a652827a72a2a7cfca7c8", "class_name": "RelatedNodeInfo"}}, "text": "87. Somashekar PV, Rathore TS, Shashidhar KS. Rapid and \n\nsimplified method of micropropagation of Pseudoxytenanthera \n\nstocksii. Ansari SA, Narayanan C, Mandal AK. Forest \nbiotechnology in India. Delhi: Satish serial publishing house. \n\n2008:165-82. \n88. Sharma P, Sarma KP. In vitro propagation of Bambusa balcooa \n\nfor a better environment. InInternational Conference on \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  67 \n\nAdvances in Biotechnology and Pharmaceutical Sciences. \nBangkok: Planetary Scientific Research Centre 2011 Dec (pp. \n\n248-252). \n89. Pratibha S, Sarma KP. In vitro propagation of Bambusa tulda: \n\nAn important plant for better environment. Journal of \nEnvironmental Research and Development. 2013 Jan;7:1216-\n\n23. \n90. Pratibha S, Sarma KP. In vitro propagation of Bambusa nutan \n\nin commercial scale in assam, India. Journal of Environmental \n\nResearch and Development. 2014 Oct 1;9(2):348.. \n91. Bag N, Chandra S, Palni LM, Nandi SK. Micropropagation of \n\nDev-ringal [Thamnocalamus spathiflorus (Trin.) Munro]-a \ntemperate bamboo, and comparison between in vitro \npropagated plants and seedlings. Plant Science. 2000 Jul \n\n28;156(2):125-35. https://doi.org/10.1016/S0168-\n9452(00)00212-0 \n\n92. Bonga JM, Aderkas P, von Aderkas P. In vitro culture of trees. \nSpringer Science & Business Media; 1992 May 31. \n\nhttps://doi.org/10.1007/978-94-015-8058-8 \n93. Venkatachalam P, Kalaiarasi K, Sreeramanan S. Influence of \n\nplant growth regulators (PGRs) and various additives on in \n\nvitro plant propagation of Bambusa arundinacea (Retz.) Wild: A \nrecalcitrant bamboo species. Journal of Genetic Engineering \n\nand Biotechnology. 2015 Dec 1;13(2):193-200. \nhttps://doi.org/10.1016/j.jgeb.2015.09.006 \n\n94. Kalaiarasi K, Sangeetha P, Subramaniam S, Venkatachalam P. \nDevelopment of an efficient protocol for plant regeneration \nfrom nodal explants of recalcitrant bamboo (Bambusa \n\narundinacea Retz. Willd) and assessment of genetic fidelity by \nDNA markers. Agroforestry systems. 2014 Jun 1;88(3):527-37. \n\nhttps://doi.org/10.1007/s10457-014-9716-3 \n95. Nadgauda RS, Parasharami VA, Mascarenhas AF. Precocious \n\nflowering and seeding behaviour in tissue-cultured bamboos. \nNature. 1990 Mar;344(6264):335-6. \nhttps://doi.org/10.1038/344335a0 \n\n96. Tuan TT, Tu HL, Du TX. The increase in in vitro shoot \nmultiplication rate of Dendrocalamus asper (Schult. f.) Back. ex \n\nHeyne. TAP CHI SINH HOC. 2012 Aug 6;34(3se):257-64. \nhttps://doi.org/10.15625/0866-7160/v34n3se.1790 \n\n97. Yasodha R, Kamala S, Kumar SA, Kumar PD, Kalaiarasi K. \n\nEffect of glucose on in vitro rooting of mature plants of \nBambusa nutans. Scientia Horticulturae.", "start_char_idx": 68116, "end_char_idx": 70809, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e0532fe4-d691-4590-8b69-ceb2a8d3ad70": {"__data__": {"id_": "e0532fe4-d691-4590-8b69-ceb2a8d3ad70", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b9db412a-4e50-4b9a-a472-9dd0c80e6f77", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "e65b5e48aed95e5cd5bd10bf2ceb702f187b021363f2256556ee8a84b833fbb4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1bd13267-a843-4622-b535-7cde974b7c0b", "node_type": "1", "metadata": {}, "hash": "c75dc8b07e39bca0d01a1ded752201f4e8c02aee902dd501f43406eb4be6bfa5", "class_name": "RelatedNodeInfo"}}, "text": "Scientia Horticulturae. 2008 Mar \n\n10;116(1):113-6. \nhttps://doi.org/10.1016/j.scienta.2007.10.025 \n\n98. Das M, Pal A. In vitro regeneration of Bambusa balcooa Roxb.: \nfactors affecting changes of morphogenetic competence in the \naxillary buds. Plant cell, tissue and organ culture. 2005 Apr \n\n1;81(1):109-12. https://doi.org/10.1007/s11240-004-3017-x \n99. Shirin F, Rana PK. In vitro plantlet regeneration from nodal \n\nexplants of field-grown culms in Bambusa glaucescens Willd. \nPlant Biotechnology Reports. 2007 Aug 1;1(3):141-7. \n\nhttps://doi.org/10.1007/s11816-007-0020-9 \n100. Lin CS, Chang WC. Micropropagation of Bambusa edulis  \n\nthrough nodal explants of field-grown culms and flowering of \n\nregenerated plantlets. Plant cell reports. 1998 May 1;17(8):617-\n20. https://doi.org/10.1007/s002990050453 \n\n101. Deb CR, Imchen T. An Efficient In vitro Hardening Technique \nof Tissue Culture Raised Plants. Biotechnology. 2010;9(1):79-\n\n83. https://doi.org/10.3923/biotech.2010.79.83 \n102. Agnihotri RK, Nandi SK. In vitro shoot cut: a high frequency \n\nmultiplication and rooting method in the bamboo \n\nDendrocalamus hamiltonii. Biotechnology. 2009;8(2):259-63. \nhttps://doi.org/10.3923/biotech.2009.259.263 \n\n103. Banerjee M, Gantait S, Pramanik BR. A two step method for \naccelerated mass propagation of Dendrocalamus asper and their \nevaluation in field. Physiology and Molecular Biology of \n\nPlants. 2011 Oct 1;17(4):387. https://doi.org/10.1007/s12298-\n011-0088-0 \n\n104. Diab EE, Mohamed SE. In Vitro Morphogenesis And Plant \nRegeneration Of Bamboos (Oxytenanthera abyssinica A. Rich. \n\nMunro). Int. J. Sustain. Crop Prod. 2008 Oct;3(6):72-9. \n\n105. Ramanayake SM, Maddegoda KM, Vitharana MC, Chaturani \nGD. Root induction in three species of bamboo with different \n\nrooting abilities. Scientia Horticulturae. 2008 Oct 1;118(3):270-\n3. https://doi.org/10.1016/j.scienta.2008.06.004 \n\n106. Ramanayake SM, Yakandawala K, Nilmini Deepika PK, Ikbal \nMC. Studies on micropropagation of Dendrocalamus gigateus  \n\nand Bambusa vulgaris var. striata. Bamboo, people and the \nenvironment. 1995;1:75-85. \n\n107. Rathore TS, Kabade U, Jagadish MR, Somashekar PV, \n\nViswanath S. Micropropagation and evaluation of growth \nperformance of the selected industrially important bamboo \n\nspecies in southern India. InProc 8th World Bamboo Congress, \nBankok, Thialand 2009 (pp. 41-55). \n\n108. Ravikumar R, Ananthakrishnan G, Kathiravan K, Ganapathi \n\nA. In vitro shoot propagation of Dendrocalamus strictus nees. \nPlant cell, tissue and organ culture. 1998 Mar 1;52(3):189-92. \n\nhttps://doi.org/10.1023/A:1006092620731 \n109. Singh M, Jaiswal U, Jaiswal VS.", "start_char_idx": 70786, "end_char_idx": 73429, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1bd13267-a843-4622-b535-7cde974b7c0b": {"__data__": {"id_": "1bd13267-a843-4622-b535-7cde974b7c0b", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e0532fe4-d691-4590-8b69-ceb2a8d3ad70", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "d1878ca1482abc78c0013fdd14c4b89308e56e69ddd8be404499b0c6385100b5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3ee28cc7-93aa-4a50-9785-82c1772f61c2", "node_type": "1", "metadata": {}, "hash": "58696c9a5e9fcbcf63341f5c7b348af8fe50d29cb620822238ea127bb8bae88b", "class_name": "RelatedNodeInfo"}}, "text": "Singh M, Jaiswal U, Jaiswal VS. Thidiazuron-induced shoot \n\nmultiplication and plant regeneration in bamboo \n(Dendrocalamus strictus Nees). Journal of Plant Biochemistry \nand Biotechnology. 2001 Jul 1;10(2):133-7. \n\nhttps://doi.org/10.1007/BF03263122 \n110. Kant A, Arya S, Arya ID. Micropropagation protocol for \n\nMelocanna baccifera using nodal explants from mature clump. \nIn8th World Bamboo Congress, Thailand 2009 (pp. 2-12). \n\n111. Beena DB, Rathore TS. In vitro cloning of Bambusa pallida \nMunro through axillary shoot proliferation and evaluation of \ngenetic fidelity by random amplified polymorphic DNA \n\nmarkers. International Journal of Plant Biology. 2012 Dec \n10;3(1):e6-. https://doi.org/10.4081/pb.2012.e6 \n\n112. Islam SA, Rahman MM. Micro-cloning in commercially \nimportant six bamboo species for mass propagation and at a \n\nlarge scale cultivation. Plant Tissue Cult Biotech. 2005;15:103-\n11. \n\n113. Ndiaye A, Diallo MS, Niang D, Gassama-Dia YK. In vitro \n\nregeneration of adult trees of Bambusa vulgaris. African \nJournal of Biotechnology. 2006;5(13). \n\n114. Nissen SJ, Sutter EG. Stability of IAA and IBA in nutrient \nmedium to several tissue culture procedures. HortScience. \n1990 Jul 1;25(7):800-2. \n\nhttps://doi.org/10.21273/HORTSCI.25.7.800 \n115. Ogita S, Kashiwagi H, Kato Y. In vitro node culture of \n\nseedlings in bamboo plant, Phyllostachys meyeri McClure. \nPlant Biotechnology. 2008 Jun 25;25(4):381-5. \n\nhttps://doi.org/10.5511/plantbiotechnology.25.381 \n116. Paranjothy K, Saxena S, Banerjee M, Jaiswal VS, Bhojwani SS. \n\nClonal multiplication of woody perennials. InDevelopments \n\nin crop science 1990 Jan 1 (Vol. 19, pp. 190-219). Elsevier. \nhttps://doi.org/10.1016/B978-0-444-88883-9.50012-1 \n\n117. Roy SS, Ali MN, Gantait S, Chakraborty S, Banerjee M. Tissue \nculture and biochemical characterization of important \n\nbamboos. Research Journal of Agricultural Sciences. 2014 \nMar;5(2):135-46. \n\n118. Thiruvengadam M, Rekha KT, Chung IM. Rapid in vitro \n\nmicropropagation of Bambusa oldhamii Munro. Philippine \nAgricultural Scientist. 2011;94(1):7-13. \n\n119. Sharma SK, Kalia S, Kalia RK. Rapid In-Vitro Regeneration \nfrom 40-Year-old Clump of Bambusa nutans wall. Ex Munro. \n\nThe Journal of Indian Botanical Society. 2012;91(4):365-78. \n120. Yasodha R, Kamala S, Kalaiarasi K. Anatomical and \n\nbiochemical changes associated with in vitro rhizogenesis in \n\nDendrocalamus giganteus. Journal of Plant Biochemistry and \nBiotechnology. 2010 Jul 1;19(2):217-22. \n\nhttps://doi.org/10.1007/BF03263343 \n121. Cruz-Mart\u00edn M, Garc\u00eda-Ram\u00edrez Y, S\u00e1nchez-Garc\u00eda C, \n\nAlvarado-Cap\u00f3 Y, Acosta-Su\u00e1rez M, Roque B, Leiva-Mora M, \n\nFreire-Seijo M. Identificaci\u00f3n y control de Bacillus sp., \ncontaminante del establecimiento in vitro de Guadua \n\nangustifolia Kunth.", "start_char_idx": 73398, "end_char_idx": 76173, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3ee28cc7-93aa-4a50-9785-82c1772f61c2": {"__data__": {"id_": "3ee28cc7-93aa-4a50-9785-82c1772f61c2", "embedding": null, "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-174", "node_type": "4", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "a1744aa869453c719b564434a06e2e9b4b8456c9039e8eeccae9c9467257b6a9", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1bd13267-a843-4622-b535-7cde974b7c0b", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "68ea1241a3d7a69a9c730128e3ec7b6976d56c002672282c69aa945cb8febc73", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "40b25736-dfac-444c-bc12-2468b7c33a68", "node_type": "1", "metadata": {}, "hash": "a623178dddfa63b6ab391cd1f2f2383da7bc371ff88d9c374587cdaa45eb1522", "class_name": "RelatedNodeInfo"}}, "text": "Biotecnolog\u00eda vegetal. 2007 Jan 5;7(1). \n122. Nadha HK, Salwan R, Kasana RC, Anand M, Sood A. \n\nIdentification and elimination of bacterial contamination \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):54-68       Suwal et al.  \n\n \n\n\u00a9NJB, BSN  68 \n\nduring in vitro propagation of Guadua angustifolia Kunth. \nPharmacognosy magazine. 2012 Apr;8(30):93. \n\nhttps://doi.org/10.4103/0973-1296.96547 \n123. Chang WC, Ho CW. Micropropagation of bamboos. InHigh-\n\nTech and Micropropagation V 1997 (pp. 203-219). Springer, \n\nBerlin, Heidelberg. https://doi.org/10.1007/978-3-662-\n07774-0_13 \n\n124. Thapa N, Gauchan DP, Suwal MM, Bhuju S, Upreti A, Byanju \nB, Lamichhane J. In Vitro Assessment of Bambusa balcooa Roxb. \n\nFor Micropropagation. Journal of Emerging Technologies and \nInnovative Research. 2018;5(12).", "start_char_idx": 76174, "end_char_idx": 76975, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "40b25736-dfac-444c-bc12-2468b7c33a68": {"__data__": {"id_": "40b25736-dfac-444c-bc12-2468b7c33a68", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3ee28cc7-93aa-4a50-9785-82c1772f61c2", "node_type": "1", "metadata": {"identifier": "njb-174", "author": "Suwal, Meena Maiya; Lamichhane, Janardan; Gauchan, Dhurva Prasad", "title": "Regeneration Technique of Bamboo Species through Nodal Segments: A Review", "date": "2020-07-31", "file": "njb-174.pdf"}, "hash": "55ea5e3bbb543490d68e67321b26477d0bc268ec6e214df1aad3a2720b5af1ad", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5b8fb342-03a4-45ff-bc53-31df83375db4", "node_type": "1", "metadata": {}, "hash": "be836e9f56516d5a456e82a3d03f1d2071b8c7b3baef93dd5813b0f48c942264", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 DOI: https://doi.org/10.3126/njb.v8i1.30208 Research article \n\n\u00a9NJB, BSN  36 \n\nStandardization of Ayurvedic Drug- Nyctanthes arbor-tristis, \nHippophae salicifolia, Ocimum tenuiflorum and Reinwardtia \nindica and Combined Herb-Herb \nRinki Kumari1             , G.P. Dubey1, 2 \n\n1Advanced Centre for Traditional and Genomic Medicine, Institute of Medical Science, Banaras Hindu \n\nUniversity, Varanasi, Uttar Pradesh -221 005, India \n2Genome foundation, Kalwari, Sikrara, Jaunpur, Uttar Pradesh - 222131, India \nArticle history:- Received: 4 Oct 2019; Revised: 11 Dec 2019; Accepted: 25 Dec 2019; Published online: 31 Jul 2020 \n\nAbstract  \nThe herbal medicines have reached extensive acceptability as therapeutic agents for various clinical diseases \ndue to global demand. Therefore, standardization is the essential and initial step to drug development. It is \nfor the establishment of consistent biological activity, a consistent chemical profile and biomarker \nidentification. It improves the safety and efficacy of herbal medicine to provide the best herbal medicine to \nsociety and increase popularity rather than non-standardized extracts.  In addition, it is essential to practice \nor maintain a quality assurance program for the production and manufacturing of herbal medicine that \nincludes the basis of organoleptic characters and photomicrographs, physicochemical, proximate analysis \nphytochemical evaluation and quality control analysis and order to assess the quality of drugs, based on the \nconcentration of their active principles. WHO has provided specific guidelines for the assessment of the \nsafety, efficacy and quality of herbal drugs as a prerequisite for global harmonization and of utmost \nimportance. In the present study, the herbal extracts were cleaned, dried in the shade and powdered by \npassing through the sieve as per the method described in the standard protocol. An overview covering \nvarious techniques employed in the extraction and characterization of Nyctanthes arbortristis, Hippophae \nsalicifolia, Ocimum tenuiflorum and Reinwardtia indica, standardization is reported in this study. The obtained \n\ndata would be very significant for future clinical aspects, as the bioactive molecules present in the extracts \nmay exhibit synergistic effect with other bioactive compound and show a better therapeutic value. Thus, this \nstudy provides standardized and therapeutically potential data of active polyherbal formulations for the \ndifferent ailments. \n\nKeywords: Physicochemical, Polyherbal formulation, Single, Herb-Herb combination, Flavonoids. \n\n    Corresponding author, email: rinkiv3@gmail.com \n\nIntroduction \nIn recent years, standardization is the essential \n\nparameter to assess the quality of any type of drugs \n\nand more important for the quality assessment of \n\nherbal formulations to justify their acceptability in \n\nthe modern medical system. It plays a crucial role \n\nto assess the quality, purity, safety and efficacy of \n\nsome drugs including herbal medicine. \n\nStandardization is more important in the drug \n\ndiscovery area due to lack of scientific evidence \n\nsuch as pharmacognostic evaluation, \n\nphytochemical study, and pharmacological \n\nevaluation of the polyherbal formulation. \n\nNowadays attention has been directed towards the \n\nutilization of Ayurvedic plants in the prevention \n\nand management of clinical diseases so that the \n\ntrend of using herbal medicines has increased in a \n\ntremendous mode in the last decade [1]. \n\nConsequently, the World Health Organization \n\n(WHO) has taken a broader step in the clinical \n\nsystem and also in phytotherapy. Sponsorship and \n\nencouragement of studies substantiating \n\nparameters of related to standardization of herbal \n\nmedicines of Ayurveda and ancient system are \n\nunder principal consideration of WHO for new \n\ndrug discovery [2].", "start_char_idx": 48, "end_char_idx": 3938, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5b8fb342-03a4-45ff-bc53-31df83375db4": {"__data__": {"id_": "5b8fb342-03a4-45ff-bc53-31df83375db4", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "40b25736-dfac-444c-bc12-2468b7c33a68", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "dede20bef2b3ceec523431d291ec463a2ec4074565690eacafe7bcd7033913ba", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5a4207cb-4890-4adb-b25d-dc14ced3bdcc", "node_type": "1", "metadata": {}, "hash": "173de9ebc93d88909a50392f18eeee9e2e4fc442195e041a83f89d4fe77729cf", "class_name": "RelatedNodeInfo"}}, "text": "Ayurvedic practitioners have developed several \n\nherbal medicines by using single herb or herb-herb \n\ncombinations or as a polyherbal formulation, which \n\nwere used in ancient traditional medical practice \n\nfor many years. These medicines offer several \n\noptions to modify the progress and symptoms of \n\nvarious diseases [3-6]. Globally, both types of \n\nmailto:rinkiv3@gmail.com\nhttps://orcid.org/0000-0003-4720-795X\nmailto:joshibalak@yahoo.com\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  37 \n\ncommercial and non-commercial single herbal \n\nmedicine or polyhedral formulations have been \n\ndeveloped, but many polyherbal formulations lack \n\nscientific evidence such as pharmacognostic, \n\nphytochemical and pharmacological evaluation [4]. \n\nNyctanthes arbor-tristis (N. arbor-tristis) Linn. is \n\nknown as Parijat belonging to the Oleaceae family. \n\nThe therapeutic utility of N. arbor-tristis has been \n\ndescribed in various classical texts of Ayurveda for \n\nthe management of various mental and physical \n\ndisorders. In Sushruta Sutra Sthana; the yoga \n\nprepared out of N. arbor-tristis, along with other \n\ndrugs is used for a good sleep, for the management \n\nof diabetes, stomach disorders, epilepsy and many \n\nmore [3-6]. N. arbor-tristis is used in combination \n\nwith other drugs for the management of \n\nksharaagada and also for the purification of \n\npoisons. According to Sushruta Uttar Tantra, N. \n\narbor-tristisis was also given for the management of \n\nvarious diseases [7-11]. \n\nThe word Hippophae (Latin word) \u2018Hippo\u2019 \n\nmeaning horse and \u2018Phaos\u2019 means \u2018shine\u2019, and \n\nHippophae salicifolia known as Sea buckthorn (SBT). \n\nIt was used as a medicinal plant in Tibet as early as \n\n900 AD. H. salicifolia have has been used as a herbal \n\nmedicine for many years globally not only as \n\ntherapeutic but also as prophylactic and health \n\npromotive agents. Recently, sea buckthorn \n\nElaeagnaceae, a unique and valuable plant has \n\ngained worldwide attention, mainly for its \n\nmedicinal,  nutritional potential and its edible fruit \n\nwhich is rich in vitamins, often made into jam.  In \n\nGreece, H. salicifolia leaves and twigs were used to \n\nfeed animals, which resulted in weight gain and a \n\nshining coat, especially in horses. It has a rich \n\nhistory of use in treating numerous medical \n\nconditions. Many of its pharmacological effects \n\nhave been recorded in classics such as SibuYidian \n\nfrom the Tang Dynasty and Jing Zhu Ben Cao from \n\nthe Qing Dynasty [12-14]. \n\nOcimum tenuiflorum, also known as, Vishnu Priya, \n\nholy basil, or tulsi (also spelt Tulasi), is an Indian \n\naromatic plant and belongs to the family \n\nLamiaceae, and cultivated throughout the \n\nSoutheast Asian tropics. Tulsi (Sanskrit:-Surasa) has \n\nbeen used for thousands of years in Ayurveda for \n\nits diverse healing properties.). From ancient times, \n\nTulsi extracts were used as ayurvedic remedies for \n\na variety of clinical ailments. It is mentioned in the \n\nCharaka Samhita, an ancient Ayurvedic text in \n\nSutrasthan for skin disorders [15], in Shwashara \n\nMahakashaya [16], for Nadisweda in Vatakapha \n\ndisorders (Neuro-muscular disorders) [17], in \n\nHarita Varga for Hikka (Hiccough), Kasa (Cough), \n\nVishavikara (Toxins), Swas (Asthma), Parshvashula \n\n(Pain in Chest) and Durgandhanashaka (for \n\nfragrance) [18].", "start_char_idx": 3941, "end_char_idx": 7275, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5a4207cb-4890-4adb-b25d-dc14ced3bdcc": {"__data__": {"id_": "5a4207cb-4890-4adb-b25d-dc14ced3bdcc", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5b8fb342-03a4-45ff-bc53-31df83375db4", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "562071fcb953748a9cd6413b498fc04daafbfba8e0f2102b3083fcb6710cb61f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "485c6bf1-0089-44d0-8252-563130558669", "node_type": "1", "metadata": {}, "hash": "8e8b5ef55d09bf1e5d6ce3224159d24897c74bdcd4394925cda9959da17a09bf", "class_name": "RelatedNodeInfo"}}, "text": "In Vimanasthanatulsi has been \n\ndescribed for the treatment of worn disorders and \n\nPanchakarma [19, 20], Katuskandha [21]. \n\nReinwardtia indica Dumort. (R. indica Dumort) is \n\nalso known as Basanti and belong to as flax family \n\n[22]. The medicinal power of R. indica mainly \n\ndepends on phytochemical constituents that have \n\ngreat pharmacological significance. R. indica extract \n\nis widely used by local communities for different \n\npurposes such as for tongue wash, to bring about \n\nan increase in - lactation period, a remedy against \n\nskin diseases, as an anti-infection agent etc. [23-26] \n\nas well as in the development of several polyherbal \n\nformulations.  \n\nThe selection of these four herbs was based upon its \n\nactivity like an antidepressant, antioxidant and \n\nanti-inflammatory. This study reports on the \n\nstandardization of polyherbal formulation, based \n\non organoleptic assessment, photomicrographs, \n\nphysicochemical properties, proximate examination \n\nphytochemical assessment and quality control \n\ninvestigation. \n\nMaterials and methods \nPlant Material  \nLeaves of N. arbor-tristis, O. tenuiflorum and R. indica \n\nwere collected from the forests of India. Fruits of H. \n\nsalicifolia was collected from Himachal Pradesh \n\n(Leh & Ladakh). These plant extracts were \n\nidentified, authenticated and voucher specimens of \n\nthe plants have been deposited (Accession No.: SH-\n\n2010, LH-2008, SH-2008 & SH-2008) in the \n\nherbarium for further reference. \n\nFresh plant materials of these plants were taken for \n\nthe medicinal purpose used for microscopic study. \n\nThe collected materials of the plants were washed \n\nwith water and dried in the shade at room \n\ntemperature and sieved. The dried parts were \n\ngrinded to a coarse powder. The powdered drug \n\nwas stored in an airtight and light-resistant \n\ncontainer for the study.  \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  38 \n\nChemicals \nAll the chemicals (Toluene: Ethyl acetate, \n\nPetroleum ether, silica gel, Chloroform, Ethanol, \n\nMethanol, NaOH, HCl, HNO3 and Nutrient agar, \n\nSalmonella Shigella Agar (SSA) and Potato \n\nDextrose Agar (PDA) Media etc. used, were of \n\nanalytical grade and were obtained from E. Merck \n\nLimited India and Hi-Media Laboratories, Mumbai, \n\nIndia. \n\nOrganoleptic evaluation \nVarious sensory parameters of the plant material \n\n(such as colour, odour, size, shape, and taste) were \n\nstudied by organoleptic evaluation [27]. \n\nPharmacognostic studies \nPharmacognostic analysis for each sample were \n\ncarried out according to the standard procedural \n\nmethods. \n\nSample preparation   \nEach sample was preserved in a fixative solution. \n\nThe fixative used was FAA (5 ml Formalin + 5 ml \n\nAcetic acid + 90 ml 70% Ethyl alcohol). The \n\nmaterials were left in the FAA for more than 48 \n\nhours. Dehydration process was followed by a \n\ngraded series of tertiary-butyl alcohol as per the \n\nschedule by Sass 1940 [28]. After dehydration, \n\nparaffin infiltration was carried out till the super-\n\nsaturation of tertiary-butyl alcohol was achieved. \n\nFollowing supersaturation, the materials were \n\ntransferred to pure paraffin wax two times and the \n\nmaterials were cast into paraffin blocks. \n\nSectioning \nThe embedded specimens were sectioned with the \n\nhelp of a rotary microtome. The thickness of the \n\nsection was 10 to 12 \u00b5m which were then/further \n\nstained with toluidine blue as per the method by \n\nO\u2019Brein et al. [29]. \n\nPhotomicrographs \nThe transverse section was photomicrographs using \n\nZeiss AXIO trinocular microscope attached to Zeiss \n\nAxio Cam camera under bright field.", "start_char_idx": 7276, "end_char_idx": 10892, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "485c6bf1-0089-44d0-8252-563130558669": {"__data__": {"id_": "485c6bf1-0089-44d0-8252-563130558669", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5a4207cb-4890-4adb-b25d-dc14ced3bdcc", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "bc8a4cd16b17098a871801ae30585ca8f1a5e2998a35b7149c95087e430d4dbd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c3cbe301-e424-4e3d-935e-b8b79c979452", "node_type": "1", "metadata": {}, "hash": "0e561faaa067c091b10b82cfa7a691d31902a2b346813f68556859e0e284ff9b", "class_name": "RelatedNodeInfo"}}, "text": "Descriptive \n\nterms of the anatomical features are as given in the \n\nstandard anatomy books [30]. \n\nPhysico-chemical evaluation  \nThe Physico-chemical evaluation of prepared \n\nsections/samples of N. arbor-tristis, H. salicifolia, O. \n\ntenuiflorum and R. indica and a mixture of the herb \n\nwere done by testing loss of weight on drying at \n\n1050C, total ash, acid insoluble ash and water-\n\nsoluble ash. \n\nPreparation of samples \nThe fresh fruit of H. salicifolia, leaf of N. arbor-tristis \n\nand root of R. indica and whole plant of O. \n\ntenuiflorum were washed, air-dried in 25\u00baC for three \n\ndays in the absence of sunlight and grinded into a \n\ncoarse powder. These samples were used for \n\nPhysico-Chemical studies and proximate analysis \n\n[31]. \n\nLoss on drying at 105\uf0b0C/Moisture content \nTo determine the weight loss in drying, 10 g of each \n\nherbal extract was placed in a tarred evaporating \n\ndish after accurately weighing it. The extract was \n\nthen dried at 105\uf0b0C for 5 hrs and weighed. After \n\ndrying, tarred evaporating dish was cooled in \n\ndesiccators for 30 mins and then weight was taken. \n\nThe % of Loss on drying at 105\uf0b0 C\n\n=\nThe difference in weight after heating\n\nWeight of the sample taken\n x 100 \n\n2.0 g of the powdered extract was incinerated in a \n\ntarred silica dish at a temperature not exceeding \n\n450\uf0b0C until free carbon was left, then cooled and \n\nfinal weight was taken. The percentage of ash was \n\ncalculated concerning the weight of the sample. \n\nThe % of Total Ash =\nWeight of ash obtained\n\nWeight of sample taken\n x 100 \n\nEstimation of Acid Insoluble Ash \nThe ash obtained as the above method was boiled \n\nfor 5 mins with 25 ml of dilute hydrochloric acid \n\nand collected the insoluble matter on an ash-less \n\nfilter paper (Whatman 41), washed with hot water \n\nand ignited to constant weight. The percentage of \n\nacid-insoluble ash concerning the air-dried drug \n\nwas calculated. \n\nThe % of Acid Insoluble Ash = \nWeight of Acid Insoluble residue\n\nWeight of the sample taken\n x 100 \n\nEstimation of Water-soluble ash: \nThe incinerated ash of the sample was dissolved in \n\n25 ml of dilute hydrochloric acid and made up to 50 \n\nml with water and boiled. The suspension was \n\nfiltered with the help of the Whatman filter paper \n\nand the residue was collected. It was kept in a \n\nweighed silica crucible and maintained in a muffle \n\nfurnace for 6 hrs at 450-650\u00bac. The crucible was \n\ntaken out and cooled at room temperature and \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  39 \n\nweighed. The percentage of ash obtained was thus \n\ncalculated. \n\nThe % of Water \u2212 soluble ash\n\n=\nWeight of ash\n\nWeight of the sample taken\n x 100 \n\nEstimation of Sulphated Ash: \nThe substance was ignited with concentrated \n\nsulphuric acid, which decomposed and oxidized \n\norganic matter, leaving a residue of inorganic \n\nsulphates. A large silica crucible was ignited to \n\nconstant weight and approximately 5 g of the \n\nsample was weighed, and moistened by adding \n\nsulfuric acid. This was then heated gently at first \n\nand more strongly later so that volatile matter \n\ncould be removed. Further, it was again ignited \n\nmore strongly to remove the cool carbon, \n\nremoistened with sulphuric acid and then re-ignite \n\nto achieve constant weight. Finally, the percentage \n\nof sulphated ash was calculated.", "start_char_idx": 10893, "end_char_idx": 14238, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c3cbe301-e424-4e3d-935e-b8b79c979452": {"__data__": {"id_": "c3cbe301-e424-4e3d-935e-b8b79c979452", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "485c6bf1-0089-44d0-8252-563130558669", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "92a1ef758b3af6a4da758d5f67049b4a77a4e3fd61cb1e0642f93a21d894b11b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d32ebf56-fe05-4874-b5d4-ac3e4cd8d31b", "node_type": "1", "metadata": {}, "hash": "1688979b79e2e0968567a18115c75ce9c13a136e97ab253a27a56dcb5a366605", "class_name": "RelatedNodeInfo"}}, "text": "Finally, the percentage \n\nof sulphated ash was calculated. \n\nThe % of sulphated ash\n\n=\nWeight of ash\n\nWeight of the sample taken\n x 100 \n\nDetermination of Extractable Matter in \nalcohol (Cold Maceration Method) \n5 g of samples were macerated with 100 ml of \n\nalcohol in a stoppered flask with frequent shaking \n\nduring the first 6 hrs and allowed to stand for 18 \n\nhrs. It was filtered after 24 hrs. 25 ml of the filtrate \n\nwas evaporated in a tarred dish at 105\u00b0C and \n\nweighed. Alcohol soluble extractive values were \n\ncalculated. The experiment was repeated twice, and \n\nthe average value was taken [32]. \n\nThe % of alcohol \u2212 soluble extractive\n\n=\nWeight of extract \u00d7  4\n\nWeight of the sample taken\n x 100 \n\nDetermination of Extractable Matter in \nwater (Cold Maceration Method)  \nAbout 5.0 g of coarsely powdered air-dried material \n\nwas accurately weighed in a glass stopper conical \n\nflask and macerated with 100 ml of distilled water \n\nspecified for the plant material for 6 hrs with \n\nfrequent shaking, then allowed to stand for 18 hrs. \n\nIt was then filtered instantly, taking care not to lose \n\nany solvent. The extracted matter was dried at \n\n105\uf0b0C for 6 hrs, cooled in desiccators for 30 mins \n\nand then weighed. The percentage of the \n\nextractable matter was calculated. \n\nThe % of water \u2212 soluble extractive\n\n=\nWeight of extract \u00d7  4\n\nWeight of sample taken\n x 100 \n\nProximate analysis   \nEstimation of fibers \nThe fiber content was estimated by the method of \n\nRaghuramulu et al. [33]. About 5 g of moisture and \n\nthe fat-free sample were weighed into a 500 ml \n\nbeaker and 200 ml of boiling 0.255 N (1.25% W/V) \n\nSulphuric acid was added. The mixture was boiled \n\nfor 30 mins, but the volume was kept constant by \n\nthe addition of water at frequent intervals. Finally, \n\nthe mixture was filtered through a muslin cloth and \n\nresidue was washed with hot water until it was free \n\nfrom acid. The material was then transferred to the \n\nsame beaker, and 200 ml of boiling 0.313 N (1.25%) \n\nNaOH was added. After boiling for 30 mins \n\n(keeping the volume constant as before) the mixture \n\nwas filtered through a muslin cloth. The residue \n\nwas washed with hot water until it was free from \n\nalkali, followed by washing with some alcohol and \n\nether. It was then transferred to a crucial, dried \n\novernight at 800-100\u00baC and weighed (We). The \n\ncrucial was heated in a muffle furnace at 600\u00baC for \n\n2-3 hours; then it was cooled and weighed again \n\n(Wa). The difference in the weights (We-Wa) \n\nrepresents the weight of fiber. \n\nFiber content =  \n{100 \u2212 (moisture +  fat)}  \u00d7  (We \u2212 Wa) \n\nWeight of sample taken\n \n\nEstimation of Total Carbohydrate \n(Anthrone method) \nThe total carbohydrate content was estimated by \n\nthe method of Hedge et al. [34]. 100 mg of the \n\nsample was weighed into a boiling tube, \n\nhydrolyzed by keeping it in a boiling water bath for \n\nthree hours with 5.0 ml of 2.5 N HCl and cooled at \n\nroom temperature. Then, the material was \n\nneutralized with solid sodium carbonate until \n\neffervesce was seen and the final volume made to \n\n100 ml. This was centrifuged, at 5000 rpm for 10 \n\nmin and the supernatant was collected. From that, \n\n0.2 to 1.0 ml was taken and the standard was \n\nprepared from the working standard. 1.0 ml of \n\nwater serves as a blank made up the volume to 1.0 \n\nml in all the tubes with distilled water, then added \n\n4.0 ml of anthrone reagent, heated for eight minutes \n\nin a boiling water bath, cooled rapidly and read the \n\ngreen to dark green colour at 630 nm.", "start_char_idx": 14180, "end_char_idx": 17700, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d32ebf56-fe05-4874-b5d4-ac3e4cd8d31b": {"__data__": {"id_": "d32ebf56-fe05-4874-b5d4-ac3e4cd8d31b", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c3cbe301-e424-4e3d-935e-b8b79c979452", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "88311a99cdbed85488569a82cf3cc1938bfb23dee099b4f2cb7e19a9c1580878", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "da77b7e2-a382-4499-b283-f73d1abef541", "node_type": "1", "metadata": {}, "hash": "dab0d6a6fb587da265240f7cf03d33264ab4d4952451d18d1752c22ee9edc550", "class_name": "RelatedNodeInfo"}}, "text": "The \n\ncarbohydrate content of the sample was compared \n\nwith the standard curve. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  40 \n\nEstimation of protein (Modified Lowry\u2019s \nMethod) \nThe total protein content was estimated by the \n\nmethod from Raghuramulu et al. [33]. 5 g extracts \n\nwas kept 3 separate tubes with 50 ml of water by \n\novernight cold percolation method. In separate \n\ntubes, 0.5 ml of each; extract, blank and standard, \n\nwere taken in duplicate and 0.5 ml of alkaline \n\ncopper reagent was added, mixed and allowed to \n\nstand undisturbed for 10 minutes. Then 2 ml of \n\nphenol reagent was added to each tube; mixed \n\nimmediately and placed at room temperature for 5 \n\nmins and absorbance of samples and standards \n\nwere taken at 615 nm against a blank. The protein \n\ncontent of the sample was calculated by comparing \n\nwith the standard curve. \n\nEstimation of Fat \n5 g of extract was placed in a soxhlet fitted with a \n\ncondenser. 90 ml of petroleum ether (boiling point \n\n40- 60\u00b0C) was taken in a 150 ml round bottom flask \n\nand boiled for 6 hrs. The extract was taken in a pre-\n\nweighed conical flask, and petroleum ether was \n\nevaporated on a water bath. The traces of \n\npetroleum ether were removed using a vacuum \n\npump [35]. \n\nPhytochemical test  \nPhytochemical analysis for each sample\u2019s crude \n\nextract and her-herb combination were carried out \n\naccording to the standard procedure methods [36, \n\n37]. \n\nPreparation of Hydroalcoholic extracts \nThe dried powder of plant parts was exclusively \n\nremoved by hydroalcoholic cold permeation \n\ntechnique. 10 g of dried powder of each plant were \n\ntaken in 100 ml of oil ether in a cone-shaped jar, \n\napplied with cotton fleece stopper and after that \n\nkept on a turning shaker at 120 rpm for 24 hrs. \n\nAfter 24 hrs, it was seived through eight layers of \n\nmuslin fabric, centrifuged and the supernatant was \n\ncollected and air-dried under decreased strain to \n\nacquire the dried buildup. Oil ether was dissipated \n\nfrom the powder. This dry powder was then taken \n\nexclusively in 100 ml of every dissolvable, for \n\nexample, ethanol (ET), 75% ET, half ET, 25% ET and \n\nwater and after that it was kept on a revolving \n\nshaker at 120 rpm for 24 hrs. At that point, the \n\nsystem pursued was equivalent to above, and the \n\ndeposits were weighed to get the extractive yield of \n\nthe considerable number of concentrates and were \n\nput away in hermetically sealed containers at 4\u00baC \n\n[38]. \n\nThe phytochemical studies were performed for \n\ntesting the different chemical groups present in the \n\ndrug. For this, 10% (w/v) solution of extract was \n\ntaken, unless otherwise mentioned in the respective \n\nindividual test. General screening of various \n\nextracts of the plant material was carried out for the \n\nqualitative determination of the groups of organic \n\ncompounds present in them as per Sethi 1966 [39]. \n\nTest for Alkaloids \nDragendorff\u2019s test:   For this test, 2 g of alcoholic \n\nand aqueous extract of the drug were dissolved in 5 \n\nml of distilled water, followed by the addition of 2 \n\nM hydrochloric acid until an acid reaction occurs, \n\nthen 1 ml of Dragendorff\u2019s reagent added. \n\nSubsequently, an orange or orange-red precipitate \n\nwas produced immediately. \n\nTest for Flavonoids \nShinoda\u2019s test: In a test tube containing 2 g of \n\nalcoholic extract of the drug, 5-10 drops of dilute \n\nHydrochloric acid as added, followed by the \n\naddition of a small piece of magnesium. In the \n\npresence of flavonoids, a pink, reddish-pink or \n\nbrown color was produced.", "start_char_idx": 17701, "end_char_idx": 21270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "da77b7e2-a382-4499-b283-f73d1abef541": {"__data__": {"id_": "da77b7e2-a382-4499-b283-f73d1abef541", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d32ebf56-fe05-4874-b5d4-ac3e4cd8d31b", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "1810f9a88df8ebb368c607a62f5739b1c9c4336078248926100c38f444216bae", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ed9206b9-d1c2-4354-8eb1-6a4bcb803842", "node_type": "1", "metadata": {}, "hash": "449db8b1c76fbe5dae268c66572141c5709a34e849cf2a201ae1eb95935b92b1", "class_name": "RelatedNodeInfo"}}, "text": "Test for Triterpenoids \nLiebermann-Burchard\u2019s test: For this 2 ml of acetic \n\nanhydride solution was added to 1 ml of petroleum \n\nether extract of the drug in chloroform followed by \n\n1 ml of conc. Sulphuric acid. A violet ring was \n\nformed indicating the presence of triterpenoids.  \n\nTest for Resins \nDissolved the extract was dissolved in acetone and \n\nsolution was poured into distilled water. Turbidity \n\nindicated the presence of resins.  \n\nTest for Saponins \nIn a test tube containing about 2 g extracts of the \n\ndrug was added a drop of sodium bicarbonate \n\nsolution, followed by shaking the mixture \n\nvigorously and left for 3 minutes. Honeycomb like \n\nforth was formed. \n\nTest for Steroids \nLiebermann-Burchard\u2019s test: In this test, 2 ml of \n\nacetic anhydride solution was added to 1 ml of \n\npetroleum ether extract of the drug in chloroform \n\nfollowed by the addition of 1 ml of Conc. Sulphuric \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  41 \n\nacid. A greenish color was developed, which turned \n\nto blue. \n\nSalkowski Reaction: For this test, 1 ml of conc. \n\nSulphuric acid was added to 2 ml of chloroform \n\nextract of the drug carefully, from the side of the \n\ntest tube. The red color was produced in the \n\nchloroform layer. \n\nTest for Tannins \nAdded a few drops of 5% FeCl3 solution in few \n\ngrams of extract and a green color indicated the \n\npresence of gallotannins while brown color tannins. \n\nTest for Starch \n0.015 g of Iodine and 0.075 g of Potassium Iodide \n\nwere dissolved in 5 ml of distilled water and few \n\ngrams of extract of the drug was added. The blue \n\ncolor was produced. \n\nTest for Glycosides \nFor the detection of glycoside in an extract sample, \n\non paper spray, solution No. 1 (0.5 % aqueous sol. \n\nof Sodium metaperiodate) was sprayed & waited \n\nfor 10 minutes. After that spray solution No. 2 [0.5 \n\n% Benzidine (w/v) was sprayed in solution of \n\nEthanol \u2013 acetic Acid (4:1)], Presence of glycoside \n\nwas detected by formation of white spot with a blue \n\nbackground.  \n\nEstimation of Total Phenolics \nMany natural antioxidants are polyphenolics in \n\nnature e.g. Flavonoids, Chalcones, Aurones and \n\nPhytoalexins. These polyphenolic compounds \n\nexhibited anti-inflammatory, antioxidant and \n\nanalgesic activity. Hence, the amount of total \n\nphenol present in the air-dried plant, as well as \n\nhydroalcoholic extracts, were estimated using \n\nFolin-Ciocalteau reagent [40]. \n\nDetection of numerous Microbial masses \nin Herbs \nThe plant material and plant extract obtained were \n\nsubjected to microbial analysis. A total 1 g of the \n\nsample was taken, and 99 ml of sterile distilled \n\nwater was added for preparing the serial dilution. \n\nThe samples in the flask were kept in a mechanical \n\nshaker for a few minutes to obtain uniform \n\nsuspension of microorganisms and diluted to 1:100 \n\nor 10-2. From that 1 ml of the 10-2 dilution was \n\ntransferred to 9 ml of sterilized distilled water. This \n\nwas 1:1000 or 10-3. This procedure was repeated up \n\nto 10-6 dilution. 0.1 ml of serially diluted samples \n\nwere inoculated into the sterile plate containing \n\nNutrient agar, Salmonella Shigella Agar (SSA) and \n\nPotato Dextrose Agar (PDA) Medium by spread \n\nplate method.", "start_char_idx": 21273, "end_char_idx": 24517, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ed9206b9-d1c2-4354-8eb1-6a4bcb803842": {"__data__": {"id_": "ed9206b9-d1c2-4354-8eb1-6a4bcb803842", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "da77b7e2-a382-4499-b283-f73d1abef541", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "274076b5651b64e61e30f01cff67dae693264d3985546478fba49d72b5916759", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1be25bed-3cbc-46d4-a941-8798628e2b7b", "node_type": "1", "metadata": {}, "hash": "990727423f3f674b446e282c546469d951d61f80e9c443fd292f1a1c362676f0", "class_name": "RelatedNodeInfo"}}, "text": "Nutrient Agar (NA) and SSA plates \n\nwere incubated at 37\u00baC for 24 hours, and PDA \n\nplates were incubated at room temperature for 3-5 \n\ndays. The bacterial and fungal colonies were \n\ncounted using a colony counter. Salmonella spp, \n\nShigella spp can be counted using SS Agar medium. \n\nEnterobacter spp. were identified by \n\nEnterobacteriaceae kits. \n\nTest for heavy metal toxicity  \nSample collection \nThe samples were cleaned and dried under shade. \n\nThe dried samples were grinded to powder form in \n\nan agate mortar-pestle, were labeled and stored in \n\npre-cleaned polyethylene bottles for further \n\nanalysis. \n\nCalibration of instruments: \nMore than three working standard solutions of \n\nanalyses were prepared, covering the concentration \n\nrange as recommended by the manufacturer of the \n\ninstrument for the elements to be determined. \n\nBefore the analysis of samples, the instruments \n\nwere calibrated with a prepared working standard \n\nsolution. The calibration curves were obtained for \n\nconcentration vs absorbance and were statically \n\nanalyzed. \n\nCadmium, Lead and Arsenic analysis \n(Flame AAS/Graphite Furnace): \nThe digested sample was subjected to analysis of \n\nCadmium, Lead and Arsenic by AAS \n\nflame/graphite Furnace with specific instrumental \n\nconditions as given by instruments manufacturer. \n\nThe solutions were introduced into flame. The \n\nmean of the three readings was recorded against \n\nthe standard calibration curve obtained from \n\nconcentration vs absorbance. \n\nAfter calibrating the instrument with prepared \n\nworking standard, 10 ml of digested sample was \n\npipette out to a specific container of Mercury \n\nHydride system analyzer followed by adding 10 ml \n\n1.5 % of HCl as diluents for each sample and blank. \n\nThe digested samples were run through the \n\nreaction flask containing reluctant (3% NaBH4 in 1 \n\n% of NaOH) to quartz cell without heating against \n\nthe calibration curve obtained from concentration \n\nvs absorbance. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  42 \n\nFluorescence analysis  \nFluorescence is an essential parameter of \n\npharmacognostical evaluation, exhibited by various \n\nchemical constituents present in plant material and \n\nconvert nonfluorescent into fluorescent derivatives \n\nby reagents. It was observed under ordinary and \n\nultraviolet light according to the procedure of \n\nKokoshi et al. [41]. For this, 10 mg of the extract of \n\neach plant was taken in a glass slide and treated \n\nwith various reagents for the presence of their \n\nfluorescence characteristics under the ultra\u2010violet \n\nlamp at 254 nm and 366 nm. \n\nHigh-performance Thin Layer \nChromatography (HPTLC)  \nHPTLC analysis was carried out by Sethi 1996 [39]. \n\nIn this technique, 5\u00b5l each of the extract was \n\napplied on a precoated silica gel 60 F254 on \n\naluminium plates to a bandwidth of 8mm using \n\nLinomat 5 TLC applicator. The plates were \n\ndeveloped in toluene-ethyl acetate (9:1) and the \n\ndeveloped plates were visualized and scanned by \n\nTLC scanner 4 Win CATS software version \n\n1.4.6.2002 under UV 254, 366. After derivatization \n\nin vanillin-sulphuric acid was sprayed as reagent at \n\n620 nm, Rf colour of the spots and densitometry \n\nscan were recorded. HPTLC plates of hydraulic \n\nextracts of plants separate mobile phases viz. \n\nEthylacetate: Dichloromethane: Formic acid: Acetic \n\nacid: Water (10:2.5:1:1:0.5) were examined. Rf value, \n\nnumbers of peaks, peak area and the peak height of \n\nplants extract in separate mobile phases viz. n-\n\nEthylacetate: Dichloromethane: Formic acid: Acetic \n\nacid: Water (10:2.5:1:1:0.5) were also analyzed \n\nunder 254 nm and 366 nm respectively.", "start_char_idx": 24518, "end_char_idx": 28183, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1be25bed-3cbc-46d4-a941-8798628e2b7b": {"__data__": {"id_": "1be25bed-3cbc-46d4-a941-8798628e2b7b", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ed9206b9-d1c2-4354-8eb1-6a4bcb803842", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "174f7b202ee17640f2b7df8c9875c169da961469008d7ca608eac2030210759a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "57df3b9a-5578-4d05-bcc6-44855c3963e5", "node_type": "1", "metadata": {}, "hash": "cb5fdc6f8a7703c4c2305ab617ca7a5dfbe6d0827eeacd3004eff647233d5a04", "class_name": "RelatedNodeInfo"}}, "text": "Further \n\nstudies was done by the help of standards for \n\nquantitative estimation and identification of the \n\ningredients peak. Present HPTLC fingerprinting \n\ndata can help in authentication and identification of \n\nformulation in the performed solvent system and \n\nextract. \n\nStatistical analysis \nStatistical analysis was done by using GraphPad \n\nPrism Version 5.02. One-way analysis of variance \n\n(ANOVA), with Tukeypost-test, was used for \n\nStatistical analysis of collected data. A probability \n\nvalue of p<0.05 was considered significant, and \n\nP<0.01 was considered highly significant. All the \n\ndata are expressed as mean \u00b1 SD (standard \n\ndeviation). \n\nResults \nThe organoleptic properties of herbs showed the \n\ncolour revealed was light brown for leaves of N. \n\narbor-tristis, O. tenuiflorum and R. indica, yellow for \n\nfruits of H. salicifolia, have a characteristic odour, \n\nbitter taste and moderately fine texture and \n\ncombined herb-herb color was yellowish-brown. \n\nPharmacognosy is the study of medicinal plants, \n\nproduced from natural sources and analysis of their \n\nbiological, chemical, biochemical, and physical \n\nproperties, or it is the study of crude drugs based \n\non their shape, size, color, and texture and cut \n\nsurface morphology. \n\nMicroscopic features of entire Transverse \nSection of the Leaf of N. arbor-tristis. \n\n \nFigure 1: Microscopy of N. arbor-tristis leaf. Figure 1a \nrepresents microscopy of the leaf TS through midrib \nwhere col=collenchyma; e=epidermis; gt=ground tissue; \nme=mesophyll; ph=pholem; sg=starch grains; \nt=trichome; xy=xylem. Figure 1b represents microscopy \nof the leaf TS through lamina where cu=cuticle; le=lower \nepidermis; mer=meristele; pal=palisade; spp=spongy \nparenchyma; t=trichome; ue=upper epidermis. \n\nThe transverse section of leaf passing through the \n\nmid-rib convexly projects on the lower side, and \n\nslightly grooved with a shallow central elevation on \n\nthe upper side.", "start_char_idx": 28184, "end_char_idx": 30128, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "57df3b9a-5578-4d05-bcc6-44855c3963e5": {"__data__": {"id_": "57df3b9a-5578-4d05-bcc6-44855c3963e5", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1be25bed-3cbc-46d4-a941-8798628e2b7b", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "4e59ad6ba25bbbc5ee8deff80702926e224a1eb106b53acb7cfc2bca6417e43f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f167ba84-136b-4e7c-96d0-04733d49ef97", "node_type": "1", "metadata": {}, "hash": "b602697c8d1713c4df688bef683a0df3ef453084ca23a5ab3881f12662c89040", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  43 \n\nLeaf lamina demonstrated an epidermis of \n\ndigressively stretched cells on the two surfaces, \n\nbigger on the upper surface, secured by striated \n\nfingernail skin, mesophyll separated into 2 or 3 \n\nlayers of palisade cells, 5 to 7 layers of \n\napproximately masterminded, fairly isodiametric \n\nsupple parenchyma, rosette gems of calcium \n\noxalate present in a couple of cells, stomata more on \n\nthe lower surface, anisocytic where anomocytic \n\ntype likewise occur on the two surfaces (Figure 1a \n\n& Figure 1b). \n\nMidrib: show a single layered epidermis, 2 or 3 \n\nlayered collenchyma on both surfaces, 4 or 5 \n\nlayered parenchyma, mostly devoid of chloroplasts, \n\ncentral zone occupied by the vascular bundles \n\ndifferentiated into xylem towards ventral side and \n\nphloem towards dorsal side, phloem consisting of \n\nsieve tubes, companion cells and phloem \n\nparenchyma, xylem consisting of radial rows of \n\nvessels with xylem parenchyma. \n\nThe total thickness of the leaf ranges from 158.08\u2013\n\n295.36 \u00b5m, the mean value being 248.46 \u00b5m. The \n\nthickness of mesophyll ranges from 110.24\u2013249.60 \n\n\u00b5m with the mean as 197.91 \u00b5m. The ratio of height \n\nto width of upper epidermal cells speaks for their \n\nbarrel-like shape. The height vis-\u00e0-vis width ratio of \n\nthe lower epidermal cells reveals that the cells are \n\nmore or less square shaped.  \n\nPetiole: In the transverse section of the petiole leaf \n\nof N. arbour-tristis have a specific feature, there are \n\ntwo projections adjacent to the ventral groove, \n\nepidermis single-layered, cells cubical covered by a \n\nthick cuticle, inner walls of epidermal cells \n\nadjoining the cortex much thickened, hairs absent. \n\nHypodermis layer composed of collenchyma 2 or 3 \n\nlayered and a broad zone of more or less rounded \n\nparenchyma cells present with intercellular spaces. \n\nIt has a few rosette crystals of calcium oxalate. \n\nResin canals are also present on the dorsal side of \n\neach vascular bundle except in the vascular bundles \n\noccurring projecting arms. In this section, vascular \n\nbundles 5 to 7 in number (Figure 2a & Figure 2b). \n\nMicroscopic features of entire Transverse \nSection of the Fruit of H. salicifolia: \nThe TS of the fleshy tissues of the fruit or \n\nhypanthium reveals various structures. Vascular \n\ntissues are oriented longitudinally through the \n\ncentral portion of the fruit flesh. Viewed cross-\n\nsectional (Figure 3), the vascular tissues are \n\nsurrounded by large parenchyma cells with round, \n\noval, and teardrop shapes and contain large \n\nquantities of red pigments, which are likely \n\ncarotenoids. Long narrow cells shaped like sclereid \n\ntissues, and containing green pigments, are found \n\non either side of the vascular and storage tissues. \n\nThese groups alternated around the middle \n\ncircumference of the mesocarp. \n\n \n\n \n\n \n \n\n2a \n\n2b \n\n2c", "start_char_idx": 30133, "end_char_idx": 33038, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f167ba84-136b-4e7c-96d0-04733d49ef97": {"__data__": {"id_": "f167ba84-136b-4e7c-96d0-04733d49ef97", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "57df3b9a-5578-4d05-bcc6-44855c3963e5", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "6804815bd97319a1ca22efaf76795f3af7610663e131b2d0fcd09cb7e8d734c2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5196bab2-1bc3-4103-a08e-899d97bfe924", "node_type": "1", "metadata": {}, "hash": "fa82c7c49b6be6148dfd36e7f12fa404f855de7e67d45fdf2deac219209631f7", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  44 \n\n \nFigure 2: The transverse section of the petiole leaf \n\nof N. arbour-tristis. Figure 2a represents transverse \n\nsection of petiole, 2b, 2c 2d represents enlarged \n\nportion of petiole \n\n Figure 3: Transverse section of fruit from H. \nsalcifolia \n\nMicroscopic features of the entire \nTransverse Section of the leave of O. \ntenuiflorum. \nLeaf: Transverse section of the leaf had a pot shape \n\nmidrib and a flimsy lamina with uneven lower \n\nepidermis appended at the sidelong sides of its \n\nupper side leaving a curved focal dorsal. Midrib \n\ncomprises of an emanating curve of xylem and \n\nphloem. Both upper and lower epidermis \n\ndemonstrated straightforward, covering, uniseriate \n\ntrichomes just as sessile short-stalked, glandular \n\ntrichomes. Powder of the air-dried leaves of this \n\nplant was seen under the magnifying instrument. \n\nThe various glandular basic trichomes of the \n\nnormal length of 101 \u03bcm were found.  \n\nPetiole: indicates to some degree cordate \n\nframework, comprising of single-layered epidermis \n\nmade out of flimsy walled, oval cells having \n\nvarious covering and glandular trichomes; covering \n\ntrichomes multicellular 1-8 celled long, rarely \n\nsomewhat reflexed at tip; glandular trichomes \n\nshort, sessile with 1-2 celled stalk and 2-8 celled \n\ninflatable formed head, estimating 22-27 in \n\ndiameterr; epidermis pursued by 1 or 2 layers and 2 \n\nor 3 layers of meager walled, stretched, \n\nparenchyma cells towards upper and lower \n\nsurfaces individually; three vascular groups \n\narranged midway, center one bigger than the other \n\ntwo; xylem encompassed by phloem. \n\nMidrib: epidermis, trichomes and vascular bundles \n\nsimilar to those of petiole except cortical layers \n\nreduced towards the apical region. \n\nLamina: epidermis and trichomes similar to those \n\nof petiole; both monocytic and diacytic type of \n\nstomata present on both surfaces, slightly raised \n\nabove the level of epidermis; palisade single \n\nlayered followed by 4-6 layers of closely packed \n\nspongy parenchyma with chloroplast and oleo-\n\nresin (Figure 4a & Figure 4b). \n\n \n\n \n\n2d \n\n4b \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  45 \n\n \n\n \nFigure 4: Transverse section of leaf from O. \ntenuiflorum. Figure 4a represents transverse section \n\nof leaf from O. tenuiflorum and Figure 4b, 4c, 4d \nrepresents enlarged section of leaf. \n\nThe physicochemical characteristics of the extracts \n\nwere determined as per WHO guidelines [34]. The \n\nphysico-chemical estimation of the drug is an \n\nimportant parameter in adulteration or improper \n\nhandling of drugs [35]. It can serve as a valuable \n\nsource of information and provide appropriate \n\nstandards to establish the quality of plant material \n\nfor further study. Equally important in the \n\nevaluation of the loss on drying, ash value, water \n\nsoluble ash, sulphated ash, acid insoluble ash value \n\nand water-soluble extractive value and alcohol \n\nsoluble extractive value determination. The low \n\nvalue of moisture content could prevent bacteria, \n\nfungal or yeast growth [40, 41, 42]. This value varies \n\nwithin fairly wide limits and is, therefore, an \n\nimportant parameter for the purpose of evaluation \n\nof crude drugs [43].", "start_char_idx": 33043, "end_char_idx": 36338, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5196bab2-1bc3-4103-a08e-899d97bfe924": {"__data__": {"id_": "5196bab2-1bc3-4103-a08e-899d97bfe924", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f167ba84-136b-4e7c-96d0-04733d49ef97", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "90e3c88b157a2bc23c651f3b6515c97db1e983e3ac28bd04f940a64aabc3a0b3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e36adaa7-6cfa-473d-a3ce-3d120ad9463c", "node_type": "1", "metadata": {}, "hash": "b5795cc91dc092a8e97e24651c1c12bea281d1be7b65c003d311ceac67c0d644", "class_name": "RelatedNodeInfo"}}, "text": "Therefore, percentage on the \n\nloss on drying, ash value, water-soluble ash, \n\nsulphated ash, acid insoluble ash value and water \n\nsoluble extractive value and alcohol soluble \n\nextractive value calculated. Results for the \n\nphysicochemical parameters are given in Table 1. \n\nAsh of any organic material contains non-volatile \n\ninorganic components. The ash content indicates \n\nthat the seed, is rich in mineral elements. \n\nControlled incineration of crud drugs result in an \n\nash residue composed of inorganic matter such as \n\nmetallic salts and/or silica. Therefore, the kind of \n\ncare that must be taken in the plant drug is \n\nimportant. Total ash value was noted in (Table 1). \n\nThe result showed that the negligible amount of \n\nacid-insoluble siliceous matter is present in these \n\nextracts. Percentage of total ash value, water-\n\nsoluble ash, sulphated ash, acid insoluble ash in \n\nthese extracts accessed falls within the margins as \n\nper. The Ayurvedic Pharmacopoeia of India, (2001), \n\nwhich states that the total ash, water-soluble ash, \n\nsulphated ash acid insoluble ash content of the \n\nsamples tested should in a limited range[44].The \n\nwater-soluble extractive value indicates the \n\npresence of sugar, acids and inorganic compounds, \n\nTable 1. Result reveals that the sugar, acids and \n\ninorganic compounds were presented with normal \n\nrange in these extract. The alcohol-soluble \n\nextractive values indicated the presence of polar \n\nconstituents like phenols, alkaloids, steroids, \n\nglycosides and flavonoids Table 1. The alcohol-\n\nsoluble extractive content of these extracts falls \n\nwithin the margins as per The Ayurvedic \n\nPharmacopoeia of India (2001), which states that \n\nthe alcohol-soluble extractive content of the \n\nsamples tested should be more than 3% [44, 45].  \n\nTable 1: The Physicochemical analysis of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica.  \n\nPhysicochemical \nparameters \n\nN. arbor-tristis H. salicifolia O. tenuiflorum R. indica Combined herbs \n \n\nLoss on drying 4.59\u00b10.05 9.87\u00b10.09 4.87 \u00b10.04 4.64\u00b10.05 5.98\u00b10.05  \n\nAsh value 19.64\u00b10.07 10.35\u00b10.03 8.61\u00b10.06 5.38 \u00b10.05 10.99\u00b10.05  \nWater soluble ash 9.29\u00b11.6 4.64\u00b10.7 3.76\u00b10.4 2.12\u00b10.5 4.95\u00b10.25  \n\nSulphated ash acid 10.82\u00b11.0 3.67\u00b10.6 2.64\u00b10.2 2.09\u00b10.3 4.80\u00b10.52  \n\nInsoluble ash value 2.11\u00b10.9 2.09\u00b10.6 1.53\u00b10.3 1.11\u00b10.1 1.71\u00b10.47  \n\nWater soluble \nextractive \n\n4.69\u00b10.3 15.26\u00b10.4 8.52\u00b10.3 8.31\u00b10.3 9.19\u00b10.32 \n \n\nAlcohol soluble \nextractive \n\n6.38\u00b10.03 6.27\u00b10.02 10.29\u00b10.3 3.64\u00b10.01 6.64\u00b10.09 \n \n\nValue are Mean \u00b1SD for 5 different preparations  \n\n4c \n\n4d \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  46 \n\n \n\nNext parameter, proximate analysis reveals the \n\nnutritive content of each extract and combined \n\nextract.", "start_char_idx": 36339, "end_char_idx": 39108, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e36adaa7-6cfa-473d-a3ce-3d120ad9463c": {"__data__": {"id_": "e36adaa7-6cfa-473d-a3ce-3d120ad9463c", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5196bab2-1bc3-4103-a08e-899d97bfe924", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "efe0e659cf9aa514ef60630e45d598ed2463401e83d67e7b4da41105c80c7901", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "255996fd-8f7d-4dfa-8daa-7d44b5ba2d5a", "node_type": "1", "metadata": {}, "hash": "965463438423aa5a02117bf787cd7d46108d17fed923effa930609e90889142b", "class_name": "RelatedNodeInfo"}}, "text": "The proximate analysis revealed that H. \n\nsalicifolia is a very good natural source of protein \n\nand all calculated values showed in (Table 2). In \n\nthe Proximate analysis, the combined herbal extract \n\npossesses the nutritive content of four herbs and \n\nfound the study presence of carbohydrate, protein, \n\nfat and crude fiber. According to the results of the \n\nenergy values which were based on the \n\ncarbohydrate, fat and fiber content in these extracts \n\nwere high. Thus, it may be used as an alternative \n\nfor food as well as medicinal. These results \n\ncorrelate with Kocchar et al. [46]. \n\nPreliminary phytochemical analysis of polyherbal \n\nformulation is of significant importance as scientist \n\nneed to understand the change upon extraction of \n\ndifferent portion of four different medicinal \n\nshowed the presence of alkaloids, glycoside, \n\nflavonoids, phenol, steroids, saponins, tannins, \n\nterpenoids and these results are presented in Table \n\n3. The investigation showed that N. arbor-tristis \n\ncontains phenolic compounds, resins, tannins, \n\nstarch, glycosides and alkaloids. Flavonoids were \n\nabsent. The H. salicifolia results revealed the \n\npresence of carbohydrate, amino acids, alkaloids, \n\nflavonoids, phenolic compounds and terpenoids \n\npresent in the extracts whereas Starch and Steroids  \n\nwere absent. The results reveal the presence of \n\nmedicinally active constituents like tannins, \n\nalkaloid, terpenoids, steroids and Flavnois, \n\nPhlobatannins, Glycosides in the leaves of O. \n\ntenuiflorum while saponins were absent in this \n\nplant. In R. indica, active constituents like steroids \n\nand Flavnois, Phlobatannins, Glycosides were \n\npresent except terpenoids, tannins and alkaloid. \n\nThe phytochemical analysis revealed that combined \n\nherb shows the strong presence of flavonoids, \n\nphenolic compounds, alkaloids and tannin. These \n\nvalues are in accordance with the results obtained \n\nby Mowl et al. and Iwalokun et al. [47, 48]. \n\nQuality Control Analysis: Numerous Microbial \n\nLoad in Herbs- The plant material and plant extract \n\nobtained was subjected to microbial analysis and \n\nresult reveals that the level of total microbial count \n\nand total yeast and mold count were not more than \n\n1000 CFU/mL in the extracts of N. arbor-tristis, H. \n\nsalicifolia, O. tenuiflorum, and R. indica and combined \n\nherb. The values were found to be within the limit \n\nof WHO standards. Also, results showed absence of \n\nvarious pathogens like Salmonella, E.coli, S. aureus \n\nand Pseudomonas (Table 4). \n\nHeavy metal toxicity evaluation in the extracts of \n\npolyherbal formulation: The growth of medicinal \n\nplants not only need nutrients for normal plant \n\ngrowth, but also can selectively uptake and \n\naccumulate some trace elements which are good \n\nand may also be toxic for human health if there not \n\nwithin the limits. \n\nTable 2: Proximate composition of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica.", "start_char_idx": 39109, "end_char_idx": 42048, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "255996fd-8f7d-4dfa-8daa-7d44b5ba2d5a": {"__data__": {"id_": "255996fd-8f7d-4dfa-8daa-7d44b5ba2d5a", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e36adaa7-6cfa-473d-a3ce-3d120ad9463c", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "01aad18d0dcfa3e67ed7ab7c6796273ef114a4035b1861109c204052f9c79aa7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f73df768-9a4f-42e3-ade3-24bdecc25789", "node_type": "1", "metadata": {}, "hash": "1b596b22c6ca6cbc5a767e5a74abb30f4123641ff9cfae9e2851ac573a8b7df4", "class_name": "RelatedNodeInfo"}}, "text": "Proximate \n\nparameters \n\nN. arbor-tristis % \n\nW/W \n\nH. salicifolia \n\n% W/W \n\nO. tenuiflorum \n\n % W/W \n\nR. indica \n\n% W/W \n\nCombined \n\nherbs \n\nCarbohydrate  9.48\u00b11.96 3.6\u00b10.53 24.71\u00b11.49 29.71\u00b11.85 16.87\u00b11.62 \n\nProtein  15.02 \u00b10.96 26.31\u00b12.02 5.01\u00b10.09 12.01\u00b12.52 11.68\u00b11.39 \n\nFat 2.10\u00b10.36 2.03\u00b10.02 3.26\u00b10.56 2.16\u00b10.23 2.38\u00b10.29 \n\nCrude fiber 15.03\u00b10.02 14.11\u00b10.36 13.20\u00b10.29 16.19\u00b10.69 14.63\u00b10.34 \n\nValues were expressed as mean \u00b1 S.D for 5 different preparations \n\nTable 3: Phytochemical analysis of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica. \n\nParameters N. arbor-tristis H. salicifolia O. tenuiflorum R. indica Combined herb \n\nAlkaloids (+) (+) (+) (-) (+++) \n\nFlavonoids (-) (+) (+) (+) (+++) \n\nTerpenoids (+) (+) (+) (+) (++++) \n\nResins (+) (+) (+) (+) (++++) \n\nSaponins (+) (+) (-) (+) (+++) \n\nSteroids (+) (-) (+) (+) (+++) \n\nTannins (+) (+) (+) (-) (+++) \n\nStarch (+) (-) (+) (+) (+++) \n\nGlycosdes (+) (+) (+) (+) (++++) \n\nTotal Phenolics (+) (+) (+) (+) (++++) \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  47 \n\n \n\n \n\nThe results revealed the presence of heavy metal \n\nand inorganic compounds in the extracts of N. \n\narbor-tristis, H. salicifolia, O. tenuiflorum and R. indica \n\nand also in combined herb and the values were \n\nfound to be within the limit of WHO standards \n\n(Table 5 and Table 6, respectively). Heavy metal \n\nand inorganic elements are present within the \n\npermissible limits. \n\nFluorescent analysis  \nIn the present study dried powder extract of each \n\nplant treated with various chemical reagents \n\n(several solvents and chemicals) showed \n\ncharacteristic fluorescence at 254 nm and 366 nm \n\nwavelength as shown in Table 7a, Table 7b, Table \n\n7c, Table 7d and Table 7e . \n\nHigh performance thin layer \nchromatography (HPTLC) of \nhydroalcoholic extracts of H. salicifolia, N. \narbor-tristis, O. tenuiflorum, R. indica   \nTLC was done for the separation of different active \n\nconstituents which are present in the extracts of H. \n\nsalicifolia, N. arbor-tristis and O.tenuiflorum. The \n\ndeveloped spots was visualized at various nm. In \n\nFigure 5a from 1 to 3 track of the plate was of leaves \n\nextract and the fourth track was of standard \n\nnyctanthoside. From the picture obtained at 254 nm \n\nassumed that standard nyctanthoside has Rf 0.47 in \n\ntrack 4 and similarly, obtained purple band from \n\ntrack 1 to 3 match with track 4 (Figure 5a).  \n\n \nFigure 5a & 5b: TLC profile of test solution of N. arbor-\ntristis leaf. In Figure 5a, 1-3:test solution, 4: N. arbor-\ntristis standard.", "start_char_idx": 42051, "end_char_idx": 44624, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f73df768-9a4f-42e3-ade3-24bdecc25789": {"__data__": {"id_": "f73df768-9a4f-42e3-ade3-24bdecc25789", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "255996fd-8f7d-4dfa-8daa-7d44b5ba2d5a", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "2c96ce4552f320b84c2e33c314bd9d8f05944fa5b583b889bdae972eed29f6b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "24de45b2-c3ea-4dd8-adcf-feab4f7d20ec", "node_type": "1", "metadata": {}, "hash": "a18c37b87fdcb68a06a4905de050677e0634b1b1a0c8262ab1f48abaa38096b8", "class_name": "RelatedNodeInfo"}}, "text": "Figure 5b represents photo \ndocumentation report of hydro-alcoholic extract of N. \narbor-tristis at 254 nm and 366 nm.  \n\nTable 4. Microbiological analysis of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica. NMT=Not More \n\nThan, A=N. arbor-tristis, B=H. salicifolia, C=O. tenuiflorum, D=R. indica, E=Combined herb \nPlant \n\nExtracts \n\nTotal microbial \n\ncount \n\ncfu/mL \n\nTotal yeast and \n\nmold count  \n\ncfu/mL \n\nPathogens \n\nSalmonella E.coli S. aureus Pseudomonas \n\nA  NMT 1000  NMT 1000  Absent  Absent  Absent  Absent  \n\nB  NMT 1000  NMT 1000  Absent  Absent  Absent  Absent  \n\nC  NMT 1000  NMT 1000  Absent  Absent  Absent  Absent  \n\nD  NMT 1000  NMT 1000  Absent  Absent Absent  Absent  \n\nE NMT 1000  NMT 1000  Absent  Absent Absent  Absent  \n\nTable 5. Heavy metal content of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica. A=N. arbor-tristis, B=H. \n\nsalicifolia, C=O. tenuiflorum, D=R. indica, E=Combined herb \n\nPlant Extracts Heavy Metals (Specification-(ppm/ml)) \n\nLead (> 10 ) Cadmium(>0.3 ) Arsenic (>5) Mercury(>0.5) Chromium (>10) \n\nA  0.036 0.017 0.054 0.0010 0.024 \n\nB  0.042 0.019 0.051 0.0011 0.027 \n\nC  0.039 0.018 0.052 0.0013 0.028 \n\nD  0.049 0.015 0.059 0.0011 0.019 \n\nE 0.041 0.017 0.054 0.0011 0.024 \n\nTable 6: Inorganic elements of N. arbor-tristis, H. salicifolia, O. tenuiflorum and R. indica. Note: A=N. arbor-\n\ntristis, B=H. salicifolia, C=O. tenuiflorum, D=R.", "start_char_idx": 44625, "end_char_idx": 46040, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "24de45b2-c3ea-4dd8-adcf-feab4f7d20ec": {"__data__": {"id_": "24de45b2-c3ea-4dd8-adcf-feab4f7d20ec", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f73df768-9a4f-42e3-ade3-24bdecc25789", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "74d743eae494c557f9c7bc6bf88e85cb77d7ae31ab8ad37bdb52bb10eae60fa3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0c60b918-5c8c-471f-b716-4c2275209cd5", "node_type": "1", "metadata": {}, "hash": "42ecfba86b89a1e0e38218f49b659cdba1ab2e8c32429186c994cb10d2db6b99", "class_name": "RelatedNodeInfo"}}, "text": "salicifolia, C=O. tenuiflorum, D=R. indica, E=Combined herb; Al=Aluminum, Co=Copper, Ca=Calcium, \n\nFe=Iron, Mg=Magnesium, K=Potassium, Na=Sodium, Mn=Manganese, Zn=Zinc, Ni=Nickel \n\nPlant \n\nExtracts  \n\nInorganic compound \n\nAl  Co   Ca   Fe Mg  K Na Mn Zn  Ni  \n\nA  32.03  0.153  0.26  4.69  0.049  0.184  0.226  0.099  0.578  0.453  \n\nB  35.78  0.192  0.22  4.703  0.043  0.192  0.217  0.094  0.679  0.441  \n\nC  36.23  0.157  0.23  4.56  0.049  0.189  0.239  0.089  0.745  0.563  \n\nD  34.15  0.195  0.29  4.26  0.042  0.163  0.215  0.062 0.697  0.438 \n\nE 34.54 0.174 0.25 4.55 0.045 0.182 0.224 0.086 0.674 0.473", "start_char_idx": 46005, "end_char_idx": 46616, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0c60b918-5c8c-471f-b716-4c2275209cd5": {"__data__": {"id_": "0c60b918-5c8c-471f-b716-4c2275209cd5", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "24de45b2-c3ea-4dd8-adcf-feab4f7d20ec", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "e23aa9bdf7875942f171df8dc38952d922c075bd1ca256f71546f9e91d55758c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e467c0cb-a884-4402-b1d8-95d1c1d80c1a", "node_type": "1", "metadata": {}, "hash": "b356f54c30f6605187f3c4c019ae5898aa57e59f535db49e488a843939af0cea", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  48 \n\nTable 7a: Fluorescence analysis of N. arbor-tristis leaf \n\nTreatment Visible light Under UV light \n\n Short wavelength (254 nm) The long wavelength  \n(365 nm) \n\nPowder Brown Light brown Green \nPowder + Methanol Brown Light brown Yellowish green \n\nPowder + 70% ethanol Brown Light brown Green \n\nPowder + Pet. ether Light brown Light green Green \n\nPowder + 50% H2SO4 Brown Greenish brown Brownish \n\nPowder + 50% HCl Dark Brown Green Green black \nPowder + 1N NaOH (aq.) Light brown Dark brown Brownish black \n\nPowder + 1N NaOH (alc.) Light brown Dark green Greenish black \n\nPowder + 50% HNO3 Brown Light brown Light green \n\nPowder + 5% KOH Brown Purplish green Dark purplish green \nPowder + Ammonia Brown Green Black \n\nPowder + Picric acid Yellowish brown Green Dark brown \nTable 7b: Fluorescence analysis of H. salicifolia fruit \n\nTreatment Visible light Under UV light \n\n Short wavelength (254 nm) The long wavelength  \n(365 nm) \n\nPowder yellow Light yellow Yellowish brown \n\nPowder + Methanol yellow Light yellow Yellow \n\nPowder + 70% ethanol yellow Light yellow Yellow \nPowder + Pet. ether Light yellow Light yellow Yellow \n\nPowder + 50% H2SO4 Light yellow Greenish yellow Yellow \nPowder + 50% HCl Dark yellow Greenish yellow Colourless \nPowder +1N NaOH \n(aq.) \n\nLight yellow Green fluorescence Yellowish brown \n\nPowder + 1N NaOH \n(alc.) \n\nLight yellow Brownish yellow Light greenish yellow \n\nPowder + 50% HNO3 yellow Greenish yellow Yellow \nPowder + 5% KOH yellow Brown Greenish yellow \n\nPowder + Ammonia yellow Light yellow Yellow \n\nPowder + Picric acid Yellowish brown Brownish yellow Light greenish yellow \n\nTable 7c: Fluorescence analysis of O. tenuiflorum leaf \n\nTreatment Visible light Under UV light \n\n Short wavelength (254 nm) The long wavelength  \n(365 nm) \n\nPowder Brown   Dark brown \nPowder + Methanol Brown Green Black \n\nPowder + 70% ethanol Yellowish brown Green Black \n\nPowder + Pet. ether Brown Green Black \n\nPowder + 50% H2SO4 Yellowish brown Green Black \n\nPowder + 50% HCl Brown Green Black \nPowder + 1N NaOH \n(aq.) \n\nYellowish brown Green Black \n\nPowder + 1N NaOH \n(alc.) \n\nBrown Green Black \n\nPowder + 50% HNO3 Yellowish brown Green Black \n\nPowder + 5% KOH Brown Green Dark brown \nPowder + Ammonia Yellowish brown Green Black \n\nPowder + Picric acid Brown Green Black", "start_char_idx": 46621, "end_char_idx": 48996, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e467c0cb-a884-4402-b1d8-95d1c1d80c1a": {"__data__": {"id_": "e467c0cb-a884-4402-b1d8-95d1c1d80c1a", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0c60b918-5c8c-471f-b716-4c2275209cd5", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "7f9f3776e0b0794634e41d91e3ee363a361f377338f0350cbaeb76f8eeefdcbc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3a158431-0381-4c99-86b8-a3f6b66a3397", "node_type": "1", "metadata": {}, "hash": "9d46e4b753eaa5aee54392b0c80dcb2167709b327175c012eeb6be148304d6ac", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  49 \n\nTable 7d: Fluorescence analysis of combined herb-herb \n\nTreatment Visible light Under UV light \n\n Short wavelength (254 nm) The long wavelength (365 nm) \nPowder Brown Brown Brown \nPowder + Methanol Brown Brown Brown \n\nPowder + 70% ethanol Yellowish Brown Brown Brown \n\nPowder + Pet. ether Dark brown Light brown Light Brown \nPowder + 50% H2SO4 Dark brown Light brown Light Brown \n\nPowder + 50% HCl Brown Yellowish green Dark Red \nPowder + 1N NaOH (aq.) Brown Yellowish green Dark Brown \n\nPowder + 1N NaOH (alc.) Green Green Reddish orange \n\nPowder + 50% HNO3 Green Yellowish brown Orange red \n\nPowder + 5% KOH Brown Reddish brown Reddish brown \nPowder + Ammonia Yellowish brown Light orange Light orange \n\nPowder + Picric acid Brown Yellowish brown Orange \n\nTable 7e: Fluorescence Analysis of R. indica leaf \n\nTreatment Visible light Under UV light \n\n Short wavelength (254 nm) The long wavelength(365 nm) \n\nPowder Yellow  Brown  Yellow  \nPowder + Methanol Brown Greenish brown Brownish \n\nPowder + 70% ethanol light Brown Green Green black \n\nPowder + Pet. ether Light brown Dark brown Brownish black \n\nPowder + 50% H2SO4 Brown Dark green Greenish black \n\nPowder + 50% HCl Brown Light brown Light green \nPowder + 1N NaOH (aq.) Brown Purplish green Dark purplish green \n\nPowder + 1N NaOH (alc.) Green  Green  Brownish green  \n\nPowder + 50% HNO3 Green  Green  Pale yellow \n\nPowder + 5% KOH Brown Green Black \nPowder + Ammonia Yellowish brown Green Dark brown \n\nPowder + Picric acid Brown Green Black \n\n \n\nFurther, the sample track (track 1, 2, 3) and \n\nstandard track (track 4) were scanned at 254 nm \n\nshowed the same Rf value 0.47 for nyctanthoside in \n\nboth the track (Table 8). Finally, this nyctanthoside \n\nband in all this track which came at 0.47 Rf were \n\nscanned at 366 nm. The spectral pattern for \n\nnyctanthoside in extract matched with the standard \n\ntrack. Thus the presence of nyctanthoside was \n\nconfirmed by overlaying the UV spectra at 366 nm \n\nand 254 nm (Figure 5b) \n\n \n\nFigure 5c, 5d & 5e: HPTLC profile of hydroalcoholic of H. \n\nsalicifolia with standard Quercetin as developed in \n\nEthylacetate: Dichloromethane: Formic acid: Acetic acid: \n\nWater (10:2.5:1:1:0.5). Figure 5c represents fruit extract of \n\nH. salicifolia and Figure 5d represents Quercetin standard. \n\nFigure 5e represents photo documentation report of \n\nhydro-alcoholic extract of H. salicifolia at 254 nm and at \n\n366 nm. FE=fruit extract; ST=Standard.  \n\nTable 8: Rf value and color of peak of fruit extract and \n\nstandard Nyctanthic acid in Toluene:Ethyl acetate \n\n(8.0:2.0) at 366 nm \n\nS.No Rf value  Colour of the band  \n\n1. 0.17  Pink  \n\n2. 0.32  Blue  \n\n3. 0.37  Brown  \n\n4. 0.47  Purple  (Nyctanthic acid)  \n\n5. 0.57  Grey  \n\n6. 0.63  Green  \n\n7. 0.70  Blue  \n\n8. 0.77  Blue \n\n\n\nNepal J Biotechnol.", "start_char_idx": 49005, "end_char_idx": 51884, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3a158431-0381-4c99-86b8-a3f6b66a3397": {"__data__": {"id_": "3a158431-0381-4c99-86b8-a3f6b66a3397", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e467c0cb-a884-4402-b1d8-95d1c1d80c1a", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "004e3d14ce063adc80d23865a33ebfe6069805fd17c4c271a7cc214a339c773d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3f630cd9-da39-4105-9a8f-48ae560065cf", "node_type": "1", "metadata": {}, "hash": "cbe7a5c0763d45aa5b1cfa9fc1726079958a9461aaaf86fc5d116755684d9edd", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  50 \n\n \n\nThe extracts of dried powder of fruits of H. salicifolia \n\nwas subjected to HPTLC analysis and illustrated \n\nthe number of active compounds. The quercetin \n\ncontent determined by HPTLC method in fruits of \n\nH. salicifolia reported in (Table 9) and showed 4 \n\npeaks at 254 nm and 8 peaks at 366 nm. At 366 nm, \n\nH. salicifolia (HA) extract showed 8 peaks and Rf \n\nvalue matches with Quercetin standard, so this \n\nconfirms the presence of Quercetin in the extract \n\nwhich is a flavonoid (Figure 5c, 5d, 5e).  \n\nThe HPTLC fingerprints of hydroalcoholic extracts \n\nof O. tenuiflorum showed 6 peaks at 254nm (Table  \n\n10a) and 9 peak at 366 nm (Table 10b) (Shown in \n\nFigure 6). HPTLC fingerprint of O. tenuiflorum \n\nshows six different peaks at maximum Rf values of \n\n0.08, 0.32, 0.46, 0.54, 0.61 and 0.94. The peak at Rf \n\nvalue of 0.46 is noticed to have an area of about \n\n26650.2, which is the highest among the other peaks \n\nobtained and similar to eugenol. \n\nHPTLC fingerprint of R. Indica points out eight \n\ndifferent peaks at starting Rf values of -0.02, 0.14, \n\n0.19, 0.26, 0.35, 0.43, 0.58 and 0.74 (Table 11 and \n\nFigure 7). Among those peaks, the peak with Rf \n\nvalue of 0.58, exhibits a larger area of 4786.3. The \n\nextract shows the presence of saponin in hydro-\n\nalcoholic extract of R. indica at Rf :0.69. \n\n \n\n \nFigure 6: HPTLC profile of extract of Ocimum \n\ntenuiflorum with standard eugnol. (a) profil at showed  6 \n\npeak at 254nm ;(b) profil at showed  9 peak at 366 nm and \n\n(c)Photo documentation report of hydro-alcoholic extract \n\nat 254nm and at 366nm LE \u2013leaf  extract;ST-Standard. \n\nTable 9: Rf value and peak area of Fruit extract and \nstandard qurecetin in Ethylacetate: Dichloromethane: \nFormic acid : Acetic acid : Water (10:2.5:1:1:0.5) at 366 nm. \n\n Rf (Max) Area(AU) \n\n -0.01 2256.7 \n 0.06 247.9 \n 0.10 142.1 \n 0.53 494.5 \n 0.57 426.9 \n 0.77 195.8 \n 0.82 127.4 \n 0.94 7453.1 (FE) \n\n 0.96 \n8189.5(Qurecetin \nstandard) \n\nTable 10a. Rf value, No. of Peaks, peak area and height of hydroalcholic extract of O. tenuiflorum in Toluene: Ethyl acetate \n\nat 254 nm.", "start_char_idx": 51865, "end_char_idx": 54042, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3f630cd9-da39-4105-9a8f-48ae560065cf": {"__data__": {"id_": "3f630cd9-da39-4105-9a8f-48ae560065cf", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3a158431-0381-4c99-86b8-a3f6b66a3397", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b08856bfb130eadeaf866bb43797161d5af0c92294b13918f4df4a8ada117396", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b6958dd5-3760-4cea-9272-27bbf684d693", "node_type": "1", "metadata": {}, "hash": "3ada27cd28bc17193fb00632772560b3be2bbd9e9de24336c6ab8c42fb8ff04a", "class_name": "RelatedNodeInfo"}}, "text": "Peak Start Rf Start Height Max Rf Max Height Height % End Rf End Height Area Area % \n\n1 0.01 302.6 0.01 324.0 47.62 0.03 0.6 2776.3 17.53 \n2 0.18 0.2 0.21 12.7 1.87 0.25 4.7 357.8 2.26 \n3 0.31 8.5 0.35 11.7 1.72 0.41 2.2 557.8 3.52 \n4 0.75 2.2 0.78 10.4 1.53 0.80 6.7 326.6 2.06 \n5 0.81 7.1 0.89 166.0 24.39 0.91 152.6 6422.5 40.55 \n6 0.91 152.9 0.92 155.6 22.87 0.97 71.1 5398.9 34.08 \n\nTable 10b. Rf value, No. of Peaks, peak area and height of hydroalcholic extract of O. tenuiflorum in Toluene: Ethyl acetate \nat 366 nm. \n\nPeak Start Rf Start Height Max Rf Max Height Height % End Rf End Height Area Area % \n\n1 0.00 0.2 0.02 265.5 31.82 0.06 157.6 6275.7 18.02 \n2 0.06 157.9 0.07 162.0 19.41 0.16 76.6 9120.9 26.19 \n3 0.16 76.7 0.18 92.8 11.12 0.24 31.6 3444.2 9.89 \n4 0.26 31.4 0.27 39.2 4.69 0.33 18.3 1545.7 4.44 \n5 0.36 16.3 0.47 67.7 8.12 0.55 12.6 5161.8 14.82 \n6 0.56 14.7 0.62 41.4 4.97 0.66 24.9 2258.7 6.49 \n7 0.66 24.9 0.71 59.5 7.13 0.79 6.5 3032.2 8.71 \n8 0.83 15.9 0.89 55.1 6.60 0.93 31.1 2453.6 7.05 \n9 0.93 31.4 0.95 51.2 6.14 0.99 1.6 1527.8 4.39 \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  51 \n\n \n\nTable 11: Rf value, No.", "start_char_idx": 54045, "end_char_idx": 55232, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b6958dd5-3760-4cea-9272-27bbf684d693": {"__data__": {"id_": "b6958dd5-3760-4cea-9272-27bbf684d693", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3f630cd9-da39-4105-9a8f-48ae560065cf", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "82594dae01b6b5b6c59c69fe38a9bdd357b17a1d7222b2b28d25e0fdd44ca125", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9bb83301-f036-440c-88d1-64acc2153b29", "node_type": "1", "metadata": {}, "hash": "8029508e3c92fd41c202bdd6fd5d1b5c9f6d7f0bc92691ecb4bf2dc8085f2d78", "class_name": "RelatedNodeInfo"}}, "text": "of Peaks, peak area and height of hydroalcholic extract of R. indica in Toluene: Ethyl acetate at \n\n254nm \nPeak Start Rf Start \n\nHeight \nMax Rf Max \n\nHeight \nHeight \n\n% \nEnd Rf End \n\nHeight \nArea Area % \n\n1 0.00 0.2 0.02 265.5 31.82 0.06 157.6 6275.7 18.02 \n2 0.06 157.9 0.07 162.0 19.41 0.16 76.6 9120.9 26.19 \n3 0.16 76.7 0.18 92.8 11.12 0.24 31.6 3444.2 9.89 \n4 0.26 31.4 0.27 39.2 4.69 0.33 18.3 1545.7 4.44 \n5 0.36 16.3 0.47 67.7 8.12 0.55 12.6 5161.8 14.82 \n6 0.56 14.7 0.62 41.4 4.97 0.66 24.9 2258.7 6.49 \n7 0.66 24.9 0.71 59.5 7.13 0.79 6.5 3032.2 8.71 \n8 0.83 15.9 0.89 55.1 6.60 0.93 31.1 2453.6 7.05 \n\n \n\n \nFigure 7: HPTLC profile of hydroalcoholic of R. \nindica with standard saponin. Photo \ndocumentation report of hydro-alcoholic extract of \nR. indica at 254 nm and at 366 nm LE; Leaf extract; \n\nST-Standard. \n\nDiscussion  \nIn the current period, there is a need for logical \n\nassessment of natural formulation for better \n\ntreatment of the clinical issues in a clinical manner. \n\nThe bioactive compound of every, one of the four \n\nrestorative plants exhibits inside homegrown \n\nconcentrate and in joined herb-herb (polyherbal \n\ndefinition) that may result in all the more dominant \n\nfor mental issue treatment. A few reports \n\ndemonstrated that the utilization of a poly-natural \n\nplan has the greatest useful power when contrasted \n\nwith a single herb. Yet different definitions couldn't \n\nbe valuable because of the absence of legitimate \n\ninstitutionalization. In India, Ayurvedic \n\nPharmacopeia is not progressively sufficient to \n\nguarantee the quality and virtue of plant extracts\u2019 \n\nuse as a herbal drug. Since the concentrates from \n\nassembling points are not in a condition that could \n\nimpact appropriately. Along these lines, for the \n\nexperimentally demonstrated, customary \n\nmedications and natural plans are to be \n\ninstitutionalized for guaranteeing and defending \n\nthe best quality, immaculateness, and genuineness \n\nof the homegrown medications convert into \n\npowerful medication [49]. Along these lines, \n\ninstitutionalization is the easiest and least \n\nexpensive approach to the investigation of \n\nhomegrown medication through synthetic, \n\ndifferent strategies, morphological, minute \n\nexamination and afterward thin layer \n\nchromatography examination as well as initiating \n\nto assigning the right ID of the source plant extract \n\n[48].  \n\nAs in the present investigation there is no \n\ncomparable work accessible on these restoratively \n\nstrong plant extricate and consolidated herb-herb. \n\nThis examination work was embraced to \n\nmastermind the norms profile for building up its \n\ncredibility. In this way, the results of the above \n\ndiscoveries will fill in,  as a promising hotspot for \n\nsetting down the pharmacopeia principles profile of \n\nN. arbortristis, H. salicifolia, O. tenuiflorum and R. \n\nindica and consolidated herb-herb for future \n\ninvestigations and research for treatment of mental \n\nmalady. \n\nConclusion \nThe present study was taken up in the view to \n\nstandardize these herbals in accordance with WHO \n\nnorms and standard laboratory procedures. \n\n\n\nNepal J Biotechnol.", "start_char_idx": 55233, "end_char_idx": 58358, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9bb83301-f036-440c-88d1-64acc2153b29": {"__data__": {"id_": "9bb83301-f036-440c-88d1-64acc2153b29", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b6958dd5-3760-4cea-9272-27bbf684d693", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "7e1947c823a6141347a110f7cffafa72a14e06f621eb5db879b33928f68d3c85", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "819131b4-5f8f-4432-a0d2-1e6fc4a5c163", "node_type": "1", "metadata": {}, "hash": "687ead461abb6bcd933d804269bf51d119adac5a04273b63cb5248dad4da115b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  52 \n\nFormulations were investigated for their \n\norganoleptic characters, physicochemical \n\nparameters, HPLC analysis and phytochemical \n\nparameters etc. The heavy metal quantity was also \n\nfound within the standard limit as given by the \n\nregulatory authorities  Hence, the pharmacognostic \n\nparameter of the plant extracts will be helpful in \n\nfurther preclinical and clinical study. The presence \n\nof phytochemicals also indicated that it can be used \n\nto develop the new lead Phyto-molecule in the \n\ntreatment and management of lifestyle disease or \n\ndisorder. The finding of this study can be used for \n\nevaluating the quality and purity of these plants as \n\na polyherbal formulation for clinical application. \n\nAuthor contributions \nAll authors have equal contributions. All authors \n\nread and approved the final manuscript. \n\nCOMPETING INTERESTS \nThe authors declare that they have no competing \n\ninterests. \n\nFunding \nThe authors declared that no grants were \n\ninvolved in supporting this work.  \n\nAcknowledgments \nThe creators stretch out their true gratitude to the \n\nDepartment of Science and Technology, Ministry of \n\nHealth, Government of India for supporting this \n\nexamination. The authors would like to thank, Dr. \n\nR.C. Satish Kumar, M.D.(Ayu) M.B.A (H.M.), \n\nInterdisciplinary Institute of Indian System of \n\nMedicine (IIISM), Sri Ramaswami Memorial \n\nUniversity (SRM) University, Kattankulathur \n\nChennai, India for identification and authentication \n\nof plants. The authors are appreciative to similarly, \n\ngrateful to SRM University, Chennai, India for the \n\noffering help in recognizable proof of bioactive \n\natoms and factual investigation and input on the \n\noutcomes. \n\nEthical approval and consent \nNot applicable. \n\nReference \n1. Aslam MS, Ahmad MS, Mamat AS, Ahmad MZ, Salam F. \n\nAntioxidant and wound healing activity of polyherbal \nfractions of Clinacanthus nutans and Elephantopus scaber. \nEvidence-based Complementary and Alternative Medicine. \n\n2016 Jan 1;2016. https://doi.org/10.1155/2016/4685246 \n2. Chaudhary A, Singh N. Contribution of world health \n\norganization in the global acceptance of Ayurveda. Journal \n\nof Ayurveda and Integrative Medicine. 2011 Oct;2(4):179. \nhttps://doi.org/10.4103/0975-9476.90769 \n\n3. Che CT, Wang ZJ, Chow MS, Lam CW. Herb-herb \ncombination for therapeutic enhancement and advancement: \ntheory, practice and future perspectives. Molecules. 2013 \n\nMay;18(5):5125-41. \nhttps://doi.org/10.3390/molecules18055125   \n\n4. Parasuraman S, Thing GS, Dhanaraj SA. Polyherbal \nformulation: Concept of Ayurveda. Pharmacognosy reviews. \n\n2014 Jul;8(16):73. https://doi.org/10.4103/0973-7847.134229   \n5. Mohd Fuat AR, Aidoo KE, Calvert TW, Candlish AA. \n\nMycoflora, cytotoxicity, and DNA interaction of polyherbal \n\nproducts from Malaysia. Pharmaceutical Biology. 2006 Jan \n1;44(1):23-31. https://doi.org/10.1080/13880200500530500 \n\n6. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \nDridhabala, Acharya VYT, editors. Chowkhamba \n\nKrishnadas Academy. 2014;4(32):49. \n7. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \n\nKrishnadas Academy. 2014;9(11):24.  \n8.", "start_char_idx": 58339, "end_char_idx": 61589, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "819131b4-5f8f-4432-a0d2-1e6fc4a5c163": {"__data__": {"id_": "819131b4-5f8f-4432-a0d2-1e6fc4a5c163", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9bb83301-f036-440c-88d1-64acc2153b29", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "519f113667689d9f6551670eabfaf11939ed24c6991d0af44ea755e67331e2aa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "786937dc-8d27-4cd7-b402-fec55a3a67db", "node_type": "1", "metadata": {}, "hash": "3fcb23ca24e600260c6ad6af551cbc496e7bb524d78ba0275a1559264b938cdf", "class_name": "RelatedNodeInfo"}}, "text": "2014;9(11):24.  \n8. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2014;13(11):25. \n\n9. Agnivesha. Charaka Samhita, Kalpa Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2000;6(3). \n\n10. Singh V. Seabuckthorn (Hippophae L.): a multipurpose \nwonder plant. 2005;2:505-21. \n\n11. Singh V. Seabuckthorn (Hippophae L.)\u2013A Wonder Plant of \nDry Temperate Himalayas. Department of Agro forestry and \nEnvironment, Himachal Pradesh Agriculture University. \n\n2001:1760-62. \n12. Goyal AK, Basistha BC, Sen A, Middha SK. Antioxidant \n\nprofiling of Hippophae salicifolia growing in sacred forests of \nSikkim, India. Functional Plant Biology. 2011 Sep 6;38(9):697-\n\n701. https://doi.org/10.1071/FP11016 \n13. Suryakumar G, Gupta A. Medicinal and therapeutic \n\npotential of Sea buckthorn (Hippophae rhamnoides L.). Journal \n\nof Ethnopharmacology. 2011 Nov 18;138(2):268-78. \nhttps://doi.org/10.1016/j.jep.2011.09.024 \n\n14. Saikia M. Antioxidant and antibacterial activity of leaf, bark, \npulp and seed extracts of seabuckthorn (Hippophae salicifolia \n\nD. Don) of Sikkim Himalayas. Journal of Medicinal Plants \nResearch. 2013 May 17;7(19):1330-8. \n\n15. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2000;3(4):59. \n\n16. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2000;4(37):90. \n\n17. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \nDridhabala, Acharya VYT, editors. Chowkhamba \n\nKrishnadas Academy. 2000;14(32):288. \n18. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2000;27(169):45. \n\n19. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \nKrishnadas Academy. 2000;7(17):729. \n\n20. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \nDridhabala, Acharya VYT, editors. Chowkhamba \n\nKrishnadas Academy. 2000;7(21):731. \n21. Agnivesha. Charaka Samhita, Sutra Sthana. Charaka, \n\nDridhabala, Acharya VYT, editors. Chowkhamba \n\nKrishnadas Academy. 2000;8(142):791. \n22. Perveen AN, Qaiser MU. Pollen flora of Pakistan-Lix. \n\nLinaceae. Pak J Bot.", "start_char_idx": 61570, "end_char_idx": 63871, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "786937dc-8d27-4cd7-b402-fec55a3a67db": {"__data__": {"id_": "786937dc-8d27-4cd7-b402-fec55a3a67db", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "819131b4-5f8f-4432-a0d2-1e6fc4a5c163", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "0837fdca10c344f53cc45d7459b28175a0a11fb058abacce43dab8b8c0ab574a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c0d5e6b1-0c33-4783-96fc-4c661a1b3626", "node_type": "1", "metadata": {}, "hash": "e11860bcc92a2b6450c7c4ac4b39675a4b4d1ddeb241c92eae944e9005923186", "class_name": "RelatedNodeInfo"}}, "text": "Pollen flora of Pakistan-Lix. \n\nLinaceae. Pak J Bot. 2008 Oct 1;40(5):1819-22. \n23. Verma S, Chauhan NS. Indigenous medicinal plants \n\nknowledge of Kunihar forest division, district Solan. Indian \nJournal of Traditional Knowledge. 2007;6(3):494-7. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):36-53 Kumari and Dubey  \n \n\n\u00a9NJB, BSN  53 \n\n24. Shah R, Pande PC, Tiwari L. Traditional veterinary herbal \nmedicines of western part of Almora district, Uttarakhand \n\nHimalaya. Indian Journal of Traditional Knowledge. \n2008;7(2)355-9. \n\n25. Phondani PC, Maikhuri RK, Bisht NS. Medicinal plants used \n\nin the health care system practiced by traditional Vaidyas in \nAlaknanda catchment of Uttarakhand, India. Ethnobotanical \n\nLeaflets. 2009;2009(12):4. \n26. Bhattarai S, Chaudhary RP, Taylor RS. Ethno-medicinal \n\nplants used by the people of Nawalparasi District, Central \nNepal. Our nature. 2009;7(1):82-99. \nhttps://doi.org/10.3126/on.v7i1.2555  \n\n27. Abha S, Swati V, Shukla RK. Preliminary phytochemical \nscreening, antibacterial and nitric oxide radical scavenging \n\nactivities of Rinwardtia indica leaves extract. Int J PharmTech \nRes. 2013;5(4):1670-8.  \n\n28. Sass JE. Elements of Botanical Microtechnique. McGraw Hill. \n1940:222. \n\n29. O'Brien T, Feder N, McCully ME. Polychromatic staining of \n\nplant cell walls by toluidine blue O. Protoplasma. 1964 Jun \n1;59(2):368-73. https://doi.org/10.1007/BF01248568 \n\n30. Easu K. Plant Anatomy. John Wiley and Sons. 1964:767. \n31. Barminas JT, James MK, Abubakar UM. Chemical \n\ncomposition of seeds and oil of Xylopia aethiopica grown in \n\nNigeria. Plant Foods for Human Nutrition. 1999 Sep \n1;53(3):193-8. https://doi.org/10.1023/A:1008028523118 \n\n32. World Health Organization. Quality control methods for \nherbal materials. World Health Organization; 2011. \n\n33. Raghuramulu N, Nair NK, Kalyansundaram S. Manual of \nlaboratory techniques, Hyderabad. National Institute of \nNutrition. 2003:56-8.  \n\n34. Hedge JE and Hofreiter BT, Whistler RL, BeMiller JN. \nMethods in Carbohydrate Chemistry. Academic Press, New \n\nYork. 1962:17. \n35. Authors AOAC. Official methods of analysis Lipids, Fats \n\nand Oils Analysis Total Fat Animal Feed-item 16. \nAssociation of Analytical Communities. 2006. \n\n36. Parekh J, Chanda S. In vitro antibacterial activity of the crude \n\nmethanol extract of Woodfordia fruticosa Kurz. Flower \n(Lythraceae). Brazilian Journal of Microbiology. 2007 \n\nJun;38(2):204-7. https://doi.org/10.1590/S1517-\n83822007000200004 \n\n37. Trease GE, Evans WC. Textbook of Pharmacognosy. Balliere \nTindall, London. 1983:343-383. \n\n38. Bray HG, Thorpe WV. Analysis of phenolic compounds of \n\ninterest in metabolism. Methods of biochemical analysis.", "start_char_idx": 63819, "end_char_idx": 66516, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c0d5e6b1-0c33-4783-96fc-4c661a1b3626": {"__data__": {"id_": "c0d5e6b1-0c33-4783-96fc-4c661a1b3626", "embedding": null, "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-175", "node_type": "4", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "b7c70f4db366febc1501a0e05e28175ea9d5ec6dbd2d9f41e4b03eaa91e06732", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "786937dc-8d27-4cd7-b402-fec55a3a67db", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "29ebb370f312c1303a676c98e281cc425ed4c62d4e765cc346a9692b80f76058", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2fafb659-ef3a-4fcb-8f5c-0be8043aec06", "node_type": "1", "metadata": {}, "hash": "1bd3069c0eb2c53258c06ef6bed861aa3074e5b06fa1ea37227c24709484eb6f", "class_name": "RelatedNodeInfo"}}, "text": "Analysis of phenolic compounds of \n\ninterest in metabolism. Methods of biochemical analysis. \n1954 Jan 1:27-52. \n\nhttps://doi.org/10.1002/9780470110171.ch2 \n\n39. Sethi PD. High-Performance Thin Layer Chromatography \nQuality Analysis of Pharmaceutical Formulations. \n\nCBS India.1996:44\u201359. \n40. Inter African Committee on Medicinal Plants and African \n\nTraditional Medicine. African Pharmacopoeia. 1st ed. Lago: \n\nOrganisation of African Unity/The Scientific, Technical and \nResearch Commission (OAU/STRC). 1986:123. \n\n41. Kokoski CJ, Kokoski RJ, Slama FJ. Fluorescence of powdered \nvegetable drugs under ultraviolet radiation. Journal of the \n\nAmerican Pharmaceutical Association (Scientific ed.). 1958 \nOct 1;47(10):715-7. https://doi.org/10.1002/jps.3030471010 \n\n42. Chase Jr CR, Pratt R. Fluorescence of powdered vegetable \n\ndrugs with particular reference to development of a system \nof identification. Journal of the American Pharmaceutical \n\nAssociation. 1949 Jun;38(6):324-31. \nhttps://doi.org/10.1002/jps.3030380612 \n\n43. Mukherjee PK. Quality control of herbal drugs: an approach \nto evaluation of botanicals. Business Horizons. 2002. \n\n44. India Department of Indian Systems of Medicine & \n\nHomoeopathy (AYUSH). The Ayurvedic Pharmacopoeia of \nIndia Part 1. New Delhi: Government of India, Ministry of \n\nHealth and Family Welfare. 2001. \n45. Krinsky NI, Beecher GR, Burk RF, Chan AC, Erdman JJ, \n\nJacob RA, Jialal I, Kolonel LN, Marshall JR, Taylor Mayne \n\nPR, Prentice RL. Dietary reference intakes for vitamin C, \nvitamin E, selenium, and carotenoids. Institute of Medicine. \n\n2000 Apr 19. \n46. Kochhar A, Nagi M, Sachdeva R. Proximate composition, \n\navailable carbohydrates, dietary fibre and anti nutritional \nfactors of selected traditional medicinal plants. Journal of \nHuman Ecology. 2006 Mar 1;19(3):195-9. \n\nhttps://doi.org/10.1080/09709274.2006.11905878 \n47. Mowl A, Alauddin M, Rahman M, Ahmed K. \n\nAntihyperglycemic effect of Trigonella foenum-graecum \n(Fenugreek) seed extract in alloxan-induced diabetic rats and \n\nits use in diabetes mellitus: a brief qualitative phytochemical \nand acute toxicity test on the extract. African Journal of \nTraditional, Complementary and Alternative Medicines. \n\n2009;6(3). https://doi.org/10.4314/ajtcam.v6i3.57165 \n48. Iwalokun BA, Usen UA, Otunba AA, Olukoya DK. \n\nComparative phytochemical evaluation, antimicrobial and \nantioxidant properties of Pleurotus ostreatus. African Journal \n\nof Biotechnology. 2007;6(15). \nhttps://doi.org/10.5897/AJB2007.000-2254 \n\n \n\n49. Verma S, Singh SP. Current and future status of herbal \n\nmedicines. Veterinary world. 2008 Nov 1;1(11):347. \nhttps://doi.org/10.5455/vetworld.2008.347-350", "start_char_idx": 66424, "end_char_idx": 69106, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2fafb659-ef3a-4fcb-8f5c-0be8043aec06": {"__data__": {"id_": "2fafb659-ef3a-4fcb-8f5c-0be8043aec06", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c0d5e6b1-0c33-4783-96fc-4c661a1b3626", "node_type": "1", "metadata": {"identifier": "njb-175", "author": "Kumari, Rinki; Dubey, G.P.", "title": "Standardization of Ayurvedic Drug- Nyctanthes arbor-tristis, Hippophae salicifolia, Ocimum tenuiflorum and Reinwardtia indica and Combined Herb-Herb", "date": "2020-07-31", "file": "njb-175.pdf"}, "hash": "cc1002ce4dbcecad5d8051a520856945e270bbfa2b4b0e4753a4413ec068aec8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "af3cc89c-a100-495a-b9a3-24961fa28e38", "node_type": "1", "metadata": {}, "hash": "d91bf6ae4cda125efd9c1a0a6f3c14be6bed210e26cd3948dc9d15f3872df1d3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35 DOI: https://doi.org/10.3126/njb.v8i1.30207   Research article  \n\n\u00a9NJB, BSN  29 \n\n  \n \n \n \n\nPhytochemicals Levels and Antioxidant Capacities of Figs Flowers \nFruits \nMohammed Messaoudi         , Maroua Merah  \n\nUniversity Dr Moulay Tahar, PO Box 138 Al-Nasr District 20000, Saida \u2013Algeria \n\nArticle history:- Received: 7  Dec 2019; Revised: 5 Apr 2020; Accepted: 16 Jun 2020; Published online: 31 Jul 2020   \n\nAbstract \nSince antiquity, phenolic compounds produced by plants were known as free radical scavengers and as powerful \nantioxidants. Huge interest has been made by researchers to the traditional uses of medicinal plants against illnesses \nrelated to oxidative stress. This study measures the correlation that can be existed between the antioxidant capacity \nand phytochemicals levels of four varieties of Ficus carica fruits, figs flowers or \"Bakor\" as called locally in Algeria. \nTherefore, extracts were assessed for determining their antioxidative potentials using both test of total antioxidant \ncapacity and DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test followed by quantitative \nphytochemical analysis to estimate the total flavonoid level (TFL), the total phenolic level (TPL), the total \nanthocyanin level (TAL) and the condensed tannins level of plants methanolic extracts. A positive correlation was \nobserved between phenolics content and the antioxidant capacity of figs flowers methanol extracts. The methanolic \nextract of Bechar (MeOH Var.2) chelated 87. 9\u00b1 1.23 % of the DPPH free radical with IC50 value equal to 0.185 mg/g \nDW. A high antioxidant ability of almost all extracts is, probably, related to the appreciable rates of flavonoids, \nphenolics and tannins showed by those fig extracts. The highest value of phenolics level was detected among the \nvariety 1 methanolic extract of Bechar (MeOH Var.1) of 10.4 mg GAE/g DW. \n\nKeywords: Antioxidant capacity; Bakor; figs flowers; Ficus carica; Methanol extracts; DPPH. \n\n    Corresponding author, email: microbiologistemed@yahoo.fr \n\nIntroduction \nPhytochemicals, including phenolics, flavonoids, \n\nflavonols, ascorbic acid, lignin, xanthones, stilbenes, \n\netc., are plant-based secondary metabolites, which \n\nare associated with the protection of human health \n\nagainst chronic diseases [1, 2, 3, 4]. The relative \n\nimportance of medicinal and food plant species can \n\nbe assessed by their use-value. Plant species with \n\nmore traditional uses exhibit high use value \n\ncompared to those which have fewer ones [5]. \n\nNowadays, medicinal plants considered as an \n\nimportant source of drugs as about 25% of the drugs \n\nprescribed worldwide derive from plants [6].  \n\nFig tree Ficus carica Linn. originated in the Middle \n\nEast areas such as Syria, Asia Minor, and Iran, then, \n\nit was spread to the Mediterranean basin countries \n\nby old humans [7, 8]. It belongs to the family of \n\nMoraceae. F. carica L. is one of the unique widely \n\nspread Ficus species that has edible fruits with high \n\ncommercial value. The production of commercial fig \n\nis situated in regions that possess a Mediterranean \n\nclimate [9].  \n\nF. carica L. has three figs yields, Early fig stays on the \n\ntree; Late fig of autumn or figs flowers carries from \n\nAugust to winter and is locally known as Bakor and \n\nthe green or winter figs [10, 11].  Oxidative stress is \n\nan inequality between prooxidants and antioxidants \n\nin favor of the first contributing to the appearance of \n\nseveral pathologies. The uncontrolled oxygen \n\nspecies resulted will have serious and severe \n\nconsequences for the human organism [12].", "start_char_idx": 52, "end_char_idx": 3691, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "af3cc89c-a100-495a-b9a3-24961fa28e38": {"__data__": {"id_": "af3cc89c-a100-495a-b9a3-24961fa28e38", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2fafb659-ef3a-4fcb-8f5c-0be8043aec06", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "a52c2afd356b2a653ed5f30f4a3edbb8a46aaa3138e30161658415d316d97fb6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "35f4e16a-9947-4ae4-9958-bb604ce2ec30", "node_type": "1", "metadata": {}, "hash": "0436ba5749a86c1528c2777167a692f9549a1a7760f8dd7c1cf62862de8059fa", "class_name": "RelatedNodeInfo"}}, "text": "Several \n\nstudies focus on natural antioxidant sources to find \n\nnew effective, safe and cheap antioxidants as there is \n\na strong relationship between the decrease of certain \n\nchronic diseases and plants-produced antioxidants \n\n[13]. Fruits are essential functional foods that \n\nmaintain the human vital functions as they \n\nproviding a well-balanced diet [14]. Viewing the \n\nbiological properties of F. carica fruits, our study \n\nfocuses on the correlation between phytochemicals \n\ncontents and antioxidant capacity of dried fruits \n\nmethanolic extracts of figs flowers or \"Bakor\" \n\noriginated of four different varieties of F. carica. Two \n\nvarieties of Bechar and the two others from Mascara. \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nmailto:microbiologistemed@yahoo.fr\nhttp://orcid.org/0000-0001-9893-5987\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  30 \n\nMaterials and Methods \nCollection of Plant Samples  \nFruits from four different varieties of F. carica figs \n\nflowers or \"Bakor\" as called locally were collected \n\nbetween May and August 2018. Two varieties from \n\nBechar located at the Southwest of Algeria: Var.1 \n\n(Lahmar), Var.2 (Ouakda). The other two ones from \n\nMascara situated at the North of Algeria: Var.3 (El \n\nBordj), Var.4 (Ghriss). Plants specimens were \n\nidentified by the Laboratory of Biotoxicology, \n\nPharmacognosy and Biological Valorisation of \n\nPlants (University of Saida).  \n\nFigure 1. Fruit of Ficus carica L. (Left: whole fruit; \nRight: cross-section) [15, 16]. \n\nPreparation of Methanolic Extracts \nFour samples of fig flowers were air-dried and \n\ncrushed. After, 1 g of F. carica fruits was soaked, \n\nunder ultrasound, using pure methanol (20 mL) for \n\n24 h. Plants extracts were filtered, concentrated and \n\nstored at 4\u00b0C until used (MeOH Var.1, MeOH Var.2, \n\nMeOH Var.3, MeOH Var.4) [17].   \n\nPhytochemical screening \nChemical products and reagents purchased from \n\nMerck Company, Darmstadt, Germany and \n\nphytochemical screening tests were repeated for \n\nthree times. \n\nDetermination of percentage yield \nThe percentage yield was calculated for each extract \n\nusing the formula:  \n\nPercentage yield (%) = a/b \u00d7 100 \n\nWhere: (a)=the dry weight of extract, (b)=soaked \n\nsamples material [18]. \n\nDetermination of Total Flavonoids Level \n(TPL) \nTo measure the total flavonoids level, volumes of 2 mL \n\nfrom both plant extracts and 2% ethanolic solution of \n\naluminum trichloride (AlCl3) were mixed and \n\nincubated 10 min at room temperature. After \n\nmeasurement of absorbance at 430 nm, TFL resulted \n\nin mg quercetin per g dry weight [19].  \n\nDetermination of Total Phenolic Level \n(TPL) \nTotal phenolics level was estimated via Folin\u2013\n\nCiocalteu's reagent. A mixture of sodium carbonate \n\nsolution (2 mL, 2%) and 0.1 mL of plant extract was \n\nincubated for 5 min. Then, a volume of 100 \u03bcL Folin\u2013\n\nCiocalteu's reagent was added. The mixture was \n\nincubated for 30 min and absorbance was read at 700 \n\nnm to determine the TPL as mg gallic acid per g dry \n\nweight (mg GAE/g) [20].", "start_char_idx": 3692, "end_char_idx": 6892, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "35f4e16a-9947-4ae4-9958-bb604ce2ec30": {"__data__": {"id_": "35f4e16a-9947-4ae4-9958-bb604ce2ec30", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "af3cc89c-a100-495a-b9a3-24961fa28e38", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "b19193527d208085dde9e1c29b8ec4a460b06eecf913a3c1dc080f1e2e8ec0d1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d2ec205c-03cb-43fc-8d97-2829be98ce2c", "node_type": "1", "metadata": {}, "hash": "4f0510bd98c003ff443a1232f94c36ae86a39627205c2401d5354b8e7e1d1c85", "class_name": "RelatedNodeInfo"}}, "text": "Determination of Total Anthocyanins Level \n(TAL) \nThe pH differential method was used to deduce total \n\nanthocyanins level, by which, the absorbance of the \n\nreaction solution was measured at both 510 nm and \n\n700 nm at two pH 1.0 then pH 4.5 using buffer \n\nsystems: hydrochloric acid (0.2 M) and sodium \n\nacetate (1 M).  \n\nA= (A510-A700)pH1,0 - (A510-A700)pH4,5 \n\nTAL=[(A x MW x DF) / MA] x 100 \n\nA: absorbance; MW: molar mass; DF: dilution factor; \n\nMA: molar absorption. \n\nTAL was expressed as mg cyanidin-3-glucoside per \n\ng of dry weight (mg C3G/g) [2, 21].  \n\nDetermination of Condensed Tannins Level \n(CTL) \nThe vanillin test was used to estimate the Condensed \n\nTannins Level (CTL). The mixture contained \n\nmethanolic solution of vanillin (4%, w/v), methanol \n\n(37%, v/v), HCl (8%, v/v), at equal volume was kept \n\nat 30 \u00b0 C until used. Then, a solution of 1500 \u03bcl of \n\nvanillin methanolic solution was added to 50 \u03bcl of \n\nplant extracts and 750 \u03bcl of concentrated HCl was \n\nincubated for 20 min. The absorbance was read at \n\n550 nm and compared to a blank of equal volumes \n\nof both 37% methanol and 8% HCl. Results were \n\nexpressed in mg of catechin equivalent per gram of \n\nthe dry weight (mg CE/g) [22, 23, 24].  \n\nTests of antioxidant capacity    \nThe antioxidant capacity of the four varieties were \n\nevaluated using free radical scavenging test (DPPH) \n\nand total antioxidant capacity (TAC). \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  31 \n\nDPPH free radical scavenging test  \nDPPH scavenging test was used to estimate the \n\ncapacity of each extract to scavenge hydrogen atom \n\ngenerated of 2,2-diphenyl-1-picrylhydrazil radical. \n\nA mixture of 1 mL of 100 \u03bcM methanol solution of \n\nthe free radical DPPH and different concentrations \n\nof each extract was incubated 20 min. The \n\nabsorbance of the solution was read at 517 nm and \n\ncompared to the blank that contained both 1 mL of \n\nDPPH methanolic solution and 0,1 mL of methanol \n\nsolvent [25, 26]. The inhibition percentage (% IP) of \n\nDPPH solution was estimated by the following \n\nformula: \n\n% IP = [(At0- At20) / At0\u00d7 100)] \n\nWhere, At0= Absorbance of the blank solution after \n\n20 min. At20= Absorbance of each sample after 20 \n\nmin. Then, the concentration of an extract that \n\nallowing to 50% inhibition of DPPH solution (IC50) \n\nwas obtained graphically. The less is the IC50, the \n\nhigher is the antioxidant capacity. A positive control \n\nwith various concentrations was prepared by the \n\nmethanolic solution of ascorbic acid [27].  \n\nTotal Antioxidant Capacity (TAC) \nThe test of phosphomolybdenum reagent allows the \n\ndetermination of Total Antioxidant Capacity (TAC) \n\nof plant samples. A mixture that contains a 3 mL \n\nvolume of a solution of ammonium molybdate \n\nreagent (4 mM), sulphuric acid (0.6 M) and sodium \n\nphosphate (28 mM) added to a volume 0.3 mL of \n\neach extract was incubated at 95\u00b0C. After, 90 min, the \n\nabsorbance was measured at 695 nm against a \n\ncontrol solution prepared under the same \n\nconditions, constituted of 0.3 mL of methanol and 3 \n\nmL of all the reagents used before. Total Antioxidant \n\nCapacity expressed as mg ascorbic acid per g dry \n\nweight (mg AAE / g).", "start_char_idx": 6896, "end_char_idx": 10113, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d2ec205c-03cb-43fc-8d97-2829be98ce2c": {"__data__": {"id_": "d2ec205c-03cb-43fc-8d97-2829be98ce2c", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "35f4e16a-9947-4ae4-9958-bb604ce2ec30", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "6966d805d2f89f76cd2472caa176bee0981a9e9212663aaf0b044f25c20ec579", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b77f0d48-70de-4243-af89-c1d0d60345a9", "node_type": "1", "metadata": {}, "hash": "1cb3dc0966dfa3c50a30a3589734b6bc7d3dc67137dba214905b41894780405a", "class_name": "RelatedNodeInfo"}}, "text": "A calibration curve with \n\nvarious concentrations was prepared using ascorbic \n\nacid [28]. \n\nStatistical analysis \nThe experimental data obtained from the TFL, TPL, \n\nTAL, CTL and antioxidant capacity tests were \n\nexpressed as means \u00b1 standard deviation. Statistical \n\nanalysis of data was performed using Microsoft \n\nExcel. Statistical differences were estimated, One-\n\nway ANOVA and student\u2019s t-test were used. The \n\ncorrelation coefficient of antioxidant capacities as \n\ndetermined by the Pearson test. Values are to be \n\nstatistically significant at p < 0.05.  \n\nResults and Discussion \nTotal flavonoids level, total anthocyanins level, total \n\nphenolics level, condensed tannins level of fig flowers \n\nfruits (Bakor) methanolic extracts are presented in \n\nFigure 2. \n\nThe total phenolics level of the four extracts varied \n\nfrom 4.7 to 10.4 mg GAE/g DW. The highest level \n\nwas significantly (p<0.05) conferring to the variety 1 \n\nmethanolic extract of Bechar (MeOH Var.1) and the \n\nlowest in the extract MeOH Var.3 of Mascara (See \n\nFigure 2). \n\n \nFigure 2. Levels of various phytochemicals of fig flowers \nfruits methanolic extracts. MeOH Var.1; MeOH Var.2: Variety \n\n1, 2 Bakor (Fig flowers) of Bechar. MeOH Var.2; MeOH Var.3: \nVariety 2 Bakor of Mascara. TFL: Total Flavonoids Level, \nTAL: Total Anthocyanins Level, TPL: Total phenolics \nLevel. CTL: Condensed Tannins Level. mg QE/g : \nquercetin equivalents; mg C3G/g : cyanidin-3-glucoside \nequivalents; mg GAE /g : acid gallic equivalents; mg \nCE/g:  catechin equivalent. \n\nThe extract of Bechar MeOH Var.2 has the highest \n\nTotal flavonoids value 7.1 mg QE/g DW while MeOH \n\nVar.4 has the lowest one of 3.2 mg QE/g DW. An \n\nimportant total anthocyanin levels were detected in \n\nMeOH Var.1 of 2.6 mg C3G/g DW compared to the \n\nmethanolic extract of mascara MeOH Var.3 which has \n\na low value (1.7 mg C3G/g DW). A statistical \n\nsignificant differences existed on phytochemicals \n\nlevels between plant samples (p < 0.05).  \n\nTotal phenolics amounts resulted in F. carica skin \n\nextracts ranged between 28.6 and 211.9 mg GAE / \n\n100 g FW and between 24 and 237 mg GAE / 100 g \n\nFW [21, 29]. The considerable phytochemicals levels \n\nobtained can be explained by the sonication \n\nextraction using pure methanol as solvent and as \n\nsuch, is not an ignored proposition as the extraction \n\nof flavones, polyphenols, anthocyanins, tannins can \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  32 \n\nbe very good using alcohol such as methanol solvent \n\n[30].  \n\nFurthermore, the special climate conditions such as \n\nthe low monthly rainfall and the high temperature \n\ncharacterizing the harvest year of plant samples can \n\nalso affect widely their phytochemicals amounts. \n\nAccording to Vallejo et al. 2012, the skin of the early \n\nfig fruit (first crop) is richer in phenolic components \n\nthan late fruit (second crop) possibly as a \n\nconsequence of climatic factors [31].  \n\nFigures 3, 4 and 5 illustrate the total antioxidant \n\ncapacity and the % inhibition of DPPH.  \n\nFigure 3. Total antioxidant capacity of fig flowers fruits \nmethanolic extracts. MeOH Var.1; MeOH Var.2: Variety 1, 2 \nBakor (Fig flowers) of Bechar. MeOH Var.3; MeOH Var.4: \nVariety 3, 4 Bakor of Mascara.  \n\n \nFigure 4.", "start_char_idx": 10114, "end_char_idx": 13397, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b77f0d48-70de-4243-af89-c1d0d60345a9": {"__data__": {"id_": "b77f0d48-70de-4243-af89-c1d0d60345a9", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d2ec205c-03cb-43fc-8d97-2829be98ce2c", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "3d71d471ccc891adfd23ba0d0bf2f144e5d96c2c3649e2c021bde6702e0cc951", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8f690923-ae05-4c24-b312-9c263f0a557f", "node_type": "1", "metadata": {}, "hash": "d93ac5633c14351bce8550da7347084db71cfddb110efc631f11bbc1142a7f7c", "class_name": "RelatedNodeInfo"}}, "text": "Figure 4. DPPH radical scavenging activity of fig \nflowers fruits methanolic extracts. MeOH Var.1; \nMeOH Var.2: Variety 1, 2 Bakor (Fig flowers) of \nBechar. MeOH Var.3; MeOH Var.4: Variety 3, 4 Bakor \nof Mascara \n\nThe inhibition concentration value that exhibits 50% \n\nof radical scavenging activity for different varieties \n\nof extracts (Table 1). Table 2 summarizes the \n\ncorrelation coefficients among antioxidant tests, \n\ntotal flavonoid level, total anthocyanins level and \n\ntotal phenolic level. \n\n \nFigure 5. % inhibition of DPPH. MeOH Var.1; MeOH \nVar.2: Variety 1, 2 Bakor (Fig flowers) of Bechar. MeOH \nVar.3; MeOH Var.4: Variety 3, 4 Bakor of Mascara  \n\nCompared to the positive control IC50 value 0.004 \n\nmg/g DW, the lowest IC50 equal to 0.185 mg/g DW \n\nwas deduced for methanolic extract of Bechar \n\n(MeOH Var.2), which chelated 87.9\u00b1 1.23 % of the \n\nDPPH free radical. However, the extract MeOH Var.4 \n\nhas presented the lowest %IP of 63.5 \u00b1 0.87% with an \n\nIC50 value of 0.448 mg/g DW. Our results showed a \n\nstatistically significant difference between studied \n\nextracts and positive controls (p < 0.05). \n\nTable 1. IC50 value of fig flowers fruits methanolic \n\nextracts. \n\nVarieties of fig flowers (Bakor) IC50 (mg/mL) \n\nMeOH Var.1 0.260A \n\nMeOH Var.2 0.185B \n\nMeOH Var.3 0.345C \n\nMeOH Var.4 0.448D \n\nA.A 0.004E \n\nMeOH Var.1; MeOH Var.2: Variety 1, 2 Bakor (Fig flowers) \n\nof Bechar. MeOH Var.3; MeOH Var.4: Variety 3, 4 Bakor of \n\nMascara. A.A: ascorbic acid. Capital letters (A\u2013B) and \n\nlowercase letters (a\u2013b) indicate significant differences at \n\np < 0.05. \n\nThe high TPL and TFL levels measured can explain \n\nwidely such a considerable percent of inhibition. \n\nPhenolics are the most effective antioxidants, which \n\nfunction as free radical scavengers, and absorb \n\noxygen radicals [32, 33], because of their acidity, \n\nability to transfer electrons and characteristic \n\nbenzene rings [34]. Solomon et al. 2006 have proven \n\nthat, compared to the fruit pulp, fruit skins have the \n\nmost contribution to phytochemicals amounts and \n\nantioxidant capacity and both dark- and brown-\n\ncolored fig varieties are the most effective ones by \n\ntheir important amounts of flavonoids,  \n\npolyphenols, and anthocyanins.  \n\n Besides, anthocyanins compounds of the dark- and \n\nbrown-colored fig skin varieties contribute to 36 and \n\n28% of their total antioxidant capacity [35].", "start_char_idx": 13388, "end_char_idx": 15776, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8f690923-ae05-4c24-b312-9c263f0a557f": {"__data__": {"id_": "8f690923-ae05-4c24-b312-9c263f0a557f", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b77f0d48-70de-4243-af89-c1d0d60345a9", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c0810252d5d32ac705b39892f7daba17a291aa40f4e0c2257a25f6dd172ef7d4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "572dbef0-204d-47a6-a12d-7fd469e27568", "node_type": "1", "metadata": {}, "hash": "85d4990e0310fe01c0e92380d18d6f2ee8733ab2b754859420adce2666fcea6e", "class_name": "RelatedNodeInfo"}}, "text": "0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n0.0625 0.125 0.25 0.5 0.75 1\n\n%\n i\n\nn\nh\n\nib\nit\n\nio\nn\n\n o\nf \n\nD\nP\n\nP\nH\n\nConcentration mg/mL\n\nMeOH Var.1\nMeOH Var.2\nMeOH Var.3\nMeOH Var.4\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\nMeOH\nVar.1\n\nMeOH\nVar.2\n\nMeOH\nVar.3\n\nMeOH\nVar.4\n\nA.A%\n i\n\nn\nh\n\nib\nit\n\nio\nn\n\n o\nf \n\nD\nP\n\nP\nH\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\nMeOH\nVar.1\n\nMeOH\nVar.2\n\nMeOH\nVar.3\n\nMeOH\nVar.4\n\nA.A\n\nT\no\n\nta\nl \n\na\nn\n\nti\no\n\nx\nid\n\na\nn\n\nt \na\n\nct\niv\n\nit\ny\n\n \n(m\n\ng\n A\n\nA\nE\n\n/\n g\n\n)\n\nhttp://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=total+antioxidant+capacity\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  33 \n\n The total antioxidant capacity ranged from 63.5 to \n\n92.1 mg AAE/g dw for MeOH Var.2 and MeOH \n\nVar.1, respectively in comparison with ascorbic acid \n\n98.2 \u00b1 1.43 mg AAE/g dw. \n\nThe total antioxidant capacity estimated for \n\nmethanolic extracts of fig varieties fruits from the \n\nsame region (Mascara) as our studied fig-flowers \n\nvarieties 3, 4 (El Bordj, Ghriss) was ranged from 68.8 \n\nto 88.6 mg AAC/g DW and the highest values were \n\nobserved for the ethanolic extracts. Also, the max of \n\nthe total tannin content of methanolic extracts was \n\nmax of 122.35 mg AAE/g DW [36].  \n\nAccording to the statistical analysis, separately, there \n\nare non-significant differences between the two fig \n\nvarieties of Bechar: Var.1 (Lahmar), Var.2 (Ouakda) \n\nand also no significant differences between fig \n\nvarieties originated of mascara Var.3 (El Bordj), Var.4 \n\n(Ghriss) (P > 0,05).  \n\nA positive and strong correlation was registered \n\nbetween the antioxidant capacity of plant extract and \n\nits total phenolic composition [37, 38, 39, 40, 41]. In \n\nvitro tests have shown that dried fig fruits possess a \n\nsignificant antioxidant capacity subsequently to \n\ntheir human consumption. As mentioned \n\npreviously, the total antioxidant capacity correlated \n\nwell with anthocyanins and phenolic compositions \n\n(R = 0.989, R = 0.515), but the correlation is low with \n\nthe flavonoid amount R = 0.248 [15, 42]. However, \n\nthe existence of other unidentified molecules with \n\nantioxidant properties in those extracts can not be \n\noverlooked. Peel extracts of fig fruits had a higher \n\nability to scavenger free radicals, at all \n\nconcentrations than pulp extracts with IC50 values \n\nof 80.04 and 28.85 mg/mL. For the pulp, IC50 values \n\nwere 105.85 and 176.88mg/mL [43]. Results showed \n\na positive and strong correlation between \n\nantioxidant capacity and different phytochemical \n\ncompounds amounts. Phenolics and tannins \n\ncomponents of MeOH Var.2 have an appreciable \n\ncorrelation with DPPH radical test R2 =0.948 and R2 \n\n=0.972, respectively. Total phenolics had a strong \n\ncorrelation with the DPPH radical test (R2 =0.948).", "start_char_idx": 15780, "end_char_idx": 18560, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "572dbef0-204d-47a6-a12d-7fd469e27568": {"__data__": {"id_": "572dbef0-204d-47a6-a12d-7fd469e27568", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8f690923-ae05-4c24-b312-9c263f0a557f", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "ae4770e15e7f40834f53d77bf624a20a56e809baa8efde337ce339bd1c76dbd3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b948d597-0be2-430b-a47a-aa17013ff471", "node_type": "1", "metadata": {}, "hash": "964d1ae01b9972ff921419e895edc5f31374cf1bb69263f56dfd7cbde75a468d", "class_name": "RelatedNodeInfo"}}, "text": "Furthermore, an important relation between both \n\ntotal flavonoids and total phenolics and DPPH \n\nradical presented by the extract MeOH Var.3 with R2 \n\n=0.954 and R2 =0.943, respectively. A high \n\ncorrelation coefficient was deduced between \n\nantioxidant capacity and the levels of polyphenols \n\nand anthocyanins with R2 = 0.985 and R2 =0.992, \n\nrespectively [34]. The differences observed in the \n\nantioxidant capacity of plant extracts can be related \n\nto certain parameters like those that the solvent used \n\nfor extraction, its polarity, and the tests used [44]. \n\nMoreover, as the mechanisms of antioxidant effect \n\nare dissimilar and the most natural antioxidants are \n\nmultifunctional, it is important to use various \n\nantioxidant capacity tests to take a global \n\nobservation of it.  \n\nConclusion \nThe important antioxidant capacity of methanolic \n\nextracts of fig flowers fruits is impressive and \n\nprobably it is the result of their exceptional richness \n\nin phenolic compounds. Such bioactive molecules \n\nreacting as natural antioxidants and then, they are \n\nwell known to display a positive impact on human \n\nhealth and can be considered for future uses as \n\nantioxidant components in agro-food industries.  \n\nThe Algerian flora known for its high richness and \n\nbiodiversity as well as Algerian folk medicine is also \n\nconsidered as an appreciable source of both new \n\ndrugs and bioactive molecules since ancient times. \n\nFuture studies will be needed to elucidate more and \n\nmore medicinal plants and traditional preparations \n\nused for therapeutic purposes.  \n\nConflict of Interest \nThe authors declare that they have no competing \n\ninterests. \n\nTable 2. The correlation coefficient among antioxidant \ncapacity tests, total flavonoid level, total anthocyanins \nlevel, total phenolic level, and tannins level. \n\nMeOH \nVar.1 \n\nDPPH      TAC  MeOH \nVar.3 \n\nDPPH      TAC  \n\nTFL 0.812A 0.531a TFL 0.954A  0.208a \nTPL 0.873A 0.715b TPL 0.943B  0.681b \nTAL 0.601A 0.224c TAL 0.176C 0.566c \nCTL 0.730A 0.478d CTL 0.658D 0.619b \n\nMeOH \nVar.2 \n\nDPPH      TAC  MeOH \nVar.4 \n\nDPPH      TAC  \n\nTFL 0.914A 0.574a TFL 0.507A 0.288a \nTPL 0.948B 0.724b TPL 0.426A 0.397b \nTAL 0.632C 0.587c TAL 0.311A 0.141c \nCTL 0.972D 0.598d CTL 0.529A 0.336b \n\nTFL: Total Flavonoids Level, TAL: Total Anthocyanins \nLevel, TPL: Total phenolics Level, CTL: Condensed \nTannins Level, TAC: Total antioxidant capacity. MeOH \nVar.1; MeOH Var.2: Variety 1, 2 Bakor (Fig flowers) of Bechar. \nMeOH Var.3; MeOH Var.4: Variety 3, 4 Bakor of Mascara. \nCapital letters (A\u2013B) and lowercase letters (a\u2013b) \nindicate significant differences at p < 0.05. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  34 \n\nAcknowledgments \nThe authors are grateful for the technical and \n\nfinancial support provided by Dr. Messaoudi \n\nMohammed. Special thanks extended to Dr. Merah \n\nMaroua for her undeniable technical and theoretical \n\nassistance. \n\nReferences  \n1. Juan ME, Gonzalez Pons, E, Planas JM. Multidrug resistance \n\nproteins restrain the intestinal absorption of trans-resveratrol \nin rats. The Journal of Nutrition. 2010 Mar 1;140(3): 489\u2013495.", "start_char_idx": 18563, "end_char_idx": 21713, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b948d597-0be2-430b-a47a-aa17013ff471": {"__data__": {"id_": "b948d597-0be2-430b-a47a-aa17013ff471", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "572dbef0-204d-47a6-a12d-7fd469e27568", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "35c36a56e20ce608ff1aa294713aaf78e6a74f4502ae746500862d06752e6877", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "21471bee-0307-4c18-8f52-0a6fc9f6c6d9", "node_type": "1", "metadata": {}, "hash": "2fa7f38909cdba921264d6cc25867df7f3b9f26537e51fd1197fd42c1a0bfd93", "class_name": "RelatedNodeInfo"}}, "text": "2010 Mar 1;140(3): 489\u2013495. \nhttps://doi.org/10.3945/jn.109.114959 \n\n2. Lako J, Trenerry VC, Wahlqvist M, Wattanapenpaiboon N, \nSotheeswaran S, Premier R. Phytochemical flavonols, \ncarotenoids and the antioxidant properties of a wide selection \nof Fijian fruit, vegetables and other readily available foods. \nFood Chemistry. 2007 Jan 1;101(4):1727-41. \nhttps://doi.org/10.1016/j.foodchem.2006.01.031 \n\n3. Liu RH. Potential synergy of phytochemicals in cancer \nprevention: mechanism of action. The Journal of nutrition. \n2004 Dec 1;134(12):3479S-85S. \nhttps://doi.org/10.1093/jn/134.12.3479S \n\n4. Fukumoto LR, Mazza G. Assessing antioxidant and \nprooxidant activities of phenolic compounds. Journal of \nagricultural and food chemistry. 2000 Aug 21;48(8):3597-604. \nhttps://doi.org/10.1021/jf000220w \n\n5. Mahmood A, Mahmood A, Malik RN. Indigenous knowledge \nof medicinal plants from Leepa valley, Azad Jammu and \nKashmir, Pakistan. Journal of ethnopharmacology. 2012 Aug \n30;143(1):338-46. https://doi.org/10.1016/j.jep.2012.06.046  \n\n6. Rates SM. Plants as source of drugs. Toxicon. 2001 May \n1;39(5):603-13. https://doi.org/10.1016/S0041-\n0101(00)00154-9 \n\n7. Mars M. Fig (Ficus carica L.) genetic resources and breeding. \nInII International Symposium on Fig 605 2001 May 7 (pp. 19-\n27). https://doi.org/10.17660/ActaHortic.2003.605.1 \n\n8. Flaishman MA, Rodov V, Stover E. The fig: botany, \nhorticulture, and breeding. Horticultural reviews. 2008;34. \nhttps://doi.org/10.1002/9780470380147.ch2 \n\n9. Patil VV, Patil VR. Ficus carica Linn.-an overview. Research \nJournal of Medicinal Plant. 2011;5(3):246-53. \nhttps://doi.org/10.3923/rjmp.2011.246.253 \n\n10. Robaine P. La figue m\u00fbre : Reproduction particuli\u00e8re du \nfiguier m\u00e9diterran\u00e9en Ficus carica L. Association des \nBotanistes Lorrains Willemetia. 2014;82: 13p.                                                         \n\n11. Paquereau J. Au Jardin des Plantes de la Bible Botanique, \nsymboles et usages. Edition Samuel six Editeur Institut pour \nle d\u00e9veloppement Forestier, Paris. 2013:416. \n\n12. Pelletier E, Campbell P, Denizeau F. Ecotoxicologie \nmol\u00e9culaire. Edition Press de l\u2019universit\u00e9 de Qu\u00e9bec. 2003:182.  \n\n13. Bag GC, Devi PG, Bhaigyabati TH. Assessment of total \nflavonoid content and antioxidant activity of methanolic \nrhizome extract of three Hedychium species of Manipur \nvalley. International Journal of Pharmaceutical Sciences \nReview and Research. 2015 Jan;30(1):154-9. \n\n14. Grigora\u015f CG. Valorisation des fruits et des sous-produits de \nl'industrie de transformation des fruits par extraction des \ncompos\u00e9s bioactifs (Doctoral dissertation, Universit\u00e9 \nd'Orl\u00e9ans). 2012;246. \n\n15. Chawla A, Kaur R, Sharma AK. Ficus carica Linn.: A review on \nits pharmacognostic, phytochemical and pharmacological \naspects.", "start_char_idx": 21686, "end_char_idx": 24465, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "21471bee-0307-4c18-8f52-0a6fc9f6c6d9": {"__data__": {"id_": "21471bee-0307-4c18-8f52-0a6fc9f6c6d9", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b948d597-0be2-430b-a47a-aa17013ff471", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "21bcd8afe8c4f5b2c2afd02c1afc5450d91e59104409c8fc4d5cd15e755d2b6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "14eff887-1823-491e-8f8e-cb0b151a3da5", "node_type": "1", "metadata": {}, "hash": "d23ff6f3752f03911526b602f90373d680922c6a6e0a8e0955b53af70515e24d", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of Pharmaceutical and \nPhytopharmacological Research. 2012;1(4):215-32. \n\n16. Nicole T, Fran\u00e7ois G. Des fruits et des graines comestibles du \nmonde entier. Lavoisier; 2013 Oct 15. 727. \n\n17. BENHAMMOU N. Activit\u00e9 antioxydante des extraits des \ncompos\u00e9s ph\u00e9noliques de dix plantes m\u00e9dicinales de l\u2019Ouest \net du Sud-Ouest Alg\u00e9rien (Doctoral dissertation). 2012. \n\n18. Sembiring EN, Elya B, Sauriasari R. Phytochemical screening, \ntotal flavonoid and total phenolic content and antioxidant \nactivity of different parts of Caesalpinia bonduc (L.) Roxb. \nPharmacognosy journal. 2018;10(1). \nhttps://doi.org/10.5530/pj.2018.1.22 \n\n19. Hebi M, Eddouks M. \u00c9valuation de l\u2019activit\u00e9 antioxydante de \nStevia rebaudiana. Phytoth\u00e9rapie. 2016 Feb 1;14(1):17-22. \nhttps://doi.org/10.1007/s10298-015-0999-y \n\n20. El-Haci IA, Atik-Bekkara F, Didi A, Gherib M, Didi MA. \nTeneurs en polyph\u00e9nols et pouvoir antioxydant d\u2019une plante \nm\u00e9dicinale end\u00e9mique du Sahara alg\u00e9rien. Phytoth\u00e9rapie. \n2012 Oct 1;10(5):280-5. https://doi.org/10.1007/s10298-012-\n0726-x \n\n21. Ercisli S, Tosun M, Karlidag H, Dzubur A, Hadziabulic S, \nAliman Y. Color and antioxidant characteristics of some fresh \nfig (Ficus carica L.) genotypes from Northeastern Turkey. Plant \nFoods for Human Nutrition. 2012 Sep 1;67(3):271-6. \nhttps://doi.org/10.1007/s11130-012-0292-2 \n\n22. Julkunen-Tiitto R. Phenolic constituents in the leaves of \nnorthern willows: methods for the analysis of certain \nphenolics. Journal of agricultural and food chemistry. 1985 \nMar;33(2):213-7. https://doi.org/10.1021/jf00062a013 \n\n23. Ba K, Tine E, Destain J, Ciss\u00e9 N, Thonart P. \u00c9tude comparative \ndes compos\u00e9s ph\u00e9noliques, du pouvoir antioxydant de \ndiff\u00e9rentes vari\u00e9t\u00e9s de sorgho s\u00e9n\u00e9galais et des enzymes \namylolytiques de leur malt. Biotechnologie, Agronomie, \nSoci\u00e9t\u00e9 et Environnement. 2010;14(1):131-9. \n\n24. Mahmoudi S, Khali M, Mahmoudi N. Etude de l'extraction des \ncompos\u00e9s ph\u00e9noliques de diff\u00e9rentes parties de la fleur \nd'artichaut (Cynara scolymus L.). Nature & Technology. 2013 \nJun 1(9):35. \n\n25. S\u00e1nchez\u2010Moreno C, Larrauri JA, Saura\u2010Calixto F. A procedure \nto measure the antiradical efficiency of polyphenols. Journal \nof the Science of Food and Agriculture. 1998 Feb;76(2):270-6. \nhttps://doi.org/10.1002/(SICI)1097-\n0010(199802)76:2<270::AID-JSFA945>3.0.CO;2-9 \n\n26. Doukani K, Tabak S, Derrriche A, Hacini Z. \u00c9tude physico-\nchimique et phyto-chimique de quelques types de miels \nAlg\u00e9riens. Revue Ecologie-Environnement. 2014;10:37-49. \n\n27.", "start_char_idx": 24466, "end_char_idx": 26977, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "14eff887-1823-491e-8f8e-cb0b151a3da5": {"__data__": {"id_": "14eff887-1823-491e-8f8e-cb0b151a3da5", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "21471bee-0307-4c18-8f52-0a6fc9f6c6d9", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "a8ba99dba93bc7f98781128141dbaf5514dd78d9d4c279d0cf71d5439531be8f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b16b6d56-3ea0-49ad-a4a6-9f529c2421e1", "node_type": "1", "metadata": {}, "hash": "7d3d8df1b5801c3a05684279bec242ca80ded8cb8d230e1721e0f0a397687038", "class_name": "RelatedNodeInfo"}}, "text": "2014;10:37-49. \n\n27. Fabri RL, Nogueira MS, Braga FG, Coimbra ES, Scio E. \nMitracarpus frigidus aerial parts exhibited potent antimicrobial, \nantileishmanial, and antioxidant effects. Bioresource \nTechnology. 2009 Jan 1;100(1):428-33. \nhttps://doi.org/10.1016/j.biortech.2008.05.053 \n\n28. Aguilar Urbano M, Pineda Priego M, Prieto P. \nSpectrophotometric quantitation of antioxidant capacity \nthrough the formation of a phosphomolybdenum complex: \nspecific application to the determination of vitamin E1. Anal \nBiochem. 1999;269:337\u2013341. \nhttps://doi.org/10.1006/abio.1999.4019 \n\n29. \u00c7ali\u015fkan O, Polat AA. Phytochemical and antioxidant \nproperties of selected fig (Ficus carica L.) accessions from the \neastern Mediterranean region of Turkey. Scientia \nHorticulturae. 2011 May 10;128(4):473-8. \nhttps://doi.org/10.1016/j.scienta.2011.02.023 \n\n30. Daniel M, Topo E. Analysis of nutrients, total phenols and \nantioxidant activity of Ficus sansibarica warb. fruits from \nEastern Botswana. Journal of Drug Delivery and \nTherapeutics. 2012 Nov 11;2(6). \nhttps://doi.org/10.22270/jddt.v2i6.318 \n\n31. Vallejo F, Mar\u00edn JG, Tom\u00e1s-Barber\u00e1n FA. Phenolic compound \ncontent of fresh and dried figs (Ficus carica L.). Food \nChemistry. 2012 Feb 1;130(3):485-92. https://doi.org/10.1016 \n/j.foodchem.2011.07.032 \n\n32. Halliwell B, Aeschbach R, Loliger J, Aruoma, OI. The \ncharacterization of antioxidants. Food Chem Tox. \n\n\n\nNepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):29-35  Messaoudi and Merah  \n\n\u00a9NJB, BSN  35 \n\n1995;33:601\u2013617. https://doi.org/10.1016/0278-\n6915(95)00024-V \n\n33. Aliyu AB, Musa AM, Oshanimi JA, Ibrahim HA, Oyewale AO. \nPhytochemical analyses and mineral elements composition of \nsome medicinal plants of Northern Nigeria. Nigerian Journal \nof Pharmaceutical Sciences. 2008 Mar;7(1):119-25. \n\n34. Brown RH. Free radicals, programmed cell death and \nmuscular dystrophy. Current opinion in neurology. 1995 Oct \n1;8(5):373-8. https://doi.org/10.1097/00019052-199510000-\n00009 \n\n35. Solomon A, Golubowicz S, Yablowicz Z, Grossman S, \nBergman M, Gottlieb HE, Altman A, Kerem Z, Flaishman MA. \nAntioxidant activities and anthocyanin content of fresh fruits \nof common fig (Ficus carica L.). Journal of agricultural and \nfood chemistry. 2006 Oct 4;54(20):7717-23. \nhttps://doi.org/10.1021/jf060497h \n\n36. Benmaghnia S, Meddah B, Tir-Touil A, Hern\u00e1ndez Ja. \nPhytochemical analysis, antioxidant and antimicrobial \nactivities of three samples of dried figs (Ficus carica L.) from \nthe region of mascara. Journal of Microbiology, Biotechnology \nand Food Sciences. 2019 Jan 12;2019:208-15. \nhttps://doi.org/10.15414/jmbfs.2019.9.2.208-215   \n\n37.", "start_char_idx": 26957, "end_char_idx": 29595, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b16b6d56-3ea0-49ad-a4a6-9f529c2421e1": {"__data__": {"id_": "b16b6d56-3ea0-49ad-a4a6-9f529c2421e1", "embedding": null, "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-176", "node_type": "4", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "c86b6687f2f00bb0b70c5dfca8b121ca51089974944109396382644bfdb03325", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "14eff887-1823-491e-8f8e-cb0b151a3da5", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "79b44fc4a951c2a6d0e503c214000ad71fb366f7d065b3025ae814529dbc40fc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "88782d64-0eec-4880-9e98-48341f9fcc6f", "node_type": "1", "metadata": {}, "hash": "23b89b46a6f970c51e567a8db3094da2b913f409d65ad1b3ecd32c347c511889", "class_name": "RelatedNodeInfo"}}, "text": "Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, \nVidal N. Antioxidant activity of some Algerian medicinal \nplants extracts containing phenolic compounds. Food \nchemistry. 2006 Aug 1;97(4):654-60. \nhttps://doi.org/10.1016/j.foodchem.2005.04.028 \n\n38. Turkmen N, Velioglu YS, Sari F, Polat G. Effect of extraction \nconditions on measured total polyphenol contents and \nantioxidant and antibacterial activities of black tea. Molecules. \n2007 Mar;12(3):484-96. https://doi.org/10.3390/12030484 \n\n39. Tawaha K, Alali FQ, Gharaibeh M, Mohammad M, El-Elimat \nT. Antioxidant activity and total phenolic content of selected \nJordanian plant species. Food chemistry. 2007 Jan \n1;104(4):1372-8. \nhttps://doi.org/10.1016/j.foodchem.2007.01.064 \n\n40. Wojdy\u0142o A, Oszmia\u0144ski J, Czemerys R. Antioxidant activity \nand phenolic compounds in 32 selected herbs. Food \nchemistry. 2007 Jan 1;105(3):940-9. \nhttps://doi.org/10.1016/j.foodchem.2007.04.038  \n\n41. Wong CC, Li HB, Cheng KW, Chen F. A systematic survey of \nantioxidant activity of 30 Chinese medicinal plants using the \nferric reducing antioxidant power assay. Food chemistry. 2006 \nAug 1;97(4):705-11. \nhttps://doi.org/10.1016/j.foodchem.2005.05.049 \n\n42. Raj SJ, Joseph B. Pharmacognostic and traditional properties \nof Cissus quadrancularis Linn-An overview. International \nJournal of Pharma and Bio Sciences2011. 2011;2(1):131-9.  \n\n43. Antolovich M, Prenzler PD, Patsalides E, McDonald S, \nRobards K. Methods for testing antioxidant activity. Analyst. \n2002;127(1):183-98. https://doi.org/10.1039/b009171p \n\n44. Fu L, Xu BT, Xu XR, Qin XS, Gan RY, Li HB. Antioxidant \ncapacities and total phenolic contents of 56 wild fruits from \nSouth China. Molecules. 2010 Dec;15(12):8602-17. \nhttps://doi.org/10.3390/molecules15128602", "start_char_idx": 29596, "end_char_idx": 31375, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "88782d64-0eec-4880-9e98-48341f9fcc6f": {"__data__": {"id_": "88782d64-0eec-4880-9e98-48341f9fcc6f", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b16b6d56-3ea0-49ad-a4a6-9f529c2421e1", "node_type": "1", "metadata": {"identifier": "njb-176", "author": "Mohammed, Messaoudi; Maroua, Merah", "title": "Phytochemicals Levels and Antioxidant Capacities of Figs Flowers Fruits", "date": "2020-07-31", "file": "njb-176.pdf"}, "hash": "15c91bed5efc925f9ccc1bd99c01acb46474f66980af191198d830d78daf7be5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fe248fa4-def7-4084-b14a-71fc9f555e05", "node_type": "1", "metadata": {}, "hash": "032ff52d522694f140cd96424590192e58e85f1808f6b74653b1898c597203e8", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):12-16 DOI: https://doi.org/10.3126/njb.v8i1.30205Research article \n\n\u00a9NJB, BSN 12 \n\nPhenolic Compounds from the Aerial Parts of Adenophora \ntriphylla (Thunb.) A. DC. var. triphylla and their Free Radical \nScavenging Activity \nKengo Hori1, Hari Prasad Devkota 1,2   \n1Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto \n\n862-0973, Japan  \n2Program for Leading Graduate Schools, Health life Sciences: Interdisciplinary and Glocal Oriented (HIGO) \n\nProgram, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan \nArticle history:-Received: 17 May 2020; Revised: 19 Jun 2020; Accepted: 25 Jun 2020; Published online: 31 Jul 2020 \n\nAbstract \nAdenophora triphylla (Thunb.) A. DC. var. triphylla (Family: Campanulaceae) is distributed in Japan, Korea, and \n\nChina. It is locally known as \u201cSaiyousyajin\u201d in Japan and the roots are used in traditional medicine to treat \n\nchronic bronchitis and whooping cough, and also as anti-inflammatory and antitussive agents. Till now, there \n\nis no report on the chemical constituents of aerial parts. Thus, the main aim of this study was to isolate and \n\nidentify major chemical constituents of aerial parts of A. triphylla var. triphylla, and to evaluate their free radical \n\nscavenging activity. The 70% methanol extract of the aerial parts was subjected to repeated column \n\nchromatography using MCI gel CHP-20P, Sephadex LH-20, ODS and silica gel columns to isolate the five \n\nphenolic components (1-5). Free radical scavenging activity of the extract and compounds was evaluated using \n\n1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity method. The structures of the isolated \n\ncompounds were elucidated as luteolin (1), luteolin 4\u2019-O-\u03b2-glucopyranoside (2), luteolin 7-O-\u03b2-\n\nglucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear \n\nmagnetic resonance (NMR) spectral data and comparison with literature values. All these compounds were \n\nisolated for the first time from A. triphylla var. triphylla. The extract showed weak free radical scavenging \n\nactivity. Among isolated compounds, luteolin (1), luteolin 7-O-\u03b2-glucopyranoside (3), luteolin 7-O-\n\nneohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. Results from this \n\nstudy suggest that the aerial parts of A. triphylla var. triphylla might be a potential plant source for the \n\ndevelopment of functional foods, however further detailed research is necessary. \n\nKeywords: Adenophora triphylla; Saiyousyajin; Phenolic compounds; Free radical scavenging \n\n Corresponding author, email: devkotah@kumamoto-u.ac.jp \n\nIntroduction \nMedicinal plants and their phytochemicals have \n\nplayed a vital role in human healthcare as an \n\nimportant source of traditional medicines, drug \n\ndiscovery and development of nutritional and \n\nfunctional foods [1\u20133]. However, many plant species \n\nare yet to be explored for their chemical constituents \n\nand potential biological activities and health-\n\npromoting effects. \n\nThe genus Adenophora belonging to belonging to \nCampanulaceae family consists of about 62 species \ndistributed in East Asia and Europe, among which, \n12 species are distributed in Japan [4].", "start_char_idx": 48, "end_char_idx": 3329, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fe248fa4-def7-4084-b14a-71fc9f555e05": {"__data__": {"id_": "fe248fa4-def7-4084-b14a-71fc9f555e05", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "88782d64-0eec-4880-9e98-48341f9fcc6f", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "a20b4879386432508e3ef907659fb3b0027243698a30a1e736efaea1804c26fa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4eed02f8-a726-4b8d-877a-b7059b2a2efb", "node_type": "1", "metadata": {}, "hash": "0550ed37e8bb4b765d060382d436dc11b2b18134fc7acc5e559fc9ca6c60795e", "class_name": "RelatedNodeInfo"}}, "text": "Adenophora \ntriphylla (Thunb.) A. DC. var. triphylla \n\n(\u201cSaiyousyajin\u201d in Japanese) and A. triphylla \n(Thunb.) A. DC. var. japonica (Regel) H. Hara \n\n(\u201cTsuriganeninjin\u201d in Japanese) are among many \nvarieties of plant A. triphylla (Thunb.) A. DC. and are \n\ndistributed in Japan, Korea, and China [4, 5]. Roots \nof both of these plants are used in traditional \nmedicine to treat chronic bronchitis and whooping \ncough and as an anti-inflammatory and antitussive \nagents [5\u20138]. There have been many studies on the \nchemical constituents/biological activities of roots \nand leaves of A. triphylla var. japonica [5, 7, 9, 10]. Kim \n\net al. [5] reported the high phenolic and flavonoid \ncontents and potent free radical scavenging activities \nof leaves of A. triphylla var. japonica, however, no  \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nmailto:devkotah@kumamoto-u.ac.jp\nhttps://orcid.org/0000-0002-0509-1621\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):12-16  Hori and Devkota \n\n\u00a9NJB, BSN 13 \n\nsuch studies are reported on the aerial parts of A. \n\ntriphylla var. triphylla for the best of our knowledge. \n\nThus, in this study, the main aim was to isolate and \n\nidentify major chemical constituents of aerial parts \n\nof A. triphylla var. triphylla and to evaluate their free \n\nradical scavenging activity. \n\nMaterials and Methods \nGeneral Experimental Procedure \n1H- and 13C- NMR spectra were measured on \n\nBRUKER AVANCE 600 NMR Spectrometer (Bruker, \n\nBillerica, MA, USA) (1H-NMR: 600 Hz and 13C-NMR: \n\n150 Hz). Chemical shift values (\u03b4H and \u03b4C) are given \n\nin ppm with reference to tetramethyl silane (TMS). \n\nColumn chromatography (CC) was carried out with \n\nMCI gel CHP20P (75 ~ 150 \u03bcm, Mitsubishi Chemical \n\nIndustries Co. Ltd., Tokyo, Japan), Sephadex LH-20 \n\n(Amersham Pharmacia Biotech, Tokyo, Japan) and \n\nsilica gel 60 (0.040-0.063 mm, Merck KGaA, \n\nDarmstadt, Germany). Thin layer chromatography \n\n(TLC) was performed on a pre-coated silica gel 60 \n\nF254 (Aluminum sheet, Merck KGaA, Darmstadt, \n\nGermany).  \n\nPlant Material \n\nThe aerial parts (stems and leaves) of A. triphylla var. \n\ntriphylla were collected from Mt. Tawarayama \n\nKumamoto, Japan in August 2015 and shade dried \n\nfor two weeks. Plant material was identified by Mr. \n\nMasato Watanabe, Technical Officer, School of \n\nPharmacy, Kumamoto University.  \n\nChemicals \n1,1-Diphenyl-2-picrylhydrazyl (DPPH) was \n\npurchased from Sigma Aldrich, Co. (Tokyo, Japan). \n\n6\u2013Hydroxy- 2, 5, 7, 8\u2013 tetramethylchroman\u20132-\n\ncarboxylic acid (Trolox) was from Wako Pure \n\nChemical Industries, Ltd. (Tokyo, Japan) and 2-\n\nmorpholinoethanesulfonic acid monohydrate (MES) \n\nwas purchased from Dojindo Chemical Research \n\n(Kumamoto, Japan).", "start_char_idx": 3330, "end_char_idx": 6153, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4eed02f8-a726-4b8d-877a-b7059b2a2efb": {"__data__": {"id_": "4eed02f8-a726-4b8d-877a-b7059b2a2efb", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fe248fa4-def7-4084-b14a-71fc9f555e05", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "d0e3ebc677ed50406a4c1ceb0ba9a1cd38c34fef483675f79b454b25274bd8f5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4047c1f2-445b-4906-a8cc-158cb7edfeed", "node_type": "1", "metadata": {}, "hash": "bf55c5602b01fca5d035776aa132464d636b25a5cd3243f86fc356d51dd25ac9", "class_name": "RelatedNodeInfo"}}, "text": "Extraction and Isolation \nThe dried aerial parts (2600 g) were then extracted \n\ntwice with 70% MeOH (18 L). The combined extract \n\nwas evaporated under reduced pressure to give \n\n414.0 g of extract. The extract was suspended in \n\nwater and subjected on MCI gel CHP20P column \n\nchromatography (CC) and eluted successively with \n\nwater, 40%, 70% and 100% MeOH to give eight \n\nfractions (1~8). Fraction 2 (109.4 g, H2O eluate) was \n\nsubjected on Sephadex LH-20 CC (H2O) to obtain \n\nfour subfractions (2-1~2-4). Subfraction 2-3 was \n\nsubjected on MCI gel CC (H2O) to afford compound \n\n5 (153.8 mg). Fraction 7 (3.05 g, 80% MeOH eluate) \n\nwas subjected on Sephadex LH-20 CC (MeOH) to \n\nafford compound 1 (174.4 mg). Fraction 6 (4.0 g, 60% \n\nMeOH eluate) was subjected to Sephadex LH-20 CC \n\n(H2O-MeOH; 1:1) to afford compound 2 (211.4 mg).  \n\nTable 1. Proton NMR spectroscopic data of compound 1 - 4 (\uf064H, mult. (J in Hz)) \nPosition 1a 2 b 3 a 4 b \n\n3 6.65, s 6.56, s 6.74, s 6.57, s \n6 6.17, d (2.1) 6.18, d (2.0) 6.43, d (2.1) 6.38, d (2.1) \n\n8 6.43, d (2.1) 6.41, d (2.0) 6.78, d (2.1) 6.73, d (2.1) \n\n2\u2019 7.40, d (2.3) 7.41, d (2.3) 7.40, d (2.1) 7.38, d (2.5) \n\n5\u2019 6.87, d (8.2) 7.28, d (8.5) \n\n)\n\n6.89, d (8.4) \n\n)\n\n6.89, d (8.5) \n\n)6\u2019 7.38, dd (8.2, 2.3) 7.39, dd (8.5, 2.3) 7.43, dd (8.4, 2.1) 7.40, dd (8.5, 2.5) \n\nGlc-1 4.93, d (7.5) 5.05, d (7.5) 5.18, d (6.7) \n\nGlc-2 3.40-4.00 3.40-4.00 3.40-4.00 \n\nGlc-3 3.40-4.00 3.40-4.00 3.40-4.00 \n\nGlc-4 3.40-4.00 3.40-4.00 3.40-4.00 \n\nGlc-5 3.40-4.00 3.40-4.00 3.40-4.00 \n\nGlc-6 3.40-4.00 3.40-4.00 3.40-4.00 \n\nRha-1 5.28, d (1.2) \n\nRha-2 3.40-4.00 \n\nRha-3 3.40-4.00 \n\nRha-4 3.40-4.00 \n\nRha-5 3.40-4.00 \n\nRha-6 1.33, d (6.2) \na in DMSO-d6, b in CD3OD \n\n\n\nNepal J Biotechnol.", "start_char_idx": 6156, "end_char_idx": 7876, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4047c1f2-445b-4906-a8cc-158cb7edfeed": {"__data__": {"id_": "4047c1f2-445b-4906-a8cc-158cb7edfeed", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4eed02f8-a726-4b8d-877a-b7059b2a2efb", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "bd993ada8eb960512c22fb918bd01e69ec2543dfc5a892df6ea33c658e0d7d23", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "caa5a1e0-3323-4e36-b69a-2b764067ceb0", "node_type": "1", "metadata": {}, "hash": "d3c094f9ea5ce0d472beaa725a7fce7963b0f84c80ef0e8636f2ee8332852371", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):12-16  Hori and Devkota \n\n\u00a9NJB, BSN 14 \n\nFraction 3 (14.3 g) and fraction 4 (2.5 g) were \n\ncombined and subjected to Sephadex LH-20 CC \n\n(50% MeOH) followed by ODS CC (20%, 30%, 40%  \n\nMeOH) and silica gel CC (CH2Cl2: MeOH: H2O \n\n=8:2:0.1) to afford compound 3 (1610.7 mg) and 4 (7.0 \n\nmg).  \n\nMeasurement of DPPH Free Radical \nScavenging Activity \nThe antioxidant potential was determined using the \n\nDPPH free radical scavenging method as described \n\npreviously [3].  \n\nResults and Discussion  \nThe 70% methanol extract of aerial parts of A. \n\ntriphylla var. triphylla was subjected to various \n\ncolumn chromatographic methods including MCI \n\ngel CHP20P, Sephadex LH-20, ODS and silica gel to \n\nafford five compounds. The structures of these \n\ncompounds were elucidated as luteolin (1) [11], \n\nluteolin 4\u2019-O- \u03b2 -glucopyranoside (2) [12], luteolin 7-\n\nO- \u03b2 -glucopyranoside (3) [11, 12], luteolin 7-O-\n\nneohesperidoside (4) [13] and chlorogenic acid (5) \n\n[14] (Figure 1) based on their NMR spectral data and \n\ncomparison with literature values. Proton and 13C \n\nNMR data for compounds 1-4 are provided in Table \n\n1 and Table 2, respectively. All of these compounds \n\nwere isolated for the first time from A. triphylla var. \n\ntriphylla. Previously, Hashiba et al. [10] had reported \n\nflavonoids including luteolin (1), luteolin 7-O- \u03b2 -\n\nglucopyranoside (3), luteolin 4\u20197-di-O-\u03b2-\n\nglucopyranoside, quercetin, quercetin 3-O-\u03b2-\n\nglucopyranoside from the leaves of A. triphylla var. \n\njaponica. Characterization of similar flavonoids in the \n\naerial parts of A. triphylla var. triphylla in this study \n\nsuggests their chemotaxonomic similarity and these \n\ncompounds can be used as the chemotaxonomic \n\nmarkers for these varieties. Further studies on other \n\nvarieties of A. triphylla or other species of Adenophora \n\nmay help explore their further similarity. \n\nThe 70% extract and all isolated compounds were \n\nevaluated for their DPPH free radical scavenging \n\nactivity (Table 3). The extract showed weak free \n\nTable 2.", "start_char_idx": 7857, "end_char_idx": 9921, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "caa5a1e0-3323-4e36-b69a-2b764067ceb0": {"__data__": {"id_": "caa5a1e0-3323-4e36-b69a-2b764067ceb0", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4047c1f2-445b-4906-a8cc-158cb7edfeed", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "b5b0f9badf3702deb922d2feabe0bfbbb6949c4a38784983efc0b56668a48e7b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9cf66d83-317b-4a67-9121-f2219d0c14d6", "node_type": "1", "metadata": {}, "hash": "cdd6b69e23b92edca00feddbbc5258c0118f5dbd49ae653019552e0b16b47fcb", "class_name": "RelatedNodeInfo"}}, "text": "The extract showed weak free \n\nTable 2. 13C NMR spectroscopic data of compound 1 - 4 \n\nPosition 1a 2 b 3 a 4 b \n\n2 146.8 165.4 164.5 166.9 \n\n3 135.7 105.1 103.2 105.6 \n\n4 175.8 183.8 182.0 184.0 \n\n5 160.7 163.2 161.1 163.0 \n\n6 98.1 100.3 99.6 102.5 \n\n7 163.8 166.1 163.0 164.4 \n\n8 93.3 95.1 94.8 97.3 \n\n9 156.1 159.4 156.7 159.0 \n\n10 102.9 105.5 105.4 107.1 \n\n1\u2019 121.9 127.2 121.4 123.5 \n\n2\u2019 115.4 118.0 113.6 114.3 \n\n3\u2019 145.0 148.6 145.8 147.1 \n\n4\u2019 147.6 150.0 150.1 151.2 \n\n5\u2019 115.5 114.9 116.0 117.0 \n\n6\u2019 119.9 120.0 119.2 120.6 \n\nGlc-1 103.3 99.9 99.6 \n\nGlc-2 74.8 73.1 78.3 \n\nGlc-3 77.5 76.4 79.0 \n\nGlc-4 71.3 69.6 71.4 \n\nGlc-5 78.5 77.2 79.1 \n\nGlc-6 62.3 60.7 62.5 \n\nRha-1 101.0 \n\nRha-2 72.2 \n\nRha-3 72.2 \n\nRha-4 74.0 \n\nRha-5 70.0 \n\nRha-6 18.3 \na in DMSO-d6,  b in CD3OD. \n\nFigure 1. Chemical\nstructures of compounds \nisolated from A. triphylla \nvar. triphylla \n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):12-16  Hori and Devkota \n\n\u00a9NJB, BSN 15 \n\nradical scavenging activity with IC50 value of 248.3 \n\n\u00b5g/mL. Compounds 1, 3, 4, and 5 showed potent \n\nfree radical scavenging activity with IC50 values of \n\n10.6, 14.3, 20.4 and 19.3 \u00b5g/mL, respectively as \n\ncompared to positive control Trolox (IC50=12.8 \n\n\u00b5g/mL). \n\nTable 3. IC50 (\u00b5g/mL) values of extract and isolated \ncompounds of A. triphylla var. triphylla for DPPH free \nradical scavenging activity \nCompounds IC50 value (\u00b5g/mL) \n\n70% Methanol Extract 248.3\u00b12.4 \n\nLuteolin (1) 10.6\u00b10.7 \n\nLuteolin 4\u2019-O- \u03b2 -\n\nglucopyranoside (2) \n62.9\u00b10.9 \n\nLuteolin 7-O- \u03b2 -\n\nglucopyranoside (3) \n14.3\u00b11.5 \n\nLuteolin 7-O-\nneohesperidoside (4) \n\n20.4\u00b12.4 \n\nChlorogenic acid (5) 19.3\u00b12.1 \n\nTrolox (positive control) 12.8\u00b1 1.1 \n\nIn recent years, there is growing attention on the \n\nplant-based functional foods. Previously, Kim et al. \n\n[5] reported the high phenolic and flavonoid \n\ncontents and free radical scavenging activities of \n\nleaves of A. triphylla var. japonica but the active \n\ncompounds were not reported.", "start_char_idx": 9882, "end_char_idx": 11844, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9cf66d83-317b-4a67-9121-f2219d0c14d6": {"__data__": {"id_": "9cf66d83-317b-4a67-9121-f2219d0c14d6", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "caa5a1e0-3323-4e36-b69a-2b764067ceb0", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "b8dcc4ddc4e8feb8f41eacab76a86c8bc10bf10ff637863cadbd1bcc9497e800", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0561f16d-8e27-4860-b4bd-cfdd88619df5", "node_type": "1", "metadata": {}, "hash": "1544a97e8a03536e8fb4b9b25c7d0888adba234285c4d488e3081d10c6af6a33", "class_name": "RelatedNodeInfo"}}, "text": "japonica but the active \n\ncompounds were not reported. In our study, the \n\nextract of aerial parts of A. triphylla var. triphylla \n\nshowed weak activity but the isolated compounds \n\nshowed strong free radical scavenging activity. \n\nHashiba et al. [10] had also reported similar \n\nflavonoids from A. triphylla var. japonica, hence, \n\nluteolin derivatives can be regarded as the active \n\nantioxidant compounds in both of these verities. \n\nFlavonoids including luteolin derivatives are well \n\nreported as strong antioxidant phytochemicals with \n\nvarious health-promoting and disease prevention \n\nactivities [15\u201320]. Further detailed research on these \n\nplants may help in the development of functional \n\nfoods. \n\nConclusion \nFive bioactive phenolic compounds; luteolin (1), \n\nluteolin 4\u2019-O- \u03b2 -glucopyranoside (2), luteolin 7-O- \u03b2 \n\n-glucopyranoside (3), luteolin 7-O-neohesperidoside \n\n(4) and chlorogenic acid (5) were isolated for the first \n\ntime from the leaves A. triphylla var. triphylla. Some \n\nof the isolated compounds showed potent free \n\nradical scavenging activity. The aerial parts of A. \n\ntriphylla var. triphylla might be an important plant \n\nsource for the development of functional foods. \n\nHowever, detailed research related to \n\npharmacological activity and safety are necessary in \n\nfuture. \n\nAuthor\u2019s Contribution \nHPD conceived the idea and designed the study. \n\nBoth authors contributed to experiments and \n\nanalysis, and wrote the manuscript. \n\nCompeting Interest \nNo competing interests were disclosed. \n\nFunding \nThis work was supported partially by Program for \n\nLeading Graduate Schools, Health life Sciences: \n\nInterdisciplinary and Glocal Oriented (HIGO) \n\nProgram, MEXT, Japan \n\nAcknowledgments \nWe are grateful to Ms. Teruo Tanaka of Institute of \n\nResource Development and Analysis, Kumamoto \n\nUniversity for the measurement of NMR.  \n\nEthical Approval and Consent \nNot applicable. \n\nReferences \n1. Belwal T, Devkota HP, Hassan HA, Ahluwalia S, Ramadan\n\nMF, Mocan A, Atanasov AG. Phytopharmacology of Acerola\n\n(Malpighia spp.) and its potential as functional food. Trends in\nFood Science & Technology. 2018 Apr 1;74:99-106. \n\nhttps://doi.org/10.1016/j.tifs.2018.01.014\n2. Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder\n\nT, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S,\nHeiss EH, Rollinger JM. Discovery and resupply of\npharmacologically active plant-derived natural products: A\n\nreview. Biotechnology advances. 2015 Dec 1;33(8):1582-614. \nhttps://doi.org /10.1016/j.biotechadv.2015.08.001\n\n3. Dirar AI, Alsaadi DH, Wada M, Mohamed MA, Watanabe T,\nDevkota HP. Effects of extraction solvents on total phenolic\n\nand flavonoid contents and biological activities of extracts \nfrom Sudanese medicinal plants. South African Journal of\nBotany. 2019 Jan 1;120:261-7. https://doi.org/ \n\n10.1016/j.sajb.2018.07.003\n4. Ohashi H, Monda Y, Murata J, Yonekura K, Kihara H. Wild\n\nFlowers of Japan. Tokyo: Heibonsha. 2017. \n5. Kim JH, Hong JY, Shin SR, Yoon KY. Comparison of\n\nantioxidant activity in wild plant (Adenophora triphylla) leaves \n\nand roots as a potential source of functional foods.\nInternational journal of food sciences and nutrition. 2009 Jan \n\n1;60(sup2):150-61. \nhttps://doi.org/10.1080/09637480902956594\n\n6.", "start_char_idx": 11790, "end_char_idx": 15045, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0561f16d-8e27-4860-b4bd-cfdd88619df5": {"__data__": {"id_": "0561f16d-8e27-4860-b4bd-cfdd88619df5", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9cf66d83-317b-4a67-9121-f2219d0c14d6", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "98b5c603706fce9d783ca453fe304ab1ad2fcb06ced5b06fd817ad018d7b862b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a0a82645-2404-4895-81db-edaa47cc75c8", "node_type": "1", "metadata": {}, "hash": "79e9c20fb37d8e63b2a07368db773162515f734de62ebe53cc0dc95e4ce720a4", "class_name": "RelatedNodeInfo"}}, "text": "Suzuki H. Encyclopedia of Traditional Oriental Medicine.\nIshiyaku Publishers. 2005. \n\n7. Ahn EK, Oh JS. Lupenone isolated from Adenophora triphylla\n\nvar. japonica extract inhibits adipogenic differentiation\nthrough the downregulation of PPAR\u03b3 in 3T3\u2010 L1 cells.\n\nPhytotherapy Research. 2013 May;2 7(5):7 61-6. https://\ndoi.org/10.1002/ptr.4779\n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):12-16  Hori and Devkota \n\n\u00a9NJB, BSN 16 \n\n8. Watanabe M, Devkota HP, Sugimura K, Watanabe T. A\nGuidebook of Medicinal Plant park. Kumamoto:School of\n\nPharmacy, Kumamoto University. 2018. \n9. Konno C, Saito T, Oshima Y, Hikino H, Kabuto C. Structure\n\nof methyl adenophorate and triphyllol, triterpenoids of\nAdenophora triphylla var. japonica roots. Planta medica. 1981\n\nJul;42(07):268-74. https://doi.org/10.1055/s-2007-971639\n10. Hashiba K, Iwashina T, Matsumoto S. Variation in the quality\n\nand quantity of flavonoids in the leaves of coastal and inland\n\nCampanula punctata. Biochemical systematics and ecology.\n2006 Dec 1;34(12):854-61. \n\nhttps://doi.org/10.1016/j.bse.2006.04.012 \n11.  Harborne JB, Mabry TJ. The flavonoids. Boston, MA: Springer\n\nUS. 1982. https://doi.org/10.1007/978-1-4899-2915-0 \n\n12.  Lee MH, Son YK, Han YN. Tissue factor inhibitory flavonoids\nfrom the fruits of Chaenomeles sinensis. Archives of pharmacal\n\nresearch. 2002 Dec 1;25(6):842. \nhttps://doi.org/10.1007/BF02977002\n\n13. Li YL, Li J, Wang NL, Yao XS. Flavonoids and a new\npolyacetylene from Bidens parviflora Willd. Molecules. 2008\nAug;13(8):1931-41. \n\nhttps://doi.org/10.3390/molecules13081931\n14. Devkota HP, Joshi KR, Ozasa T, Fukunaga S, Yoshida N,\n\nYahara S. Steroidal glycosides from the fruits, aerial parts and\ntubers of potato (Solanum tuberosum). J. Pharmacognosy &\n\nPhytochemistry. 2015;3:252-5. \n\n15.  Khan H, Sureda A, Belwal T, \u00c7etinkaya S, S\u00fcntar \u0130, Tejada S,\nDevkota HP, Ullah H, Aschner M. Polyphenols in the\n\ntreatment of autoimmune diseases. Autoimmunity reviews.\n2019 Jul 1;18(7):647-57. \n\nhttps://doi.org/10.1016/j.autrev.2019.05.001 \n16. Rice-Evans C. Flavonoids and isoflavones: absorption,\n\nmetabolism, and bioactivity. Free Radical Biology and\nMedicine. 2004;7(36):827-8. \nhttps://doi.org/10.1016/j.freeradbiomed.2003.12.012\n\n17. Raffa D, Maggio B, Raimondi MV, Plescia F, Daidone G.\nRecent discoveries of anticancer flavonoids. European journal\n\nof medicinal chemistry.", "start_char_idx": 15046, "end_char_idx": 17415, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a0a82645-2404-4895-81db-edaa47cc75c8": {"__data__": {"id_": "a0a82645-2404-4895-81db-edaa47cc75c8", "embedding": null, "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-178", "node_type": "4", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "0926cd724634841725057a22d4756584a4de44ab80c1b450e35a435c3a4cb3da", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0561f16d-8e27-4860-b4bd-cfdd88619df5", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "c9a3d092d0ee2eba943086e65a4123bd6152deb9d86bfd671dbf94fb91f5b954", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0a90a85e-2ef6-4961-af80-acb0fa8043a4", "node_type": "1", "metadata": {}, "hash": "a504450c39ef3616b5d611cce0d8a092059b57bb894520a12cb1e1c807040817", "class_name": "RelatedNodeInfo"}}, "text": "European journal\n\nof medicinal chemistry. 2017 Dec 15 ; 142 :2 13-28. \nhttps://doi.org /10.1016/j.ejmech.2017.07.034\n\n18. Rodriguez-Mateos A, Vauzour D, Krueger CG,\n\nShanmuganayagam D, Reed J, Calani L, Mena P, Del Rio D,\nCrozier A. Bioavailability, bioactivity and impact on health of \n\ndietary flavonoids and related compounds: an update.\nArchives of toxicology. 2014 Oct 1;88(10):1803-53. \n\nhttps://doi.org /10.1007/s00204-014-1330-7\n19. L\u00f3pez-L\u00e1zaro M. Distribution and biological activities of the\n\nflavonoid luteolin. Mini reviews in medicinal chemistry. 2009\n\nJan 1;9(1):31-59. \nhttps://doi.org/10.2174/138955709787001712\n\n20. Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with\npotential for cancer prevention and therapy. Current cancer\n\ndrug targets. 2008 Nov 1;8(7):634-46. https://doi.org/\n10.2174/156800908786241050", "start_char_idx": 17374, "end_char_idx": 18208, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0a90a85e-2ef6-4961-af80-acb0fa8043a4": {"__data__": {"id_": "0a90a85e-2ef6-4961-af80-acb0fa8043a4", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a0a82645-2404-4895-81db-edaa47cc75c8", "node_type": "1", "metadata": {"identifier": "njb-178", "author": "Hori, Kengo; Devkota, Hari Prasad", "title": "Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity", "date": "2020-07-31", "file": "njb-178.pdf"}, "hash": "c6c1377cd2568fb28c90145c063c92d5962f6833ab8921ec87984b0f3f347b77", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aa69c529-7e74-44fa-a364-1b5143c374e7", "node_type": "1", "metadata": {}, "hash": "f56faa2bd8d7e4357cc43461622a48cac022da8594181a7bed2ebc02dabc822b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2020 Dec; 8 (3): 82-86 DOI: https://doi.org/10.3126/njb.v8i3.30080 Research article \n\n\u00a9NJB, BSN 82 \n\nAntibiogram of Escherichia coli and Staphylococcus aureus Isolated \nfrom Milk Sold in Kathmandu District \nShyamala Rai, Barsha Karki, Sujita Humagain, Sandesh Rimal, Sandhya Adhikari, Shilpa Adhikari, \n\nSuchitra Thapa   . \n\nAmrit Campus, Tribhuvan University, Department of Microbiology, Thamel, Kathmandu \n\nArticle history:- Received: 15 Jun 2020; Revised: 17 Dec 2020; Accepted: 18 Dec 2020; Published online: 30 Dec 2020\n\nAbstract \nThe emergence of antibiotic resistance in microorganisms and the presence of such isolates in milk pose a great risk to public \nhealth. Therefore, this study aims to determine the antibiotic susceptibility pattern of Escherichia coli and Staphylococcus aureus \nisolated from milk and assess the microbial quality of milk. For this, a total of 70 milk samples were collected and the total \nbacterial count (TBC) was determined. E. coli and S. aureus were isolated using their respective selective media while antibiotic \nsusceptibility testing was carried out by Kirby Bauer Disc Diffusion method. The TBC showed that the raw milk samples \ncontained two-fold higher microbial load while the pasteurized milk samples contained four-fold higher microbial loads than \nthe standard guidelines. A total of 62 isolates were identified from culture-positive milk samples of which 32 were E. coli and \n30 were S. aureus. A significant correlation was observed between microbial load and the organism isolated (r = 0.339, p<0.01). \nAll S. aureus isolates were susceptible to Chloramphenicol while 40% were resistant to Cefoxitin, indicating the presence of \nMethicillin resistant S. aureus (MRSA). Also, 12 multidrug resistant (MDR) S. aureus were identified. While for E. coli, all were \nsusceptible to Chloramphenicol but resistant to Ampicillin. Also, 9 MDR E. coli were detected. Higher resistance was observed \namong isolates from the raw milk samples than the pasteurized milk. It can be concluded that the milk produced by small-\nscale farms and dairy industries of Kathmandu district are of poor quality. Hence, routine microbial quality assessment and \nantimicrobial resistance monitoring should be followed to safeguard public health. \n\nKeywords: Antibiotic resistance, Milk-borne infection, Multidrug resistance, E. coli, S. aureus, Total bacterial count, MRSA \n\n Corresponding author, email: suchitrathapa69@gmail.com \n\nIntroduction \nMilk, a daily diet requirement of people, can become \n\nmicrobiologically hazardous to consumers when the \n\nprinciples of hygiene and sanitation are not met. Such \n\nconditions may become a vehicle for transmission of \n\nfood-borne infections [1]. Among all microorganism, \n\nEscherichia coli and Staphylococcus aureus are the most \n\ncommon food contaminants [1]; and in recent years, both \n\nare observed to cause a number of significant illnesses in \n\nanimals and humans [2]. S. aureus is specifically a \n\nversatile pathogen capable of causing numerous diseases \n\nin humans [2]. In addition, it is also a major causative \n\npathogen of clinical or subclinical mastitis of dairy \n\nanimals [1]. Further, MRSA together with Extended \n\nSpectrum \u03b2-Lactamase (ESBL) producing E. coli, are \nconsidered as serious threats to human health [3]. \n\nTherefore, the relative importance of these pathogens \n\nalong with other pathogenic microorganisms in milk is \n\ninevitable. But the lack of awareness about milk-borne \n\ninfections in many developing countries and \n\nconsumption of contaminated milk predisposes \n\nconsumers at risk of contracting infections with these \n\npathogens [4].", "start_char_idx": 48, "end_char_idx": 3706, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aa69c529-7e74-44fa-a364-1b5143c374e7": {"__data__": {"id_": "aa69c529-7e74-44fa-a364-1b5143c374e7", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0a90a85e-2ef6-4961-af80-acb0fa8043a4", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "ccb8b144a4f851e47136eb977874c020917fcd8bba01e606045510fcaa216bfa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0a4cf60d-46d8-40d6-905a-abf8747c899f", "node_type": "1", "metadata": {}, "hash": "792ce2a6d5a2157d5493a41dc1301266677ec8d4a123e8ad166f3c4a6ac52f75", "class_name": "RelatedNodeInfo"}}, "text": "Antibiotics are essential to treat infections caused by \n\npathogenic bacteria, both in humans and animals. \n\nHowever, their overuse and misuse in veterinary and \n\nhuman medicine has been linked to the emergence and \n\nspread of resistant bacteria, rendering the treatment of \n\ninfectious diseases ineffective in animals and humans. \n\nAnd, now antimicrobial resistance is one of the main \n\nthreats to modern medicine [5]. Further, the escalating \n\nprevalence of antimicrobial resistance among foodborne \n\npathogens [6, 7] has exaggerated the public health \n\nhazards including milk-borne infections. Available \n\nstudies around the world have reported about the \n\npresence of multidrug resistant E. coli [8], ESBL [9, 10], \n\ntoxin-producing S. aureus [11] along with MRSA strains \n\n[7, 11] in milk samples but in the case of Nepal very less \n\nstudies are found on antibiotic resistance of milk isolates. \n\nTherefore, it is necessary to know the microbial quality of \n\nmarketed milk and understand the recent trend of \n\nantimicrobial resistance among milk pathogens so as to \n\nproperly diagnose and treat the infection. This study was \n\nconducted to determine the current trend of antibiotic \n\nresistance of E. coli and S. aureus isolated from milk and \n\nassess the microbial quality of milk sold in Kathmandu \n\ndistrict.  \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nmailto:suchitrathapa69@gmail.com\nhttps://orcid.org/0000-0001-5995-1909\nmailto:suchitrathapa69@gmail.com\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 82-86   Rai et al. \n\n\u00a9NJB, BSN 83 \n\nMaterials and Methods \nSampling site and sample \nA total of 70 milk samples were tested in this study. Forty \n\nraw milk samples (30 ml each) from 4 different \n\nmunicipalities of Kathmandu district (Kageshwori-\n\nManohara, Chandragiri, Tarkeshwor and Dakshinkali \n\nMunicipality) were collected in a sterile screw capped \n\nbottle and transported to laboratory in an icebox within 2 \n\nhours. Further, 30 pasteurized milk pack (500ml) was \n\nbought from local vendors from the same municipalities. \n\nThe collection of the sample was done from December \n\n2018 to March 2019 in compliance with the guidelines \n\nstated in Bacteriological Analytical Manual [12]. \n\nTotal bacterial count of milk samples \nThe collected milk samples were serially diluted up to- \n\n10-8 dilution. The bacterial count was determined by \n\nculturing the serially diluted milk samples. Conventional \n\npour plate technique was employed for culturing the \n\ndiluted milk samples [13]. \n\nIsolation and identification of E. coli and S. \naureus \nIsolation of E. coli and S. aureus was done by enrichment \n\nin buffered peptone water and cultured in EMB (Eosin \n\nMethylene Blue) and MSA (Mannitol Salt Agar) media \n\nrespectively [14]. The distinct colonies were identified \n\nand confirmed by following their respective biochemical \n\ncharacteristics [13].  \n\nAntibiotic susceptibility testing of milk \nisolates \nIsolates of E. coli and S. aureus were subjected to antibiotic \n\nsusceptibility testing using Kirby Bauer disk diffusion \n\nmethod as recommended by CLSI [15].", "start_char_idx": 3711, "end_char_idx": 6912, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0a4cf60d-46d8-40d6-905a-abf8747c899f": {"__data__": {"id_": "0a4cf60d-46d8-40d6-905a-abf8747c899f", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aa69c529-7e74-44fa-a364-1b5143c374e7", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "39dcfe130f1027808b2ed0de4f4845bf8728c2f5f4fa7fc580054c3117ae86ae", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4e69aa5f-575e-48aa-8d8d-581138de2ca1", "node_type": "1", "metadata": {}, "hash": "a0fadf596a4f400c427be65c8dff60bbef4997b5a0590238b0201f5c557eeee2", "class_name": "RelatedNodeInfo"}}, "text": "The antibiotics \n\nused for E. coli were Ampicillin (10\u00b5g), Chloramphenicol \n\n(30\u00b5g), Ciprofloxacin (5\u00b5g), Nalidixic acid (30\u00b5g), \n\nTetracycline (30\u00b5g) and Ceftriaxone (5\u00b5g); and for S. \n\naureus, Cefoxitin (30\u00b5g), Tetracycline (3\u00b5g), \n\nChloramphenicol (30\u00b5g), Ceftriaxone (30\u00b5g) and \n\nCiprofloxacin (5\u00b5g) were used. \n\nData analysis \nThe data was initially entered in MS Excel and exported \n\nto SPSS. The frequency distribution, normal distribution \n\ntesting, variance analysis and correlation were done \n\nusing SPSS (version 20). The significance was measured \n\nat both 95% and 99% confidence interval. The resistance \n\nprofile was analyzed using WHONET2019 (32-bit version \n\n19.13.21) and the outcome was interpreted accordingly. \n\nResults \nMicrobial load of milk samples \nThe TBC of raw milk ranged from 0.31\u00d7105 CFU/ml to \n\n1000\u00d7105 CFU/ml with mean TBC of 8.13\u00d7106 CFU/ml \n\n(S.D = 18.9x 106). Likewise, the TBC of pasteurized milk \n\nranged from 0.35\u00d7103 CFU/ml to 1600\u00d7103 CFU/ml with \n\nmean TBC of 14.25\u00d7104 CFU/ml (S.D = 29.71 x 104).  \n\nFigure 1. Quality of milk samples according to BIS \nstandard guideline. \n\nFor raw milk, the TBC exceeding 50\u00d7105 CFU/ml \n\nis graded as poor while below it is graded either fair, \n\ngood and very good according to Bureau of Indian \n\nStandards (BIS) [16] and are considered within safe \n\nlimit. Following this Standard, among the total raw \n\nmilk samples 14 samples were observed to fall under \n\n\"very good\" grading, 6 under \"good\" grading, 10 under \n\n\"fair\" grading and rest 10 under \"poor\" grading.  \n\nIn case of pasteurized milk, 9 samples had TBC below \n3\u00d7104 CFU/ml and 21 samples had higher than that. The\ndistribution of milk samples in terms of their standard \n\nlimit of TBC is given in Figure 1. Statistically, a highly \n\nsignificant difference was observed in the distribution of \n\nTBC across the raw and pasteurized milk sample \n\n(p<0.01). \n\nPrevalence of E. coli and S. aureus in Milk \nE. coli and S. aureus were isolated from raw and \n\npasteurized milk samples using selective media (Figure \n\n3A and 3B). Out of 70 samples, 46 (65.71%) samples \n\nshowed culture positivity towards E. coli or S. aureus or \n\nto both. The percentage distribution of organisms among \n\nthe raw and pasteurized milk sample is given in Figure \n\n2.  It was observed that the distribution of microbial count\n\nis not the same across culture positivity (p<0.05) and \n\nisolated organism (p<0.01).  \n\nA total of 32 E. coli isolates were subjected to antibiotic \n\nsusceptibility test using Ciprofloxacin, Ceftriaxone, \n\nChloramphenicol, Nalidixic acid and Ampicillin \n\nantibiotic disc. \n\n30%\n\n75%\n70%\n\n25%\n\n0%\n\n10%\n\n20%\n\n30%\n\n40%\n\n50%\n\n60%\n\n70%\n\n80%\n\nPasteurised Milk Raw milk\nP\n\ne\nrc\n\ne\nn\n\nta\ng\n\ne\no\n\nf\nsa\n\nm\np\n\nle\ns\n\nType of milk sample\n\nWithin Limit Above limit\n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 82-86   Rai et al. \n\n\u00a9NJB, BSN 84 \n\nFigure 2.", "start_char_idx": 6913, "end_char_idx": 9785, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4e69aa5f-575e-48aa-8d8d-581138de2ca1": {"__data__": {"id_": "4e69aa5f-575e-48aa-8d8d-581138de2ca1", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0a4cf60d-46d8-40d6-905a-abf8747c899f", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "c8351c1cce8882e87590ee8614e1c1b6519c66fa427ffe789b1205d92fcd04c8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9948f832-70d6-4d71-87ff-6d0a8d1d1c36", "node_type": "1", "metadata": {}, "hash": "0ef1258322c0d13446cc70f928e98e00019114bfebbfb4e3ccbf34600ed0aca1", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN 84 \n\nFigure 2. Distribution of E. coli and S. aureus in the milk sample \n(n=46) \n\nFigure 3A: E. coli Culture on EMB media plate and 3B: S. \naureus culture on MSA media plate \n\nAntibiogram of E. coli \nAll the isolates showed 100% susceptibility towards \n\nChloramphenicol while none of the isolates showed \n\nsusceptibility towards Ampicillin. Also, the susceptibility \n\nfor Ciprofloxacin, Ceftriaxone, Tetracycline and Nalidixic \n\nacid were 96.88%, 87.5%, 81.25% and 78.13%, \n\nrespectively. Of the 9 MDR E. coli identified, 4 isolates of \n\nimportant antibiotic resistance was recognized through \n\nantibiogram analysis. A detail resistance profile of these \n\nisolates is provided in Table 1.  \n\nAntibiogram of S. aureus \nA total of 30 S. aureus isolates were subjected to antibiotic \n\nsusceptibility test using Cefoxitin, Ciprofloxacin, \n\nCeftriaxone, Chloramphenicol and Nalidixic acid \n\nantibiotic disc. All the isolates showed 100% \n\nsusceptibility towards Chloramphenicol, while the \n\nsusceptibility for Tetracycline, Ciprofloxacin, Cefoxitin \n\nand Ceftriaxone were 93.33%, 70%, 60% and 30%, \n\nrespectively. A total of 12 MDR S. aureus (40%) were \n\nidentified and they were confirmed as MRSA due to their \n\nresistance towards Cefoxitin. A detail resistance profile of \n\nimportant isolates of S. aureus is provided in Table 2.   \n\nTable 2 Antibiotic resistance profile of S. aureus (n=12) \n\n Milk sample \ntype \n\nResistance \nantibiotics \n\nNo of \nS. aureus \n\nImportance \nResistance \n\nPriority \n\nRaw milk CIPR CXR 2 Yes* Medium*\n\nRaw milk CTRR CX R 6 Yes* Medium*\n\nRaw milk \nCTRR CX R \nTER \n\n1 Yes* Medium*\n\nRaw milk \nCTRR CX R \nCIPR \n\n1 Yes* Medium*\n\nPasteurized \nmilk \n\nCTRR CX R \nCIPR \n\n2 Yes* Medium* \n\n* According to the WHONET 2020 software interpretation\nLegends: CX-Cefoxitin, CTR- Ceftriaxone, TE-Tetracycline, \nCIP-Ciprofloxacin \n\nDiscussion \nThis study, which examined milk samples from four \n\ndifferent municipality of Kathmandu districts, showed \n\nhigh significance (p<0.01) in the distribution of microbial \n\nload among the raw and pasteurized milk samples. This \n\nsignificance may comply with the fact that pasteurized \n\nmilks are heat treated and ought to have lower microbial \n\nload. However, the majority of pasteurized milk samples \n\n(70%) were below the standard recommended guidelines \n\nof Indian standards [16] compared to raw milk (25%) \n\n(Figure 1). Also, the raw milk samples showed two-fold \n\nhigher microbial load than the recommended value of BIS \n\nwhile pasteurized milk showed four-fold higher \n\nmicrobial load than the standard recommended value of \n\nBIS. This indicates that the pasteurized milk samples in \n\nour study were of bad quality. Such results of high \n\nmicrobial load may be due to inefficient pasteurization, \n\npoor packaging material and pipeline, post \n\npasteurization contaminants, presence of heat resistant \n\nbacteria, poor air and storage condition, etc. However, as \n\nboth harmful and beneficial microbes can reside in milk \n\nand higher microbial load does not necessarily indicate \n\nthe exact type of microbes present in the milk, it may not \n\nbe appropriate to gauge the quality of milk solely based \n\non microbial load unless each of the microbial strain in \n\nthe milk are identified.", "start_char_idx": 9761, "end_char_idx": 13005, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9948f832-70d6-4d71-87ff-6d0a8d1d1c36": {"__data__": {"id_": "9948f832-70d6-4d71-87ff-6d0a8d1d1c36", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4e69aa5f-575e-48aa-8d8d-581138de2ca1", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "0315889b959fd3b00777f292a134a6aac9cdd9335fea2f71af3af355c406a276", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9d68c8b4-fc2c-4cdf-9584-a8164f4d62dc", "node_type": "1", "metadata": {}, "hash": "6c9a6d660332451babe7e192ddddfb3128b5994195a4defecb4ca11665167d87", "class_name": "RelatedNodeInfo"}}, "text": "The quality issue aside, \n\napparently similar prevalence of higher microbial load in \n\npasteurized milk in Kathmandu was documented in a \n\nTable 1 Antibiotic resistance profile of E. coli (n=4) \n\nMilk sample type Resistance antibiotics Number of E. coli Importance Resistance Priority Inference \n\nRaw milk AMPR NARCTRR 1 Yes* Medium* Possible ESBL \n\nPasteurized milk AMPR TER NARCTRR 2 Yes* Medium* Possible ESBL \n\nRaw milk AMPR TER CIPRCTRR 1 Yes* Medium* Possible ESBL \n* According to the WHONET 2020 software interpretation\nLegends: AMP-Ampicillin, NA-Nalidixic acid, CTR- Ceftriaxone, TE-Tetracycline, CIP-Ciprofloxacin \n\n9\n\n1 2\n\n7\n\n13\n14\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\nE. coli S. aureus Both\n\nN\nu\n\nm\nb\n\ne\nr\n\no\nf\n\nm\nil\n\nk\nsa\n\nm\np\n\nle\n\nOrganism Isolated from milk samples\n\nPasteurized Milk Raw Milk\n\nA B \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 82-86   Rai et al. \n\n\u00a9NJB, BSN 85 \n\nstudy conducted by Acharya et al. (2017) [17]. Even the \n\nDFTQC, Nepal reported the higher rate of non-\n\ncompliance among milk and milk product in the annual \n\nbulletin of 2019 [18]. The presence of microbes in high \n\nnumber in treated milk sample is a risk to the consumers \n\nas the microbes present may be pathogenic strains.  \n\nIn this study, almost half of the milk sample showed the \n\npresence of E. coli (45.71%) and similar results were \n\nreported in other studies [8, 19]. Also, a study conducted \n\nby Arjyal et al. (2004), has reported E. coli prevalence rate \n\nas high as 92%  [20]. Understandably, E. coli is a \n\ncommensal enteric microorganism; yet, as their \n\npathogenic strains are associated with a range of illness \n\nin animal and humans especially the toxin producers \n\n(Shiga toxin-producing E.coli) and thus their presence in \n\nmilk should not be overlooked. In case of S.aureus, their \n\npresence in milk is of greater concern as they also \n\nproduce heat-stable toxin which causes food poisoning \n\n[1]. The finding in this study related to S. aureus \u2013 almost \n\nhalf of the milk sample (42.86%) had the presence of S. \n\naureus \u2013is comparable to the results of several studies \n\nconducted in Nepal [20, 21] as well as in the rest of the \n\nworld [22, 23, 24]. Statistically, a significant positive \n\ncorrelation (p<0.05) was observed in the distribution \n\npattern of TBC across the culture; it signifies that the \n\nmicrobial load affects the presence of E. coli or S. aureus \n\nand vice versa. Similar correlation but with higher \n\nsignificance (p<0.01) was observed among microbial load \n\nand the type of organism which suggests that the \n\nmicrobial load affects the type of organism present in the \n\nsample and vice versa. This justifies the existence of \n\nhigher prevalence of S. aureus (90%) and E. coli (65.63%) \n\nin raw milk as they have higher microbial load (Figure 2). \n\nRegarding the antibiotic susceptibility test of E. coli and \n\nS. aureus, the result showed an emerging antibiotic \n\nresistance among both isolates. All the E. coli isolates in \n\nthis study were resistant to Ampicillin which was in \n\ncompliance with the findings of Badri et al. (2017) [9] and \n\nSingh et al. (2018) [25].  Besides ampicillin resistance, the \n\nresults revealed higher resistance among E. coli towards \n\nNalidixic acid, Tetracycline, Ceftriaxone and \n\nCiprofloxacin in descending order.", "start_char_idx": 13006, "end_char_idx": 16286, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9d68c8b4-fc2c-4cdf-9584-a8164f4d62dc": {"__data__": {"id_": "9d68c8b4-fc2c-4cdf-9584-a8164f4d62dc", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9948f832-70d6-4d71-87ff-6d0a8d1d1c36", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "5be73fa4e9dfd529729fba76a096763d1b1bf33f6d51f5898d2cf14fc043cc49", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4dc01977-2a84-4a19-bbf5-f7c904e93dda", "node_type": "1", "metadata": {}, "hash": "e7c4d356dfe330cec2477fa5ba4559d7bf6e9f30e0cd4d707e291d74c0ab5091", "class_name": "RelatedNodeInfo"}}, "text": "Even though XDR \n\nwas not detected, 9 MDR was present and their \n\nantibiogram analysis indicated that 4 out of 9 isolates \n\nwere important resistance of medium priority (Table 1). \n\nSuch presence of MDR in milk samples were also \n\nreported in similar studies [8, 10]. This presence of MDR \n\nis of greater concern to public health and indicates an \n\nalarming situation. \n\nWhile in case of S. aureus, the resistance was higher for \n\nmost of the antibiotics except for Chloramphenicol. Such \n\nfull susceptibility was also reported in various other \n\nstudies of milk samples [22, 17]. Besides \n\nChloramphenicol susceptibility, the results revealed \n\nhigher resistance among S. aureus towards Ceftriaxone, \n\nCefoxitin, Ciprofloxacin and Tetracycline in descending \n\norder. Further, 12 MDR (40%) were detected which were \n\nMRSA as well, and their antibiogram analysis indicated \n\nthat these are important resistance of medium priority \n\n(Table 2). Several other studies have also reported the \n\nhigher prevalence of MDR [7, 11, 26] and MRSA [7, 11, \n\n17]. Simultaneous presence of MDR in MRSA reported in \n\nthis study also resembled the study of Aliyu et al. (2020) \n\n[27]. This result indicates an emerging trend of \n\nantimicrobial resistance among S. aureus.  \n\nThis emerging antibiotic resistance among both E. coli \n\nand S. aureus isolates was observed in higher number in \n\nraw milk sample compared to pasteurized milk sample. \n\nAlso, the multidrug resistant isolate was found to be \n\nhigher in raw milk than pasteurized milk. Since the \n\nexposure to environment is more in raw milk compared \n\nto pasteurized milk, the chances of resistant isolates \n\nfinding its way to raw milk is more likely. Further the \n\nextensive misuse of antibiotics for the treatment of farm \n\nanimals may have created selective pressure and resulted \n\nin the survival and persistence of resistant isolates. This \n\nemerging resistance may lead to treatment failure of the \n\nlast resort drug. Thus, routine monitoring of resistant \n\nprofile of milk pathogens should be implemented in \n\norder to properly diagnose and treat milk-borne \n\ninfections effectively, along with the assessment of \n\nmicrobial quality of milk with the purpose of \n\nsafeguarding the public health.  \n\nConclusion \nTo conclude, the resistance towards common \n\nantimicrobials is emerging among milk isolates and \n\ninfections by these isolates pose a serious threat to animal \n\nand public health. Therefore, regular monitoring \n\nprograms, good farming practice training, improved \n\nstandard guidelines, antibiotic surveillance program on \n\nfood isolates and rational use of antibiotics are needed to \n\nimprove sustainable food production and avoid the \n\nemergence of antibiotic-resistant strains.  \n\nAuthors' Contribution \nThe development of concept, preliminary work and \n\nlaboratory analysis, was done by SR, BK, SH, SR and \n\nSaA under the guidance of SuT and ShA. Further, SuT\nperformed data analysis, prepared, reviewed and edited \n\nthe manuscript. All authors read and approved the final \n\nmanuscript. \n\n\n\nNepal J Biotechnol. 2020 Dec; 8 (3): 82-86   Rai et al. \n\n\u00a9NJB, BSN 86 \n\nCompeting Interests \nThe authors declare that they have no conflicts of interest. \n\nFunding \nThis study was not funded by any agency or institution. \n\nAcknowledgments \nAll authors are grateful to the faculty and laboratory staff \n\nof Microbiology department of Amrit Campus for their \n\ncontinuous support in this research work, and extent \n\nespecial mention to the farm owners of Kathmandu \n\ndistrict who supported us by providing the milk sample. \n\nEthical Approval and Consent \nA brief detail of this research study was provided to the \n\nfarm owner, and a verbal consent was obtained before \n\nsampling. This study was carried out with the approval \n\nfrom the concerned authorities. \n\nData Availability  \nThe data can be made available upon request. \n\nReferences \n1. Tamime AY, editor. Milk processing and quality management.", "start_char_idx": 16287, "end_char_idx": 20239, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4dc01977-2a84-4a19-bbf5-f7c904e93dda": {"__data__": {"id_": "4dc01977-2a84-4a19-bbf5-f7c904e93dda", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9d68c8b4-fc2c-4cdf-9584-a8164f4d62dc", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "0cd6d1725706a6ad7f4e9d8f28f4c06c9dc7d516d04272cb091a8ac19e6caa64", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "06984f70-1cda-4e7a-bccd-fcc649acb6f3", "node_type": "1", "metadata": {}, "hash": "fd42ab75877616a7ece84caa9246c59be782d1ec27ff1e5c2e9d31b4a9ed7df0", "class_name": "RelatedNodeInfo"}}, "text": "Tamime AY, editor. Milk processing and quality management. John\n\nWiley & Sons; 2009 Jan 30. \nhttp://www.foodtechnologist.yolasite.com/resources/Milk%20P\nrocessing%20 and%20Quality%20Management.pdf\n\n2. Food and Drug Administration. The Bad Bug Book: Foodborne \nPathogenic Microorganisms and Natural Toxins Handbook, 2nd\nedn. 2012. https://www.fda.gov/downloads/Food/ \nFoodborneIllnessContaminants/UCM297627.pdf\n\n3. Centres for Disease Control and Prevention (US). Antibiotic \nresistance threats in the United States, 2019. Centres for Disease\nControl and Prevention, US Department of Health and Human\nServices; 2019. http://www.cdc.gov/drugresistance/threat-\nreport-2019.\n\n4. Mosalagae D, Pfukenyi DM, Matope G. Milk producers\u2019 awareness\nof milk-borne zoonoses in selected smallholder and commercial\ndairy farms of Zimbabwe. Tropical animal health and production.\n2011 Mar 1;43(3):733-9. https://doi.org/10.1007/s11250-010-9761-5 \n\n5. World Health Organization. Antimicrobial resistance: global report\non surveillance. World Health Organization;2014. \nhttps://apps.who.int/iris/bitstream/handle/10665/112642/9789\n241564748eng.pdf\n\n6. Threlfall EJ, Ward LR, Frost JA, Willshaw GA. The emergence and \nspread of antibiotic resistance in food-borne bacteria. International\njournal of food microbiology. 2000 Dec 5;62(1-2):1-5.\nhttps://doi.org/10.1016/S0168-1605(00)00351-2\n\n7. Joshi LR, Tiwari A, Devkota SP, Khatiwada S, Paudyal S, Pande KR.\nPrevalence of methicillin-resistant Staphylococcus aureus (MRSA) in\ndairy farms of Pokhara, Nepal. International Journal of Veterinary\nScience. 2014;3(2):87-90. http://www.ijvets.com/pdf-\nfiles/Volume-3-no-2-2014/87-90.pdf \n\n8. Rasheed MU, Thajuddin N, Ahamed P, Teklemariam Z, Jamil K.\nAntimicrobial drug resistance in strains of Escherichia coli isolated\nfrom food sources. Revista do Instituto de Medicina Tropical de S\u00e3o \nPaulo. 2014 Aug;56(4):341-6. https://10.1590/s0036-\n46652014000400012  \n\n9. Badri AM, Ibrahim IT, Mohamed SG, Garbi MI, Kabbashi AS,\nArbab MH. Prevalence of extended-spectrum beta-lactamase\n(ESBL) producing Escherichia coli and Klebsiella pneumoniae isolated\nfrom raw milk samples in Al Jazirah State, Sudan. Mol. Biol.\n2017;7(1):201.https://10.4172/2168-9547.1000201\n\n10. Batabyal K, Banerjee A, Pal S, Dey S, Joardar SN, Samanta I, Isore\nDP, Singh AD. Detection, characterization, and antibiogram of\nextended-spectrum beta-lactamase Escherichia coli isolated from\n\nbovine milk samples in West Bengal, India. Veterinary World. 2018 \nNov;11(10):1423. https://10.14202/vetworld.2018.1423-1427   \n\n11. Riva A, Borghi E, Cirasola D, Colmegna S, Borgo F, Amato E,\nPONTELLO M, Morace G. Methicillin-resistant Staphylococcus\naureus in raw milk: prevalence, SCCmec typing, enterotoxin \ncharacterization, and antimicrobial resistance patterns.", "start_char_idx": 20181, "end_char_idx": 22980, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "06984f70-1cda-4e7a-bccd-fcc649acb6f3": {"__data__": {"id_": "06984f70-1cda-4e7a-bccd-fcc649acb6f3", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4dc01977-2a84-4a19-bbf5-f7c904e93dda", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "3a04f89c1be55c5dd772a723b12f903816b9821659aa5b4c81515f25d5cd10f9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b30bbe49-d901-46fb-915e-63d119c03ff5", "node_type": "1", "metadata": {}, "hash": "a4a3d208aba0db08207fef6f0d1b2b4dfd707cfa45a12f0e6db9df0492dc51a4", "class_name": "RelatedNodeInfo"}}, "text": "Journal of\nfood protection. 2015 Jun 1;78(6):1142-6. \nhttps://doi.org/10.4315/0362-028X.JFP-14-531\n\n12. Andrews WH, Hammack TS. BAM: Food sampling/preparation of \nsample homogenate. Retrieved July. 2003 Apr;19:2016. \nhttps://www.fda.gov/food/laboratory-methods-food/bam-\nchapter-1-food-samplingpreparation-sample-homogenate\n\n13. Cheesbrough M. District laboratory practice in tropical countries,\npart 2. Cambridge university press; 2006 Mar 2.\n\n14. Singh P, Prakash A. Isolation of Escherichia coli, Staphylococcus aureus\nand Listeria monocytogenes from milk products sold under market\nconditions at Agra region. Acta agriculturae Slovenica. 2008\nNov;92(1):83-8. http://aas.bf.uni-lj.si\n\n15. Clinical and Laboratory Standards Institute. Performance\nstandards for antimicrobial susceptibility testing. CLSI supplement\nM100. 2013.\n\n16. BIS DW. 10500: 1991. First Revision, Bureau of Indian Standards,\nIndia. 1992.\n\n17. Acharya S, Bimali NK, Shrestha S, Lekhak B. Bacterial Analysis of\nDifferent Types of Milk (Pasteurized, Unpasteurized and Raw \nMilk) Consumed in Kathmandu Valley. Tribhuvan University\nJournal of Microbiology. 2017;4:32-8. \nhttps://doi.org/10.3126/tujm.v4i0.21674\n\n18. DFTQC . Annual Bulletin. Department of Food Technology and\nQuality Control, Babar Mahal, Kathmandu, Nepal,8-9. 2019\nhttp://www.dftqc.gov.np/downloadfile/DFTQC_English_Annu\nal_Buletin_Book_1591495040.pdf\n\n19. Parajuli A, Rimal P, Maharjan R, Chaudhary R, Chaturwedi SB.\nQuality Analysis of Milk in Kathmandu Valley. Tribhuvan\nUniversity Journal of Microbiology. 2018 Sep 26;5:7-10.\nhttps://doi.org/10.3126/tujm.v5i0.22295\n\n20. Arjyal C, Dahal BN, Khadka B. Microbial quality of milk available \nin Kathmandu valley. Journal of the Nepal Medical Association.\n2004 May 1;43(153).https://doi.org/10.31729/jnma.475\n\n21. Shrestha S, Bindari YR. Prevalence of sub-clinical mastitis among\ndairy cattle in Bhaktapur District, Nepal. International Journal of \nAgriculture and Biosciences. 2012;1(1):16-9.\nhttp://www.ijagbio.com/wp-content/uploads/pdf-files/\nVolume- 1-Issue-1-2012/16-19.pdf \n\n22. Uddin MA, Motazzim-ul-Haque HM, Noor R. Isolation and\nidentification of pathogenic Escherichia coli, Klebsiella spp. and\nStaphylococcus spp. in raw milk samples collected from different\nareas of Dhaka City, Bangladesh. Stamford Journal of\nMicrobiology. 2011;1(1):19-23. \nhttps://doi.org/10.3329/sjm.v1i1.9098\n\n23. Titouche Y, Hakem A, Salmi D, Yabrir B, Chenouf N, Chergui A, \nChenouf A, Houali K. Assessment of microbiological quality of raw\nmilk produced at Tizi Ouzou area (Algeria). \nhttps://10.3923/ajava.2016.854.860\n\n24.", "start_char_idx": 22981, "end_char_idx": 25579, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b30bbe49-d901-46fb-915e-63d119c03ff5": {"__data__": {"id_": "b30bbe49-d901-46fb-915e-63d119c03ff5", "embedding": null, "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-180", "node_type": "4", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "6d75fd301314dc7046282d96fe487c84aa161be331a3d888c8a3fa705d5fc512", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "06984f70-1cda-4e7a-bccd-fcc649acb6f3", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "13e7d4ac9a8d9dca5147f6a6a27683cf4d290104316ddfb04edf5a13355a3019", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a3cde2df-3ce5-44aa-8879-868017bb6a83", "node_type": "1", "metadata": {}, "hash": "cdf2ee3cf60c08a3b6d59225ba6e8b11db7d2283218cc304ed70d4560a19cbb1", "class_name": "RelatedNodeInfo"}}, "text": "https://10.3923/ajava.2016.854.860\n\n24. Bano S, Hayat M, Samreen T, Asif M, Habiba U, Uzair B. Detection\nof Pathogenic Bacteria Staphylococcus aureus and Salmonella sp. from \nRaw Milk Samples of Different Cities of Pakistan. Natural Science.\n2020; 12, 295-306. https://doi.org/10.4236/ns.2020.125026\n\n25. Singh A, Chhabra D, Sikrodia R, Shukla S, Sharda R, Audarya S.\nIsolation of E. coli from Bovine Mastitis and their Antibiotic\nSensitivity Pattern. International Journal of Current Microbiology \nand Applied Sciences. 2018; 7(10): 11-18. \nhttps://doi.org/10.20546/ijcmas.2018.710.002\n\n26. Massawe HF, Mdegela RH, Kurwijila LR. Antibiotic resistance of\nStaphylococcus aureus isolates from milk produced by smallholder\ndairy farmers in Mbeya Region, Tanzania. Int. J. One Health.\n2019;5:31-7. https://doi:10.14202/ijoh.2019.31-37\n\n27. Aliyu Y, Abdullahi IO, Whong CZ, Olalekan BO, Reuben RC.\nOccurrence and antibiotic susceptibility of methicillin-resistant\nStaphylococcus aureus in fresh milk and milk products in Nasarawa\nState, North-Central Nigeria. Journal of Microbiology and\nAntimicrobials. 2020 Jan 31;12(1):32-41. \nhttps://doi.org/10.5897/JMA2020.0424", "start_char_idx": 25540, "end_char_idx": 26701, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a3cde2df-3ce5-44aa-8879-868017bb6a83": {"__data__": {"id_": "a3cde2df-3ce5-44aa-8879-868017bb6a83", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b30bbe49-d901-46fb-915e-63d119c03ff5", "node_type": "1", "metadata": {"identifier": "njb-180", "author": "Rai, Shyamala; Karki, Barsha; Humagain, Sujita; Rimal, Sandesh; Adhikari, Sandhya; Adhikari, Shilpa; Thapa, Suchitra", "title": "Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District", "date": "2020-12-30", "file": "njb-180.pdf"}, "hash": "656538f9363d7c8f23605b79b4568a7f88b0050b5add72da9a1ac4b1b5c7f3d5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70e184db-e5f1-4759-8161-46be95bd935b", "node_type": "1", "metadata": {}, "hash": "cfc119826369bfc2cda5579ebd479d5a0143a3f4016fd107a368a7ba9a7cde5a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1:90-95  DOI: https://doi.org/10.3126/njb.v7i1.26958 \n\n\u00a9NJB, Biotechnology Society of Nepal  90 Nepjol.info/index.php/njb. \n\nORIGINAL RESEARCH ARTICLE \n\n \n\nVancomycin Intermediate MRSA Isolates Obtained from \nRetail Chicken Meat and Eggs Collected at Pokhara, Nepal \n\nSurya Prasad Devkota 1, 2, 3*, Ashmita Paudel 3, Krishna Gurung 1, 4 \n\n1Pokhara Bigyan Tatha Prabidhi Campus, Nayabazzar, Pokhara \n2 School of Health and Allied Sciences, Pokhara University \n\n3Regional College of Health Science and Technology, Nayabazzar, Pokhara \n4Prithvi Narayan Campus, Pokhara \n\nAbstract \nAntimicrobial resistance among food animal isolates is increasing as a result of their uncontrolled \n\nuses. The monitoring of antibiotic resistance among these isolates is very necessary. S aureus was \n\nisolated from eggshells and chicken meat samples collected from different retail outlets of the \n\nPokhara metropolitan. Samples were inoculated on Mannitol salt agar aseptically and inoculated \n\novernight. Isolated yellow colonies were further examined by Gram-staining, catalase, and \n\ncoagulase test to detect S aureus. Methicillin resistance was screened using cefoxitin disc. \n\nVancomycin minimum inhibitory concentration (MIC) of Methicillin-resistant S aureus (MRSA) \n\nisolates were determined by the agar dilution method following CLSI guidelines. 139 S aureus \n\nwere isolated from 205 samples. Among them, 89 were from egg samples (out of 125 samples) and \n\n50 from chicken (out of 80 samples). The overall prevalence of MRSA was 12.94%. Antibiotic \n\nresistance was significantly higher in MRSA isolates compared to Methicillin sensitive S aureus \n\n(MSSA) isolates. The highest rate of resistance was noted for ampicillin, amoxicillin, and \n\nerythromycin while the least resistance was noted for gentamicin and amikacin. Vancomycin \n\nminimum inhibitory concentration (MIC) range of the MRSA isolates was 0.25-8\u00b5g/ml indicating \n\nthe detection of both vancomycin-intermediate and sensitive isolates from the samples. This is the \n\nfirst study reporting vancomycin-intermediate S aureus (VISA) isolates from Nepal and indicates \n\nthe increasing drug resistance among animal isolates. Further surveillance studies about the \n\ntransmission of these pathogens to humans as well as detail molecular analyses are imminent. \n\nKeywords: MRSA, vancomycin-intermediate, S aureus, eggs, meat \n\n*Corresponding Author \n\nEmail: devkotasp1@gmail.com \n\nIntroduction \nStaphylococcus aureus is a significant pathogen \n\nthat causes diseases both in humans and animals \n\nand their treatment is gradually becoming \n\ndifficult due to their increasing resistance to \n\nantimicrobial agents [1]. Dry resistance \n\nproperties of the organism assist them to thrive \n\nin adverse environmental conditions including \n\nvarious food items, human skin, nose and other \n\ninanimate objects for a longer period [2]. The \n\npresence of these organisms in animals has \n\nstarted gaining importance these days as they are \n\nthe cause of increasing livestock infections and \n\ntheir ability to cause zoonotic infections [3]. \n\nStaphylococcus associated with animals may be \n\nhazardous to humans not only due to their \n\nantibiotic resistance but also as the causative \n\nagents of food-related infections [4]. The presence \n\nof drug-resistant bacteria in meat is a significant \n\npublic health challenge and the incidence of \n\nresistance has risen in this decade among \n\npathogens that are transmitted via food [5].", "start_char_idx": 47, "end_char_idx": 3565, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70e184db-e5f1-4759-8161-46be95bd935b": {"__data__": {"id_": "70e184db-e5f1-4759-8161-46be95bd935b", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a3cde2df-3ce5-44aa-8879-868017bb6a83", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "ae2fa548b220ec240185d444d0fca6a07ec8f88e3c6ddd7575727755649b0d98", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c5aa02f8-8976-41a1-abe5-201d19475d2b", "node_type": "1", "metadata": {}, "hash": "0a505c30f49908672f3ebb9a210404b04820f74b97e938e58d324571ec754b16", "class_name": "RelatedNodeInfo"}}, "text": "Methicillin-resistant S. aureus (MRSA) has been \n\ndetected from various animals used for food \n\nproduction and from animal-related food items \n\nincluding milk, meat, and dairy products which \n\nnecessitate the targeted researches to determine \n\nthe prevalence of these isolates in food to assess \n\nthe probable risk of these foods as sources for \n\nhuman infection [6]. In addition to two common \n\ntypes of MRSA i.e community-associated MRSA \n\nand hospital-associated MRSA, a third form of \n\nMRSA known as livestock-associated MRSA has \n\nnow emerged which infects various animals [7]. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 90-95                   Devkota et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  91 Nepjol.info/index.php/njb. \n\n \n\n \n\nThere is the possibility of transmission of MRSA \n\nisolates associated with livestock not only \n\nbetween animals and humans but also between \n\nhumans [8]. \n\nThere are reports of decreasing vancomycin \n\nsensitivity among S aureus isolated in Nepal in \n\nrecent times [9]. The study of Adhikari et al have \n\nreported that vancomycin MIC range for S aureus \n\nisolated from pus samples was 0.016\u00b5g/ml to \n\n1\u00b5g/ml [10]. Very recently, vancomycin-resistant \n\nand vancomycin-intermediate S aureus have been \n\nisolated from clinical samples [11]. Many studies \n\nhave reported that all clinical S aureus isolates \n\nfrom Nepal were absolutely sensitive to \n\nvancomycin [12, 13]. However, there have not \n\nbeen any studies that focus on vancomycin MIC \n\nvalue of S aureus isolated from non-clinical \n\nsamples including food animals, food samples, \n\nand environmental samples in Nepal.  \n\nThere is a huge and uncontrolled use of \n\nantibiotics in animal farms in Nepal. This might \n\nhave led to the development of resistance among \n\nanimal isolates. In this study, we particularly \n\nfocused on S. aureus isolates as they are one of the \n\nfrequent isolates from eggs and meat samples. \n\nHowever, there are no studies about the effects of \n\nthis unscientific use of antibiotics on these animal \n\nisolates.  This is the first study focusing on \n\nvancomycin MIC values of MRSA isolates \n\nobtained from these samples in this region and \n\nthe country as well. \n\nMaterials and methods \nSamples \nChicken eggs and chicken meat samples were \n\ncollected in sterile zipped plastic bags from \n\ndifferent retail outlets of Pokhara Metropolitan \n\nfrom January to February and July to August \n\n2019. The number of samples collected was 125 \n\nand 80 respectively for eggs and chicken meat.  \n\nSample processing \nEggshells were swabbed thoroughly using the \n\nsterile cotton swabs moistened with sterile \n\nnutrient broth and inoculated on Mannitol salt \n\nagar using aseptic technique. Similarly, 5 grams \n\nof each meat sample was transferred to 50 ml of \n\nsterile nutrient broth in a sterile environment and \n\nincubated overnight. Then one loopful of the \n\nbroth was inoculated on mannitol salt agar \n\naseptically. Sterile forceps, mask, and gloves \n\nwere used during sample processing to prevent \n\ncontamination during the procedure. The \n\ninoculated plates were then incubated at 37\u02daC for \n\n18-24 hrs.      \n\nIdentification of S aureus \nAfter incubation, the mannitol salt agar plates \n\nwere observed for yellow colonies. Isolated \n\nyellow colonies on mannitol salt agar were \n\nfurther tested using Gram-staining, catalase test, \n\noxidase test, and coagulase test. Gram-positive \n\ncocci in a cluster that was catalase-positive, \n\ncoagulase-positive were confirmed as S aureus as \n\nreported earlier [4].   \n\nAST of S. aureus isolates and MRSA \n\nscreening \nKirby Bauer disc diffusion method was used to \n\nstudy the antimicrobial susceptibility pattern of \n\nthe S. aureus isolates as per CLSI guidelines [14].", "start_char_idx": 3568, "end_char_idx": 7320, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c5aa02f8-8976-41a1-abe5-201d19475d2b": {"__data__": {"id_": "c5aa02f8-8976-41a1-abe5-201d19475d2b", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70e184db-e5f1-4759-8161-46be95bd935b", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "48c59fee61ffeccba728b15cfa19408202cb81326dd1a6d9c4982d3e95b0d984", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2dea3def-aead-4d22-9d80-9e9b80516e8c", "node_type": "1", "metadata": {}, "hash": "7ebcd24db6cf8eacff42e2d3f3675e0711932db21b07430a3f3a926d415828ff", "class_name": "RelatedNodeInfo"}}, "text": "Antibiotics used were amikacin (30\u00b5g), \n\nciprofloxacin (5\u00b5g), ampicillin (10\u00b5g), \n\namoxycillin (10\u00b5g), gentamicin (10\u00b5g), \n\nerythromycin (15\u00b5g), and cefoxitin (30\u00b5g). \n\nCefoxitin was used for the screening of MRSA \n\nisolates. Isolates with a zone of inhibition 21 mm \n\nor less were confirmed as MRSA isolates as per \n\nCLSI guidelines [14]. MRSA isolates were \n\npreserved using glycerol stock preparation and \n\nby stabbing on semi-solid media for vancomycin \n\nMIC detection. \n\nVancomycin MIC detection \nThe agar dilution method was used to detect MIC \n\nof the isolates. 526 mg of vancomycin powder \n\n(Hi-Media, India with potency 950 \u00b5g/mg) was \n\ndissolved in 10 ml sterile distilled water to \n\nprepare the stock solution (50 mg/ml). Then ten-\n\nfold dilutions (two times) of the stock were \n\nprepared to prepare working solutions having a \n\nconcentration of 0.5 mg/ml. From the working \n\nsolution, further dilutions were made on Brain \n\nheart infusion agar. Various dilutions of \n\nvancomycin prepared were 0.25\u00b5g/ml, \n\n0.5\u00b5g/ml, 1\u00b5g/ml, 2\u00b5g/ml, 4\u00b5g/ml, 8\u00b5g/ml, \n\n16\u00b5g/ml, and 32\u00b5g/ml as reported earlier [15]. \n\nStaphylococcus aureus ATCC 25923 was used as a \n\nnegative control. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 90-95                   Devkota et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  92 Nepjol.info/index.php/njb. \n\n \n\n \n\nMac-Farland standard (0.5) suspension of each \n\ntest isolates was prepared by mixing one or two \n\nisolated colonies on blood agar.  Each isolate was \n\nthen inoculated on Brain heart infusion agar \n\ncontaining various dilutions of vancomycin and \n\nincubated at 37\u00b0c for 24 hours and observed for \n\npresence or absence of growth as indicated \n\nearlier [16]. Lowest dilution of vancomycin \n\npreventing notable growth was considered as the  \n\n MIC value for that isolate. Isolates were \n\nclassified as vancomycin sensitive, intermediate, \n\nand resistant if the MIC value were \u22642\u00b5g/ml, 4-\n\n8\u00b5g/ml, and \u226516\u00b5g/ml respectively based on \n\nCLSI guidelines [14]. \n\nResults \n A total of 125 chicken eggs were processed \n\nduring the study period. Among them, 50 were \n\nanalyzed during winter and 75 during the \n\nsummer season. Similarly, 80 meat samples were \n\nincluded for the study (35 in winter and 45 in \n\nsummer). In winter isolation rate of S aureus from \n\neggs was 68 % (34 from 50 samples). Only 2 \n\nisolates (5.88%) were confirmed as MRSA. \n\nLikewise, in the same season, 21 S aureus was \n\nisolated from the meat sample. Among them, 5 \n\n(23.8%) were confirmed as MRSA (Table 1). \n\nThe prevalence of both S aureus and MRSA was \n\nslightly higher in summer. During this period \n\n73.33 % of egg samples were positive for S aureus. \n\nOnly four S aureus isolates (out of 55 total \n\nisolates) from egg were MRSA. Similarly, 64.44 % \n\nof chicken samples yielded S aureus. The \n\nincidence of MRSA was 24.13% in chicken S \n\naureus isolates (Table 1). \n\n Both vancomycin sensitive and vancomycin-\n\nintermediate MRSA isolates were detected both \n\nfrom egg and chicken samples. However, the \n\nMIC value was higher for meat isolates. MIC \n\nrange of vancomycin for egg isolates was 0.25 to \n\n4\u00b5g/ml.", "start_char_idx": 7323, "end_char_idx": 10475, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2dea3def-aead-4d22-9d80-9e9b80516e8c": {"__data__": {"id_": "2dea3def-aead-4d22-9d80-9e9b80516e8c", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c5aa02f8-8976-41a1-abe5-201d19475d2b", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "53194107abc352697abcb99eac74b212e2b99f3fd10f37837191776374dee7dc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "03907237-eed6-4301-9d4b-499094c3ea14", "node_type": "1", "metadata": {}, "hash": "af997a73d2af8d12b59ab7f0beecd45faebdc193bc0a271ff6f525a5c20e509d", "class_name": "RelatedNodeInfo"}}, "text": "Similarly, the MIC range was 0.25 to \n\n8\u00b5g/ml for chicken isolates (Table 3). The \n\nmajority of egg MRSA isolates were vancomycin \n\nsensitive while only 1 isolate (16.6%) isolates \n\nwere vancomycin-intermediate. 8 (66.66%) of the \n\nchicken meat isolates were sensitive to \n\nvancomycin and the remaining 4(33.33%) isolates \n\nwere vancomycin-intermediate. This result \n\nindicates that there is reduced vancomycin \n\nsusceptibility among MRSA isolates obtained \n\nfrom meat and egg. This increase in resistance \n\nmay be either due to the direct use of vancomycin \n\nin animal farms or due to the use of similar \n\nantibiotics for various purposes.  If we consume \n\nthese food products without proper cooking they \n\nmay cause infections that are difficult to treat. In \n\naddition to consumers, the workers working in \n\nslaughterhouse and retail meat shops are at high \n\nrisk of acquiring these pathogens as they work \n\nthere without proper safety measures. If the \n\ncurrent practices of animal farming, processing, \n\nand distribution of meat and egg samples \n\ncontinue, vancomycin resistance may be \n\nobserved in S aureus isolates from these food \n\nitems soon. Hence, all the concerned sectors \n\nshould be converged to minimize the increasing \n\ndrug-resistance among various food isolates. \n\nDiscussion \nAntibiotic resistance among animal and food \n\nanimal isolates has been increasing throughout \n\nthe world. This problem is more severe in \n\ndeveloping countries due to the lack of proper \n\nmonitoring of antibiotics use and the lack of \n\nawareness about the adverse effects of antibiotic  \n\nTable 1. Prevalence of MRSA isolates from \nmeat and egg samples \nStudy \n\nperiod \n\nSample Number S \n\naureus \n\nMRSA \n\nNo (%) \n\nJan-\n\nFeb \n\n2019 \n\nChicken \n\neggs \n50 34 \n\n2 \n\n(5.88%) \n\nChicken \n35 21 5 \n\n(23.8%) \n\nJul-\n\nAug \n\n2019 \n\nChicken \n\neggs \n75 55 \n\n4 \n\n(7.27%) \n\nChicken 45 29 \n\n7 \n\n(24.13%) \n\n \n\nTable 2: Resistance pattern of MRSA and \nMSSA isolates \n\nAntibiotics MRSA \n(N=18) \n\nMSSA \n(N=121) \n\nAmpicillin 11(61.1%) 21(17.3%) \nAmoxycillin 9(50%) 19(15.7%) \n   \nAmikacin 7 (38.8%) 12(9.9%) \nGentamicin 8 (44.4%) 11 (9%) \nCiprofloxacin 10 (55.5%) 13 (10.7%) \nErythromycin 10 (55.5%) 15 (12.3%) \nCo-trimoxazole 9 (50%) 14 (11.5%) \nCefoxitin 18 (100%) 0 (0%) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 90-95                   Devkota et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  93 Nepjol.info/index.php/njb. \n\n \n\n \n\nresistance. This problem is further complicated \n\nby the lack of proper hygiene during animal \n\nslaughter and the use of contaminated  \n\n equipment and water during the processing of \n\nmeat and eggs before sending it to the retail  \n\n market. Hence this study was designed to \n\nmonitor the antibiotic resistance pattern of MRSA \n\nand MSSA isolates obtained from meat and egg \n\nsamples as well as to access the vancomycin MIC \n\nvalue of the MRSA isolates obtained from these \n\nsamples. \n\nThe overall prevalence of S aureus among egg and \n\nchicken samples was 59.02% (121 out of 205 \n\nsamples). Among the isolates, 89 were from eggs \n\nand the remaining 50 isolates were from meat \n\nsamples. This result indicates gross \n\ncontamination of these food products with S \n\naureus and is the indication of poor hygiene \n\npractices. The very similar isolation rate of S \n\naureus was reported in Nigeria from meat and \n\nother food items [17].", "start_char_idx": 10476, "end_char_idx": 13848, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "03907237-eed6-4301-9d4b-499094c3ea14": {"__data__": {"id_": "03907237-eed6-4301-9d4b-499094c3ea14", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2dea3def-aead-4d22-9d80-9e9b80516e8c", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "ff29ccc1c4d5a72c1d096581a0d51cfbde20731e9c237cefa18581affbd593f9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "de24e966-d499-4c59-a936-4a5730d05316", "node_type": "1", "metadata": {}, "hash": "b43384daa034d20a570ad4cdbddd83e84fab8edde1d9c0a0b24d822a150c0b24", "class_name": "RelatedNodeInfo"}}, "text": "While the prevalence was \n\nsignificantly different in other studies [18, 19]. \n\nThe prevalence of this bacteria varies based on \n\ngeographic location and sanitation status.  The \n\naverage incidence of MRSA from both samples \n\nwas 12.94% (6.74% among egg isolates and 24% \n\namong chicken isolates). Almost identical \n\nincidence of MRSA was noted among chicken \n\nsamples from Nigeria [20]. On the other hand, a \n\nlow prevalence of MRSA was also noted from \n\nmeat and other food items in China [21], and no \n\nMRSA isolates at all from meat, egg, and other \n\nfood isolates in China [22] as indicated by the \n\nabsence of mecA gene in all isolates. The \n\nprevalence of MRSA from various meat and \n\nother food samples was comparatively high in \n\nAlgeria compared to this study [23]. \n\nAntibiotic resistance was quite high among \n\nMRSA isolates compared to MSSA isolates as the \n\nresistance range for the tested antibiotics was 9-\n\n17.3% for MSSA while the range was 38-61% for \n\nMRSA isolates.  Resistance was high for \n\nampicillin (61.1%), ciprofloxacin (55.5%), and \n\nerythromycin (55.5%) while low for gentamicin \n\n(44.4%) and amikacin (38.8%) for MRSA isolates. \n\nOne study from China [22] has shown increased \n\ndrug resistance of S aureus isolated from food \n\nitems as these isolates were resistant to penicillin \n\n(92.47%), erythromycin (58.06%), and kanamycin \n\n(25.8%). Another study has also reported \n\nelevated antibiotic resistance among S aureus \n\nisolated from raw meat samples collected from \n\nvarious provinces of China where the resistance \n\npattern was 83.7 % for penicillin, 52.1% for \n\nerythromycin, and 17.4 % for ciprofloxacin [24]. \n\nThese findings are comparable with the \n\nresistance pattern of isolates obtained in the \n\ncurrent study. \n\nTo the best of our knowledge, this is the first \n\nstudy reporting vancomycin MIC value of MRSA \n\nisolates obtained from food samples in Nepal. \n\nSimilarly, this is the first report indicating the \n\ndetection of vancomycin intermediate methicillin \n\nresistant S aureus isolated from retail chicken \n\nmeat and egg samples.  Similar to the finding of \n\nthis study various authors also have reported \n\nvancomycin-intermediate S aureus from various \n\nfood samples obtained from animals.  \n\nVancomycin intermediate isolates (MIC 8\u00b5g/ml) \n\nhave been detected in India from milk samples \n\n[25]. Not only this, several reports are indicating \n\nthe presence of vancomycin-resistant S aureus \n\n(VRSA) isolates from various food samples. The \n\nstudy of Bhattacharyya et al [25] reported \n\nvancomycin-resistant Staphylococcus from milk \n\nsamples. Likewise, VRSA isolates were reported \n\nfrom Egypt from diverse samples including \n\nminced meat, mastitis milk, and sausage samples \n\nwith vancomycin MIC range of 64-1024\u00b5g/ml \n\n[26].  VRSA also has been detected from camel \n\nmeat and slaughterhouse workers in Egypt with \n\na prevalence of 35% among S aureus isolates [27].  \n\nThough vancomycin resistance was not detected \n\nin this study further studies are very necessary \n\nthroughout the nation including more sample \n\nsize and diverse samples as only large scale study \n\ncan provide an actual picture of this problem.", "start_char_idx": 13849, "end_char_idx": 17010, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "de24e966-d499-4c59-a936-4a5730d05316": {"__data__": {"id_": "de24e966-d499-4c59-a936-4a5730d05316", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "03907237-eed6-4301-9d4b-499094c3ea14", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "e55ee993745bb985ed63442e4be27bf59ed211f2a20c0ab41686c0ebcb0fc60c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a79caca0-cb69-4f9c-b1ff-ab72ac9e93bb", "node_type": "1", "metadata": {}, "hash": "daf1370eddb71ec282f4e9c35c2adbf8eee9122716aed9deaecea665f428d039", "class_name": "RelatedNodeInfo"}}, "text": "Timely surveillance of these pathogens is also \n\nTable 3: Vancomycin Minimum inhibitory concentration (MIC) values of MRSA isolates \n\nSample Total \nisolates \n\n0.25\u00b5g/ml \nNo (%) \n\n0.5\u00b5g/ml \nNo (%) \n\n1\u00b5g/ml \nNo (%) \n\n2\u00b5g/ml \nNo (%) \n\n4\u00b5g/ml \nNo (%) \n\n8\u00b5g/ml \nNo (%) \n\n16\u00b5g/ml \nNo (%) \n\n32\u00b5g/ml \nNo (%) \n\nEgg  6 1 (16.6) 1(16.6) 2 (33.3) 1(16.6) 1(16.6) 0 (0) 0 (0) 0 (0) \n\nMeat 12 1 (8.33) 2 (16.6) 3 (25) 2 (16.6) 2 (16.6) 2 (16.6) 0 (0) 0 (0) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 90-95                   Devkota et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  94 Nepjol.info/index.php/njb. \n\n \n\n \n\nnecessary. Physician and microbiology labs \n\nshould not completely rely on vancomycin disc \n\ntest for the susceptibility of MRSA isolates and \n\nmust determine the vancomycin MIC values \n\nbefore treatment. They should be updated with \n\nthe recent susceptibility trend of these isolates of \n\nthe particular locality as well.  \n\nConclusion \nThis study reported the existence of vancomycin-\n\nintermediate S aureus isolates in Nepal and \n\ntypically in Pokhara from meat and egg samples. \n\nRise of vancomycin-intermediate S aureus \n\nisolates among various food isolates is a serious \n\nconcern as there is a possibility of transmission of \n\nthese isolates to the consumers. Hence, further \n\nepidemiological and molecular analyses are \n\nneeded to prevent their spread to humans. \n\nSimilarly, the prudent use of antibiotics in animal \n\nfarms is urgent. \n\nLimitations of the study \nVancomycin MIC of the MSSA isolates couldn\u2019t \n\nbe performed. Similarly, molecular analysis \n\n(mecA gene detection) of the MRSA isolates was \n\nnot done due to limited laboratory facilities and \n\neconomical constrain. \n\nAcknowledgment \nWe are very thankful to Pokhara Bigyan Tatha \n\nPrabdhi Campus, Pokhara for allowing us to \n\nconduct this research in their laboratory and all \n\nthe Microbiology lab staff for their continuous \n\nsupport.  \n\nFunding  \nThis study was carried out without any financial \n\nsupport.  \n\nReference \n1. Sato T, Usui M, Konishi N, Kai A, Matsui H, \n\nHanaki H, et al: Closely related methicillin-\nresistant Staphylococcus aureus isolates from \nretail meat, cows with mastitis, and humans in \nJapan. PLoS ONE. 2017 12(10): e0187319. \n\n2. Kadariya J, Smith TC, Thapaliya D: \nStaphylococcus aureus and Staphylococcal \nFood-Borne Disease: An Ongoing Challenge in \nPublic Health.  BioMed Res Int. 2014 Article \nID 827965, 9 pages \n\n3. Lozano C, Gharsa H, Slama KB, Zarazaga \nM, Torres C: Staphylococcus aureus in Animals \nand Food: Methicillin Resistance, Prevalence \nand Population Structure. A Review in the \n\nAfrican Continent. Microorganisms. 2016, 4(1): \n12. \n\n4. Pondit A, Haque ZF, Al MSA, Khan SR, Saha S: \nCharacterization of Staphylococcus \naureus isolated from chicken and quail \neggshell. J Adv Vet Anim Res. 2018 5(4): 466-471. \n\n5.", "start_char_idx": 17013, "end_char_idx": 19884, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a79caca0-cb69-4f9c-b1ff-ab72ac9e93bb": {"__data__": {"id_": "a79caca0-cb69-4f9c-b1ff-ab72ac9e93bb", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "de24e966-d499-4c59-a936-4a5730d05316", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "7c2397660654bc1fff2426e2090e929712293c397a964b02bb67cafbae9b78e5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c5fcbdcf-0c44-4d00-b6b6-2c029aa62739", "node_type": "1", "metadata": {}, "hash": "e9a31a7d4ec7ce2d472f8d079cf4ef44747b96dd803c768efe6b9042cfa4a9e1", "class_name": "RelatedNodeInfo"}}, "text": "2018 5(4): 466-471. \n\n5. Ruban SW, Babu RN, Abraham RJJ, \nSenthilkumar TMA, Kumraswamy P,bRao VA: \nPrevalence of Methicillin Resistant \nStaphylococcus aureus in retail Buffalo meat in \nChennai, India. Buffalo Bull. 2018 37(1): 51-58. \n\n6. Sergelidis D, Abrahim A, Anagnostou V: \nPrevalence, Distribution, and Antimicrobial \nSusceptibility of Staphylococcus aureus in \nReady-to-Eat Salads and in the Environment of \na Salad Manufacturing Plant in Northern \nGreece. Czech J. Food Sci. 2012, 30(3): 285-291. \n\n7. Chon J, Sung K, Khan S: Methicillin-\nresistant Staphylococcus aureus (MRSA) in \nfood-producing and companion animals and \nfood products. Eds Enany S, Alexander LC In \nFrontiers in Staphylococcus aureus. 2017 48-101. \n\n8. Fan Y, Li SM, Deng BG and Zhao YX:  Prevalence \nand relevance analysis of multidrug-resistant \nStaphylococcus aureus of meat, poultry and \nhuman origin. Indian J Anim Res. 2015 49 (1): 86-\n90. \n\n9. Kshetry AO, Pant ND, Bhandari R, Khatri S, \nShrestha KL, Upadhaya SK et al: Minimum \ninhibitory concentration of vancomycin to \nmethicillin resistant Staphylococcus aureus \nisolated from different clinical samples at a \ntertiary care hospital in Nepal. Antimicrob Resist \nInfect Control. 2016 5:27. \n\n10. Adhikari R, Pant ND, Neupane S, Neupane M, \nBhattarai R, Bhatta S, et al: Detection of MRSA \nand determination of MIC concentration of \nvancomycin for S aureus isolated fron pus/ \nwound swab samples of the patient attending a \ntertiary care hosprtal in Kathmandu, Nepal. Can \nJ Infect Dis Med Microbiol. 2017 2017: 2191532. \n\n11. Lama U, Shah D, Shrestha U: Vancomycin \nResistant Staphylococcus aureus Reported \nfrom Tertiary Care Hospital in Nepal. TUJM. \n2018, 4: 63-72. \n\n12. Khanal LK, Adhikari RM, Guragain A: \nPrevalence of MRSA and Antibiotic \nsusceptibility pattern in a tertiary care Hospital \nin Nepal. J Nepal Health Res Counc. 2018 16(2):172-\n174. \n\n13. Bhatta DR, Cavaco LM, Nath G, Gaur A, Gokhale \nS, Bhatta DR: Threat of multidrug resistant \nStaphylococcus aureus in Western Nepal. Asian \nPac J Trop Dis. 2015 5(8): 617-21. \n\n14. Clinical and Laboratory Standards Institute: \nPerformance standards for antimicrobial \nsusceptibility testing, twenty-second \ninformational supplement, CLSI, Document \nM100 S27, CLSI, Wayne, Pa, USA, 2017. \n\n15. Othman HB, Halim RMA, Gomaa FAM, Amer \nMZ: Vancomycin MIC distribution among \nMethicillin-Resistant Staphylococcus aureus.", "start_char_idx": 19860, "end_char_idx": 22274, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c5fcbdcf-0c44-4d00-b6b6-2c029aa62739": {"__data__": {"id_": "c5fcbdcf-0c44-4d00-b6b6-2c029aa62739", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a79caca0-cb69-4f9c-b1ff-ab72ac9e93bb", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "424e9250634351ce9c2036339bf661941bd2f9d497a52eca1cf2a149b7d7c963", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "00f26fa0-4843-4589-b43f-19ca18c12a3d", "node_type": "1", "metadata": {}, "hash": "241f18b39d806501e9113e1b0695f002c7f77de2bdedd89e3c063522cb53bb15", "class_name": "RelatedNodeInfo"}}, "text": "Is \n\nhttps://www.hindawi.com/83904342/\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Lozano%20C%5BAuthor%5D&cauthor=true&cauthor_uid=27681906\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Gharsa%20H%5BAuthor%5D&cauthor=true&cauthor_uid=27681906\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Ben%20Slama%20K%5BAuthor%5D&cauthor=true&cauthor_uid=27681906\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Zarazaga%20M%5BAuthor%5D&cauthor=true&cauthor_uid=27681906\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Torres%20C%5BAuthor%5D&cauthor=true&cauthor_uid=27681906\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029517/\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Kshetry%20AO%5BAuthor%5D&cauthor=true&cauthor_uid=27446531\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Pant%20ND%5BAuthor%5D&cauthor=true&cauthor_uid=27446531\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Bhandari%20R%5BAuthor%5D&cauthor=true&cauthor_uid=27446531\nhttps://www.ncbi.nlm.nih.gov/pubmed/27446531\nhttps://www.ncbi.nlm.nih.gov/pubmed/27446531\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Adhikari%20R%5BAuthor%5D&cauthor=true&cauthor_uid=28154581\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Pant%20ND%5BAuthor%5D&cauthor=true&cauthor_uid=28154581\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Neupane%20S%5BAuthor%5D&cauthor=true&cauthor_uid=28154581\nhttps://www.ncbi.nlm.nih.gov/pubmed/28154581\nhttps://www.ncbi.nlm.nih.gov/pubmed/28154581\nhttps://www.ncbi.nlm.nih.gov/pubmed/29983432\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Othman%20HB%5BAuthor%5D&cauthor=true&cauthor_uid=30740152\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Halim%20RMA%5BAuthor%5D&cauthor=true&cauthor_uid=30740152\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Gomaa%20FAM%5BAuthor%5D&cauthor=true&cauthor_uid=30740152\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Amer%20MZ%5BAuthor%5D&cauthor=true&cauthor_uid=30740152\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Amer%20MZ%5BAuthor%5D&cauthor=true&cauthor_uid=30740152\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 90-95                   Devkota et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  95 Nepjol.info/index.php/njb. \n\n \n\n \n\nreduced vancomycin susceptibility related to \nMIC creep?  Maced J Med Sci. 2018 7(1):12-18 \n\n16. Chaudhari CN, Tandel K, Grover N, Bhatt P, \nSahni AK, Sen S, et al: In vitro vancomycin \nsusceptibility amongst methicillin resistant \nStaphylococcus aureus. Med J Armed Forces India. \n2014 70(3): 215-9. \n\n17. Sokari T: Distribution of entero-\ntoxigenic Staphylococcus aureus in ready-to-eat \nfoods in eastern Nigeria. Int J Food \nMicrobiol. 1991, 12:275-279. \n\n18.", "start_char_idx": 22275, "end_char_idx": 24844, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "00f26fa0-4843-4589-b43f-19ca18c12a3d": {"__data__": {"id_": "00f26fa0-4843-4589-b43f-19ca18c12a3d", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c5fcbdcf-0c44-4d00-b6b6-2c029aa62739", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "9723f0f15521ac22d84c3fdc484cd666e969f6bbd0a8cc78e2a0d3bc6cf5d4fb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f5526779-b1d8-4508-824b-0b43e9694c76", "node_type": "1", "metadata": {}, "hash": "0fb226a96ada7c11549fed66ed17d84df336c8746eccff467e74eaf995d66ec5", "class_name": "RelatedNodeInfo"}}, "text": "1991, 12:275-279. \n\n18. Wu S, Huang J, Wu Q, Zhang J, Zhang F, Yang X \net al: Staphylococcus aureus Isolated From \nRetail Meat and Meat Products in China: \nIncidence, Antibiotic Resistance and Genetic \nDiversity. Front Microbiol. 2018 9: 2767. \n\n19. Higenyi J, Kabasa JD: Microbial contamination \nload of hatching eggs in Butaleja, eastern \nUganda. Anim Vet Sci. 2014 2: 22-30. \n\n20. Igbinosa EO, Beshiru A, Akporehe LU, Oviasogie \nFE, Igbinosa OO: Prevalence of Methicillin-\nResistant Staphylococcus aureus and \nOther Staphylococcus Species in Raw Meat \nSamples Intended for Human Consumption in \nBenin City, Nigeria: Implications for Public \nHealth. Int J Environ Res Public Health. 2016 \n13(10): 949. \n\n21. Wu S, Huang J, Zhang F, Wu Q, Zhang J, Pang R \net al: Prevalence and characterization of food \nrelated methicillin resistant Staphylococcus \n\naureus (MRSA) in China. Front Microbiol. 2019 \n10:1-13. \n\n22. Ma Y, Zhao Y, Tang J, et al: Antimicrobial \nsusceptibility and presence of resistance & \nenterotoxins/enterotoxin-likes genes \nin Staphylococcus aureus from food. CyTA - \nJournal of Food. 2018, 16(1): 76-84. \n\n23. Chaalal W, Chaalal N, Bourafa N, et al:  \nCharacterization of Staphylococcus \naureus isolated from food products in Western \nAlgeria. Foodborne Pathog. Dis. 2018, 15: 353\u2013\n360. \n\n24. Wang W, Baloch Z, Jiang T, et al: \nEnterotoxigenicity and antimicrobial resistance \nof Staphylococcus aureus isolated from retail \nfood in China. Front. Microbiol. 2017, 8:2256.  \n\n25. Bhattacharyya D, Banerjee J, Bandyopadhyay S, \net al: First report on vancomycin-resistant \nStaphylococcus in Bovine and Caprine milk. \nMicrob Drug Resist. 2016, 22(8): 675-681. \n\n26. Abd El-Aziz NK, Abd El-Hamid MI, Bendary \nMM, et al: Existence of vancomycin resistance \namong methicillin resistant S aurues recovered \nfrom animal and human sources in Egypt. Slov \nVet Res. 2018, 55 (Suppl 20): 221-30. \n\n27. Al-Amery K, Elhariri M, Elsayed A, et al: \nVancomycin-resistant Staphyloocus aureus \nisolated from camel meat and slaughterhouse \nworkers in Egypt. Antimicrob Resist Infect \nControl.", "start_char_idx": 24821, "end_char_idx": 26910, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f5526779-b1d8-4508-824b-0b43e9694c76": {"__data__": {"id_": "f5526779-b1d8-4508-824b-0b43e9694c76", "embedding": null, "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-182", "node_type": "4", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "2a5d02eaf8a2d8e4a33e2f9070d403a0bd4ae35c8a0b3dc2e45041c34ef08710", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "00f26fa0-4843-4589-b43f-19ca18c12a3d", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "53193293c0fcdd62b0b6c25fcff17ec9dd04981803b317533da8d58de1e56470", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bf090bd4-6fb3-4df5-90ae-2671bcb785b7", "node_type": "1", "metadata": {}, "hash": "fca889aeec8dc2dc4abcfe13b79d3b1045bcc9cc10c8e733903c9d3d4b833287", "class_name": "RelatedNodeInfo"}}, "text": "Antimicrob Resist Infect \nControl. 2019 5(8):129 \n\n \n\nhttps://www.ncbi.nlm.nih.gov/pubmed/30740152\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Wu%20S%5BAuthor%5D&cauthor=true&cauthor_uid=30498486\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Huang%20J%5BAuthor%5D&cauthor=true&cauthor_uid=30498486\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Wu%20Q%5BAuthor%5D&cauthor=true&cauthor_uid=30498486\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249422/\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Igbinosa%20EO%5BAuthor%5D&cauthor=true&cauthor_uid=27669285\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Beshiru%20A%5BAuthor%5D&cauthor=true&cauthor_uid=27669285\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Akporehe%20LU%5BAuthor%5D&cauthor=true&cauthor_uid=27669285\nhttps://www.ncbi.nlm.nih.gov/pubmed/27669285\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Bhattacharyya%20D%5BAuthor%5D&cauthor=true&cauthor_uid=26990514\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Banerjee%20J%5BAuthor%5D&cauthor=true&cauthor_uid=26990514\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Bandyopadhyay%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26990514\nhttps://www.ncbi.nlm.nih.gov/pubmed/26990514\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Al-Amery%20K%5BAuthor%5D&cauthor=true&cauthor_uid=31404199\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Elhariri%20M%5BAuthor%5D&cauthor=true&cauthor_uid=31404199\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Elsayed%20A%5BAuthor%5D&cauthor=true&cauthor_uid=31404199\nhttps://www.ncbi.nlm.nih.gov/pubmed/31404199\nhttps://www.ncbi.nlm.nih.gov/pubmed/31404199", "start_char_idx": 26876, "end_char_idx": 28415, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bf090bd4-6fb3-4df5-90ae-2671bcb785b7": {"__data__": {"id_": "bf090bd4-6fb3-4df5-90ae-2671bcb785b7", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f5526779-b1d8-4508-824b-0b43e9694c76", "node_type": "1", "metadata": {"identifier": "njb-182", "author": "Devkota, Surya Prasad; Paudel, Ashmita; Gurung, Krishna", "title": "Vancomycin Intermediate MRSA Isolates Obtained from Retail Chicken Meat and Eggs Collected at Pokhara, Nepal", "date": "2019-12-29", "file": "njb-182.pdf"}, "hash": "90a41d02db7072e53b37103446869f2115538ff9dc09469f021a71dc09a30989", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "658baf2c-e52c-4b48-86bd-10f8c2b935c4", "node_type": "1", "metadata": {}, "hash": "62afdc60f7278da956f3f61c87eae3f45d55e686b975c0b783825b876bbbad99", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, Biotechnology Society of Nepal  39 Nepjol.info/index.php/njb. \n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49   DOI: https://doi.org/10.3126/njb.v7i1.26950 \n\nORIGINAL RESEARCH ARTICLE \n\n \n\nIsolation, Identification and Production of Encapsulated \nBradyrhizobium japonicum and Study on their Viability \n\nTil Kumari Chhetri1, Bijay Raj Subedee2, Bijaya Pant1* \n\n1Central Department of Botany, Tribhuvan University, Kathmandu, Nepal \n2Research Centre for Applied Science and Technology, Tribhuvan University, Kathmandu, Nepal \n\nAbstract \nRhizobium, a nitrogen-fixing bacteria is the essential feature of leguminous plants which is \n\nessential for the regeneration of nutrient-deficient soil. This study was aimed to isolate, identify, \n\nmass culture and immobilize Bradyrhizoium japonicum in encapsulated form and test their \n\nviability. Root nodules were sterilized, grinded and cultured aseptically in YEMA media \n\ncontaining Congo red. The obtained colon was sub-cultured to get a pure culture and different \n\nbiochemical tests were conducted which proved Bradyrhizobium japonicum as the slow-growing \n\nspecies. The test shows a positive result of catalase production and nodulation test whereas the \n\npH tolerance test shows more tolerance to the acidic pH. Similarly, Bradyrhizaobium japonicum can \n\ntolerate 1% and 2% NaCl concentration and it doesn\u2019t show resistance to the penicillin disc of \n\n10mg. The mass culture and encapsulation with sodium alginate adding sucrose as nutrient \n\nproved the simplicity for handling. Altogether 548 beads were prepared from the 100ml of the \n\ncultured broths which were viable for more than 190 days at 1%, 2% and 3% sucrose concentration \n\nbut less viable at 5% and 10% sucrose concentration under room temperature. \n\nKeywords: Bradyrhizobium, encapsulation, immobilization, viability, Legumes, symbiotic bacteria \n\n*Corresponding Author \n\nEmail: bijayapant@gmail.com\n\nIntroduction \nA distinctive characteristic of the majority of \n\nlegumes is their ability to enter into a nitrogen-\n\nfixing symbiosis with a distinct group of soil \n\nbacteria collectively called root nodules bacteria \n\nor the Rhizobia [1,2]. The Rhizobia reduce the \n\natmospheric nitrogen into ammonium which is \n\ntermed as the biological nitrogen fixation and is \n\nmore advantageous in the perspective of soil \n\nquality. The productivity and sustainability of \n\nagriculture throughout the globe are being \n\nsignificantly enhanced through nitrogen fixation \n\nfrom effectively nodulated legumes [3]. \n\nHowever, only certain combinations of legumes \n\nand Rhizobia result in the formation of effective \n\nnitrogen-fixing nodules even though many \n\nmoderately effective and ineffective \n\ncombinations may and do arise. Thus the \n\nBradirhizobium japonicum is host specific and \n\nnodulate only the species of soybean. Apart from \n\ndirect benefit from effective nitrogen fixation [4] \n\nlegumes and Rhizobium provides added value in \n\nweed, pathogen and insect control when rotated \n\nwith crop in farming system [5] together with \n\nimproving soil structure and increasing soil \n\norganic matter content [6]. \n\nThe importance of legume crops to world \n\nproduction and compelling needs to exploit the \n\nnitrogen fixing potential of those crops have \n\nfocused attention on technologies for the \n\nproduction of more effective legume inoculants. \n\nMost legume inoculants have been prepared by \n\nadsorbing broth culture of selected Rhizobia on a \n\nsuitable carrier such as peat, clays, charcoal, \n\nlignite, cellulose powder, various powdered crop \n\nresidues or soil compost mixtures. In 1979, \n\nDommergues et al. [7] proposed to entrap rather \n\nthan adsorb Rhizobium cells by incorporating the \n\nbacteria in a polymeric gel. The encapsulation of \n\nthe inoculants with polyacrylamide maintained \n\nthe suitable moisture content.", "start_char_idx": 47, "end_char_idx": 3899, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "658baf2c-e52c-4b48-86bd-10f8c2b935c4": {"__data__": {"id_": "658baf2c-e52c-4b48-86bd-10f8c2b935c4", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bf090bd4-6fb3-4df5-90ae-2671bcb785b7", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "8a2f0bb2a13f2ceb6f2c08ad3a5a4f516008a9534621637246b9a6f4dc76502d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b8c1b6b3-2d0b-4f44-a065-825a9a71c50a", "node_type": "1", "metadata": {}, "hash": "fd3e0d5594e0c89b201a13ee88af7c3b811e2db9e7f409573f1a9081fada7ce0", "class_name": "RelatedNodeInfo"}}, "text": "These formulations \n\nof immobilized cells protect the microorganism \n\nagainst the environmental stresses and release \n\nthem to the soil gradually when the polymers are \n\ndegraded [8]. Increasing the efficiency of the use \n\nof available soil nitrogen can meet the additional \n\nNitrogen demand by making cereal plants \n\ncapable of fixing its own nitrogen through close \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  40 Nepjol.info/index.php/njb. \n\n \n\n \n\nassociation with diazotrophic bacteria will pay \n\noff in term of increasing cereal production and \n\nhelping resource poor farmers as well as saving \n\nthe environment [9]. \n\nSymbiotic Nitrogen fixation is an important \n\nsource of nitrogen and the various legumes crops \n\nand pasture species often fix as much as 200-300 \n\nkg Nitrogen per hectare [10]. Globally, symbiotic \n\nnitrogen fixation has been estimated to amount to \n\nat least 70 million metric tons of nitrogen per year \n\n[11]. In 1990, world consumption of fertilizer \n\nNitrogen is 88 million tones and apart from the \n\nconsumption of nonrenewable energy sources, \n\nenvironmental pollution from fertilizer Nitrogen \n\nescaping the root zones is high because in many \n\ncases Nitrogen fertilizers are not used efficiently \n\nby crops [10]. Therefore biological nitrogen \n\nfixation is an important and integral component \n\nof sustainable agricultural system. Furthermore, \n\nbiological nitrogen fixation from legumes offers \n\nmore flexible management than fertilizer \n\nnitrogen because the pool of the organic Nitrogen \n\nbecomes slowly available to non-legumes species \n\n[10]. Concomitant with Nitrogen fixation, the \n\nlegumes in rotation offers the control of crop \n\ndisease and pests [3,12]. The Bellagio conference \n\non N2 fixation [13] acknowledged that with the \n\ndecline in the price of manufactured fertilizer in \n\n1990s, biological nitrogen fixation with legumes \n\nand Rhizobia, was most likely to remain in \n\nextensive rather than intensive agricultural \n\nsystems. Thus the present study is emphasized \n\nfor the mass production and immobilization of \n\nRhizobial inoculants in the most effective and \n\ncost effective ways of encapsulation. \n\n Rhizobia are the gram negative, rod shaped, \n\naerobic and heterotrophic soil bacteria, which \n\nincludes genera Rhizobium, Bradyrhizobium, \n\nSinorhizobium, Mesorhizobium, Allorhizobium, and \n\nAzorhizobium, which are able to form symbiosis \n\nwith leguminous plants. They are facultative \n\nsymbionts that have adapted to persist for long \n\nperiod in soil in a free living state if the suitable \n\nlegume host is absent [2].  They could form the \n\nspecialized organs, called nodules, on roots or \n\nstems of their hosts. Rhizobia inside nodule could \n\nreduce atmospheric nitrogen and make it \n\navailable to the plant. Symbiotic rhizobia are \n\ncommon colonizers of the rhizosphere of both \n\nlegume and non-legume plants and in addition to \n\nlegumes they are also endophytes of several non-\n\nlegumes like rice and maize [14]. However, non-\n\nsymbiotic rhizobia can also be present in soil [15].  \n\nIn the old system of classification the Rhizobium \n\nfall into two groups based on their growth \n\ncharacteristics i.e. fast growing and the slow \n\ngrowing Rhizobium. Fast growing Rhizobium are \n\nacid producers which develop pronounced \n\nturbidity in liquid media within 2-3 days and \n\nhave the mean doubling time of 2-4 hours. The \n\ncells are rod shaped to pleomorphic, 0.5-0.9 \n\nmicrons in diameter and 1.2 to 3.0 micron long, \n\nand are motile by 2-6 peritrichous flagella. \n\nWhereas slow growing Rhizobium are alkali \n\nproducing Rhizobia and require 3-5 days to \n\nproduce moderate turbidity in liquid media and \n\nhave the mean doubling time of 6-7 hours.", "start_char_idx": 3900, "end_char_idx": 7711, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b8c1b6b3-2d0b-4f44-a065-825a9a71c50a": {"__data__": {"id_": "b8c1b6b3-2d0b-4f44-a065-825a9a71c50a", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "658baf2c-e52c-4b48-86bd-10f8c2b935c4", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "9736ca184f9cd123de3f181547416eefe63c6ddada9130c753ba3a1a0c161765", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "548db623-3f04-416e-a4c8-1953ad27358d", "node_type": "1", "metadata": {}, "hash": "0721c85b7316ca69ffc7c397b80af485df7e91559d6e9bb32f19d29d0dbdb65c", "class_name": "RelatedNodeInfo"}}, "text": "The \n\ncells are predominantly rod shaped and motile \n\nby a single polar or sub-polar flagellum [16]. \n\nSince 1886, when it was discovered that bacteria \n\ncaused the formation of the nitrogen-fixing \n\nnodules; then, the isolation of rhizobia from the \n\nnodules as pure cultures opened the way for \n\nartificial inoculation to replace the \u2018soil transfer\u2019 \n\nmethod, in which dry soil, from a location where \n\nthe legume had been grown previously, was \n\ncoated onto the seed just before sowing [17]. This \n\ndust method was modified to the \u201csoil-paste or \n\nmuddy water process\u201d, in which the soil was \n\nmixed with water before pouring over the seed \n\n[18]. The first commercial pure (agar) culture \n\ninoculants have been patented by Nobbe and \n\nHiltner in 1896 [44]. Their patented culture was \n\nplaced on the market under the name Nitragin, \n\nwhich consist of a pure culture of desired strain \n\nof rhizobia grown in flat glass bottle containing \n\nonly a small amount of solid gelatin medium. \n\nThis material was either to be applied to seed or \n\nmixed with soil and spread over the field [19]. \n\nThen, solid carrier such as soil or peat was first \n\nsuggested in 1914 [17]. Present day inoculants \n\nproduction techniques have been changed from \n\nthose of the early 1900s. Even many types of \n\ninoculant have been investigated; peat is the best \n\ncarrier and is widely accepted in the inoculant \n\nindustry. However, the challenge today is to \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  41 Nepjol.info/index.php/njb. \n\n \n\n \n\ndevelop further improved inoculant \n\nformulations and methods of application. \n\nIn Nepal, Rhizobial inoculants has been used \n\nfrom few years. Rhizobial inoculants had been \n\nproduced in soil science department of Nepal \n\nAgricultural Research Council. This was studied \n\nand conducted by Sanu kesheri Bajracharya. \n\nPowder inoculums were made in soil and goal \n\nmixture in 3:1 ratio. But for the research proposes \n\nliquid inoculums is being used. The work was \n\nperformed under the supervision of Soil Science \n\nDepartment and Farmer Centered Agricultural \n\nResource Management (FARM), Asian Bio-\n\nTechnology and Bio- Diversity Sub\u2013Program \n\nNepal (Annual reports of Soil Science \n\nDepartment of Nepal Agricultural Research \n\nCouncil). Some research has been done on the \n\neffect of the peat based inoculums of the \n\nBradyrhizobium japonicum on the Glycine max in \n\nthe university researches. \n\nAlthough rhizobia seem to be widely distributed \n\nin the soil, however soil in different places \n\ncontains different strain of rhizobia and these \n\nrhizobia may not be effective for nitrogen \n\nfixation, and may not be appropriate for all \n\nlegume. Some soil may have effective rhizobial \n\nstrain, but the number of rhizobia is low or \n\ncontaining higher number of ineffective strain \n\n[20]. Inoculation of legume seed is a simple and \n\npractical means of ensuring effective nitrogen \n\nfixation. However, to answer the question \u201cIs \n\ninoculation of seed necessary?\u201d is critical, even \n\nthe use of rhizobial inoculant is not necessary in \n\nthat area. Therefore, Allen [21] has listed four \n\nindicators that, if positive, the inoculation would \n\nbe beneficial i.e. the absence of the same or \n\nsymbiotically-related legume in the Immediate \n\npast history of   the land ; Poor nodulation when \n\nthe same crop was grown on the land previously; \n\nwhen the legume followed a non-legume in the \n\nrotation and when the land was undergoing \n\nreclamation. \n\nRhizobial inoculants can be immobilized in \n\ndifferent materials. The material for peat based \n\ncarrier is obtained from a naturally occurring \n\norganic material. The supply of peat based \n\norganic material is limited.", "start_char_idx": 7712, "end_char_idx": 11505, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "548db623-3f04-416e-a4c8-1953ad27358d": {"__data__": {"id_": "548db623-3f04-416e-a4c8-1953ad27358d", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b8c1b6b3-2d0b-4f44-a065-825a9a71c50a", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "247301a040426db7c955ceb908334f8fce35f6127b677adb62e05efe1d3e67d2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1a4a4de7-d53c-43ed-879f-ef840fe536a2", "node_type": "1", "metadata": {}, "hash": "54cc0a42b65c1ed7b38067da6065f766ab6cc981a2938286548002582e99987d", "class_name": "RelatedNodeInfo"}}, "text": "The supply of peat based \n\norganic material is limited. Even other solid \n\nmaterials such as lignite, charcoal, coir dust and \n\ncompost of various agricultural wastes have been \n\nused instead of peat but their performance \n\ncharacteristics are not equivalent to peat based \n\ninoculants product [22]. Therefore it is important \n\nto immobilize the Rhizobium in any other suitable \n\nform as sodium alginate encapsulation. In solid \n\nand liquid inoculants three basic contaminant \n\ntypes were observed, such as bacteria, \n\nactinomycetes, and fungi. These include the \n\npossibilities of pathogenicity to human, animal, \n\nplant or rhizobia, which reduce the effectiveness \n\nof inoculant [23]. Thus it is necessary to \n\nimmobilize the bacterial cells in the form of \n\nencapsulated beads made in aseptic condition \n\nwhich prevents the contaminants and well as \n\npreserved the cells for several months without \n\nlosing their viability. Also the encapsulated \n\nbeads are easy to handle, to use and to do \n\npackaging and distribute to the farmers. \n\nUnbalanced use of chemical fertilizers had led to \n\na reduction in soil fertility and to environmental \n\ndegradation [24] and the cost of chemical \n\nfertilizers has increased so that it is unaffordable \n\nfor farmer of developing country such as Nepal. \n\nAs a consequent, there has recently been a \n\ngrowing level of interest in environmentally \n\nfriendly sustainable agricultural practices \n\nincluding organic farming systems [25]. For \n\nexample, Rhizobium and phosphate solubilizing \n\nmicroorganisms would reduce the need for N2 \n\nand P chemical fertilizers and decrease adverse \n\nenvironmental effects. Therefore, in the \n\ndevelopment and implementation of sustainable \n\nagriculture techniques, bio-fertilization is of great \n\nimportance in alleviating environmental \n\npollution and the deterioration of nature [26]. A \n\ntightening of the agricultural N2 cycling to reduce \n\nN losses and an increase of N2 inputs through \n\nBNF to replace artificial fertilizer N2 use can help \n\nachieve this goal while at the same time \n\nmaintaining agricultural production and \n\nreducing greenhouse gas emissions and energy \n\nconsumption for the production of artificial N \n\nfertilizers [27-29]. \n\nMaterials and Methods \nThe materials used for the present study were \n\nroot nodules of Glycine max (white seeded \n\nspecies) grown at the earthen pot. The seed of \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  42 Nepjol.info/index.php/njb. \n\n \n\n \n\nGlycine max were taken from the market for the \n\ntest. The necessary equipment and the chemicals \n\nrequired for the completion of the research were \n\nprovided from the Biotechnology and \n\nBiochemistry unit of Central Department of \n\nBotany. \n\nPreparation of YEMA media [30]: All the \n\ningredients required for the preparation of the \n\nYEMA media except agar and Congo red were \n\ndissolved in the 950 ml of the sterilized water. \n\nCongo red was dissolved separately in the next \n\nconical flask in 50 ml of water and sterilized them \n\nseparately. Then pH was maintained to 6.8-7.0. \n\nAgar was added in the mixture of the ingredients \n\nand sterilized in Autoclave at 121 degree Celsius \n\nand 15 lb. pressure for fifteen minutes. From the \n\nAutoclave, media was directly taken to the \n\nLaminar Air Flow Chamber and Congo red was \n\nmixed with the ingredients mixture and poured \n\nin the sterilized petri plates and allowed it to cool \n\ndown. Finally the media was ready for the \n\ninoculation of the Rhizobia. \n\nIsolation of Bradyrhizobium japonicum \nCollection of root nodules: The roots of the \n\nsoybean (white seeded species) were collected \n\nfrom Putalisadak, Kathmandu which were \n\ncultivated in the pots at the rooftops. The soil \n\nfrom the root was removed by washing with tap \n\nwater. Then only the fresh, turbid, matured and \n\npinkish colored nodules were selected and \n\ncollected on the beaker. Only 0.2 gm.", "start_char_idx": 11450, "end_char_idx": 15442, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1a4a4de7-d53c-43ed-879f-ef840fe536a2": {"__data__": {"id_": "1a4a4de7-d53c-43ed-879f-ef840fe536a2", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "548db623-3f04-416e-a4c8-1953ad27358d", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "5f95de618492fb7daf20599327b0e2ab87bdc8b9d2ff24317ac837643c4e3504", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6e0a0c3c-aa47-4e18-bf19-23807470aa56", "node_type": "1", "metadata": {}, "hash": "1f3ec8fed6b219aad1c2fe2605aeb80bef9a94621d65f88f443d11ea39bd2c5a", "class_name": "RelatedNodeInfo"}}, "text": "Only 0.2 gm. of nodules \n\nwere taken for the present study. \n\nSurface sterilization of the root nodules: Root \n\nnodules were rinsed with tap water to remove \n\nthe soil particle followed by rinsing with \n\ndetergent and few drops of tween-20 for 1 hour \n\nin the running tap water. Roots nodules were \n\ndipped in 95% ethanol for 5-10 seconds under \n\nlaminar air flow chamber and transferred to 2.5% \n\nsodium hypochlorite for 2-4 minutes. Then \n\nrinsed with sterile water for five times. \n\nPreparation of the inoculants: The root nodules \n\nwere crushed in 1ml of sterile water in the test \n\ntube with the sterile glass rod. Then the solution \n\nwas made 10 ml by adding sterile water. With the \n\nhelp of the pipette, 1 ml of the solution was taken \n\nin the next test tube and the final volume was \n\nmade 10 ml by adding 9 ml of the sterile water to \n\nmake 10-1 dilution of the solution. Similarly, the \n\nsolution was serial diluted upto 10-6 by \n\ntransferring 1ml solution from the former test \n\ntube to the next one. From each of the serial \n\ndilution, 0.5 ml solution was taken and \n\ninoculated in the YEMA media by spreading with \n\nthe help of L-shaped glass rod. Finally, the plates \n\nwere incubated at 300C in dark in inverted \n\nposition for 4 days. To isolate the pure culture of \n\nRhizobia, only red colony from 4th day cultures \n\nwere taken with the inoculating loop and \n\nstreaked in the YEMA media with Congo red and \n\nincubated at same condition as before. \n\nIdentification of Rhizobium:  \nThe species of Rhizobium were identified on the \n\nbasis of its host as well as some biochemical tests \n\nas mentioned below: \n\nCatalase production test [31]: The dark red \n\nportion of 18 to 24 hours pure colony was picked \n\nwith the help of an inoculating loop and placed \n\nin the clean glass watch. Then few drops of the \n\n3% H2O2 were added over the organism on the \n\nwatch glass with the help of the Pasteur pipette. \n\nThe immediate emergence of bubbles shows the \n\nproduction of catalase. \n\npH tolerance test: YEM broth was prepared \n\nwithout adding the agar in the YEMA media and \n\nadjusted to different pH as 4.5, 7, 9 and 9.5 by \n\nadding HCl and NaOH. Then the media was \n\nsterilized and Rhizobium strain was inoculated \n\nand incubated for 14 days at 30 degree Celsius \n\nand observed the growth of the rhizobia. \n\nNaCl tolerance test: YEMA plates with different \n\nconcentration of NaCl (1%, 2%, and 4%) was \n\nprepared, sterilized and inoculated with \n\nRhizobium and incubated for 14 days at 30 degree \n\nCelsius and observed the specific growth of the \n\nRhizobium. \n\nPenicillin resistance test (Kirby-Bauer Method) \n\n[32]: YEMA plates were prepared and placed \n\nright side up in an incubator at 37 0C for 10 to 20 \n\nminutes with the cover adjusted so that the slides \n\nare slightly opened. Each plates were labeled \n\nwith the name of test organism to be inoculated. \n\nA sterile cotton swab was dipped into a test \n\nculture and removes excess inoculums by \n\npressing the saturated swab against the inner \n\nwall of the beaker containing the test organism. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  43 Nepjol.info/index.php/njb. \n\n \n\n \n\nUsing the swab, the entire agar surface was \n\nstreaked horizontally and vertically to ensure a \n\nheavy growth over the entire surface. The culture \n\nplates were allowed to dry for about 5 minutes. \n\nUsing the aseptic technique the penicillin disc \n\nwas applied on the agar surface by using sterile \n\nforceps.", "start_char_idx": 15430, "end_char_idx": 18989, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6e0a0c3c-aa47-4e18-bf19-23807470aa56": {"__data__": {"id_": "6e0a0c3c-aa47-4e18-bf19-23807470aa56", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1a4a4de7-d53c-43ed-879f-ef840fe536a2", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "50b9a69b1d296373f434583effdefd2342544f489fdf4fa5436745d1d813b5c4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ced0fc5c-74b4-4dc4-b9df-65373288c2a7", "node_type": "1", "metadata": {}, "hash": "2907512ff66423298cf258af5897a065d3e30aa684c00643b8eb7867bf9e9836", "class_name": "RelatedNodeInfo"}}, "text": "Each disc were kept at least 15 mm from \n\nthe edge of plate. Each disc were gently pressed \n\ndown with the sterile forceps to endure that the \n\ndisc adhere to the surface of the media. The plate \n\ncultures were then incubated in an inverted \n\nposition for 24 to 48 hours at 300C. Finally all the \n\nplates were examined for the presence or absence \n\nof a zone of inhibition surrounding each disc. \n\nNodulation test [33]: The seeds of soya bean were \n\ntaken and surface sterilized in running tap water \n\nfollowed by dipping in 95% ethanol for 1 \n\nminutes. Seeds were then washed with 6 \n\nconsecutive washing with sterilized water. Then \n\nthe earthen pots along with 1:1 ratio of sand and \n\nsoil were sterilized in Hot Air Oven at 1600C for \n\nthree hours. The sticking solution was made by \n\nadding 10% sucrose in distilled water which was \n\nfirst heated and then cooled to make sticky. The \n\nRhizobial inoculants of 4 days culture were \n\nadded in the sticker solution to make slurry. The \n\nseeds of soybean were mixed in that slurry and \n\nstirred completely to make the inoculants \n\nattached on the seeds. They were then taken out \n\nand rolled on the CaCO3 to maintain the \n\nalkalinity, the process is called pelleting. The \n\nseeds were then dried in the air and sown in the \n\nsterilized earthen pots at the depth of one inch. \n\nSimilarly the seeds without inoculating the \n\nRhizobia are also sown in the next pot. Finally the \n\npots were watered and covered with transparent \n\npolyethylene sheet and tied around the pots. The \n\npolyethylene were made to have lots of holes for \n\nwatering as well as for providing ventilation and \n\nkept in the green house. They were watered \n\nregularly and observed for the nodulation when \n\nthe plant becomes 10-15 cm high. The presence of \n\nnodules in the inoculated plants and absence in \n\nun-inoculated plants shows the positive result of \n\nthe respective Rhizobial strain. \n\nColor change of BTB: YEMA plates containing \n\nBTB were prepared similarly as the YEMA plates \n\nwith Bromothymol Blue and inoculated with test \n\norganism and incubated at 300 C and observed \n\nthe color change after 4-5 days. The appearance \n\nof blue color shows that the rhizobial strain is \n\nslow growing and the appearance of the yellow \n\ncolor shows that the rhizobial strain is of fast \n\ngrowing type. \n\nMass production of Rhizobium \nStarter culture of Rhizobium: YEM broth \n\nmedium (100 ml) was prepared and autoclaved \n\nby transferring in a flask. Thereafter, pure \n\nrhizobium colony was transferred into sterilized \n\nYEM broth. Inoculated YEM broth was incubated \n\nat the water bath at 300C for four days. This was \n\nthe starter culture of the Rhizobium. \n\nMass culture of Rhizobium: For the mass culture \n\nof Rhizobium, YEM broth was prepared in the \n\nlarge quantity in the conical flask and sterilized \n\nas mentioned before. The PH was maintained 6.5 \n\nto 7.0. Then the sterilized YEM broth was \n\ninoculated with the broth of starter culture \n\nprepared in advance. This was incubated for 3-4 \n\ndays on the water bath at 300C. The culture was \n\ntested for the purity by inoculating in the YEMA \n\nplates staining with Congo red. The broth culture \n\nwas then transferred to the large flask and \n\nincubated for 4-9 days for the bacterial growth. \n\nEncapsulation of Rhizobium [34]: The Rhizobium \n\nwere immobilized by encapsulating with sodium \n\nalginate along with different concentration of \n\nsucrose as their nutrient. Beads were prepared \n\naseptically in laminar air flow chamber by using \n\ndropper and the micropipette. From the mass \n\nculture of Rhizobium of 4-9 days, 25ml of broth \n\nwas taken in four different beaker. Then the 2% \n\nsodium alginate was weighted and mixed in the \n\nbroth in each beaker.", "start_char_idx": 18990, "end_char_idx": 22715, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ced0fc5c-74b4-4dc4-b9df-65373288c2a7": {"__data__": {"id_": "ced0fc5c-74b4-4dc4-b9df-65373288c2a7", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6e0a0c3c-aa47-4e18-bf19-23807470aa56", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "beab49f87e8aa60e52c4abcd553731e51668f2d1a6b4d0812cff38d1253ff4d5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a014db75-0fc8-4656-a16e-166d41b638e5", "node_type": "1", "metadata": {}, "hash": "965872e749600eabe98b6eac865a6ed5959a597468e29d19f9732ed5e6e5b535", "class_name": "RelatedNodeInfo"}}, "text": "The sucrose concentration \n\nof 1%, 2%, 5% and 10% was added in different \n\nbeaker and leveled them. The sterilized magnet \n\nwas kept in the beaker and covered with the \n\naluminum foil. Then the beaker was kept on \n\nmagnetic stirrer at 250 rpm for 8 minutes in order \n\nto dissolve the sodium alginate and the sucrose. \n\nOn the other hand the solution of the 0.2 M CaCl2 \n\nwas prepared in the 1 liter beaker. The solution of \n\nthe inoculums, sucrose and the sodium alginate \n\nwas allowed to settle down for few minutes so \n\nthat the air bubbles get disappeared.  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  44 Nepjol.info/index.php/njb. \n\n \n\n \n\nThen the mixture was dropped from about 30 cm \n\nheight by using the blunt ended pipette and \n\ncollected in beaker containing 0.2 M CaCl2 \n\nsolution. The rounded beads being formed in the \n\nbeaker were  \n\nstirred regularly to prevent them from being \n\nattached with each other. After 30 minutes beads \n\nwere formed which were taken out from the \n\nCaCl2 solution by filtering with a muslin cloth \n\nand kept in the filter paper to be air-dried and left \n\novernight. Finally, the air-dried beads were kept \n\nin the lead closed culture tubes for further use to \n\ntest their viability. \n\nViability tests of the encapsulated beads of \n\nBradyrhizobium japonicum (modified from [35]): \n\nThe sodium alginate encapsulated beads hence \n\nprepared were stored in the airtight culture tube \n\nat room temperature for the further viability test. \n\nFor the viability test YEMA media was prepared \n\nand sterilized as mentioned before. With the \n\nsterilized forceps the beads were inoculated in \n\nthe surface of the media. The beads with different \n\nsucrose concentration were inoculated in \n\ndifferent plates for testing the viability. Then they \n\nwere incubated at 300C for 48 to 72 hours in the \n\nincubator. The plates were observed for the \n\nformation of the Rhizobial colony in the surface \n\nof the media. The same process was repeated at \n\nthe interval of 2 weeks, 4 weeks, 6 weeks, 8 \n\nweeks, 10 weeks, and so on up to 7 months. \n\nResults  \n\nIsolation and enumeration of the colonies \nThe Bradyrhizobium were isolated in the YEMA \n\nmedia and the number of colonies formed in the \n\nplates were enumerated and the average number \n\nof the cell forming unit were calculated by using \n\nfollowing formula: \n\n \n\nDilution factor = \n\n \nvolume of the sample used\n\n\ud835\udc47\ud835\udc5c\ud835\udc61\ud835\udc4e\ud835\udc59 \ud835\udc63\ud835\udc5c\ud835\udc59\ud835\udc62\ud835\udc5a\ud835\udc52 \ud835\udc5c\ud835\udc53 \ud835\udc60\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52 \ud835\udc4e\ud835\udc5b\ud835\udc51 \ud835\udc61\u210e\ud835\udc52 \ud835\udc51\ud835\udc56\ud835\udc59\ud835\udc62\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc60\n \n\nNumber of organism = dilution \u00d7 number of \n\ncolonies \n\nNumber of cfu per ml = \n\n  \nNumber of organisms formed in average\n\nInoculums size \u00d7 dilution\n \n\n \n\nHere one colony is considered as one colony \n\nforming unit (cfu). \n\nThe enumeration by spread plate method shows \n\nrandom result where 1st dilution have more \n\nnumber of colonies and the 6th dilution have the \n\nlowest number of colonies but the other have the \n\nascending number of the colonies except the 4th \n\nserial dilution which have the less number of the \n\ncolonies than the 5th dilution which alter from the \n\nprinciple of the serial dilution. The calculation \n\nshows that altogether 1156\u00d710-2colony forming \n\nunits are present in the 1 ml of the original sample \n\nobtained from the root nodules.", "start_char_idx": 22716, "end_char_idx": 26015, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a014db75-0fc8-4656-a16e-166d41b638e5": {"__data__": {"id_": "a014db75-0fc8-4656-a16e-166d41b638e5", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ced0fc5c-74b4-4dc4-b9df-65373288c2a7", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "dde023ce5cef94d9bf4bac67d55fa867f46fecc85f998700b6bd939bfbcd881c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d282287f-4c5a-49c0-ae73-4667d0fcf21a", "node_type": "1", "metadata": {}, "hash": "26d5066d8a1045cc0c131ab2e48ba49df65264c171cebc67c4802da5bcdb486a", "class_name": "RelatedNodeInfo"}}, "text": "Shape, size, color and texture of organism on \n\nthe plate:  In the first plate many colonies were \n\nformed by the inoculation of the Bradyrhizobium \n\ninoculums which were irregular shaped and \n\nsome were concentric and spreading. Some \n\ncolonies were large enough and some were too \n\nsmall. The colony formed after re-streaked had \n\nshown smooth, the raised and convex shape at \n\nthe place of the streak. The size of the colonies in \n\naverage was 4-5 mm and the maximum colony \n\nwas achieved in 6-8 days of culturing as it was \n\nnoticed under visual estimation. The colonies \n\nwere watery translucent with dark red rib like \n\nmarking in the center of the streak. Their color \n\nwas noticed pinkish red on the plate of re\u2013streak. \n\nBiochemical tests: Many biochemical tests have \n\nperformed which confirmed the isolated bacterial \n\nstrains as the Bradyrhizobium japonicum.  \n\nTable 1: Enumeration of organism by spread plate technique. \n\nS.N Dilution \nfactor  \n\nInoculums \nsize(ml) \n\nNo. of \ncolonies \nper plate  \n\nNo. of \norganisms  \n\nAverage  no. of \norganisms  \n\nNumber of c.f.u. \nper ml \n\n1. 10-1 0.5 339 33.9   \n \n1156\u00d7 10-2 \n\n2. 10-2 0.5 76 0.76  \n3. 10-3 0.5 17 0.017 578\u00d710-2 \n4. 10-4 0.5 2 0.0002  \n5. 10-5 0.5 5 0.00005  \n6. 10-6 0.5 1 0.000001", "start_char_idx": 26018, "end_char_idx": 27270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d282287f-4c5a-49c0-ae73-4667d0fcf21a": {"__data__": {"id_": "d282287f-4c5a-49c0-ae73-4667d0fcf21a", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a014db75-0fc8-4656-a16e-166d41b638e5", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "d70ca631e1b40ab5bd031cb50b82dbd2298a53cfc563f59fa97e5ff91ffa3244", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "dc0981dd-965c-4989-b1f9-c92d50068776", "node_type": "1", "metadata": {}, "hash": "3625f80d491357d80d9742c790bb832b34fa525c2ca487c1b30746498e5062a7", "class_name": "RelatedNodeInfo"}}, "text": "10-6 0.5 1 0.000001  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  45 Nepjol.info/index.php/njb. \n\n \n\n \n\n \n\n \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n\n \n \n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nTable 4: viability test of the encapsulated beads of \nRhizobium japonicum \n\nS.N Periods of \nviability \ntest (In \nDays) \n\nConcentration of the sucrose \n1% 2% 3% 5% 10% \n\n1 7 + + + + + \n2 20 + + + + + \n3 50 + + + + + \n4 75 + + + + + \n5 100 + + + + + \n6 120 + + + + + \n7 145 + + + + + \n8 170 + + + -  - \n9 190 + + + - - \n\n*(+)means viable and (\u2013) means not viable \nImmobilization of Rhizobial strain: As the \n\nRhizobial strain was immobilized by \n\nencapsulating in the beaded form with sodium \n\nalginate hardened by CaCl2 and mixing the \n\nsucrose as the additives, the number of beads \n\nformed from every 25 ml of broth were \n\nenumerated and the beads formed per liter of the \n\nbroth was calculated which is mentioned in the \n\nTable 3. \n\nThe number of beads formed from every 25 ml of \n\nthe cultured broth was different. An average of \n\n137 beads were formed from 25 ml of the \n\nsolution.  \n\nViability tests of the encapsulated beads: The \n\nencapsulated beads of the Bradyrhizobium \n\njaponicum were kept in the sealed bottle and they \n\nwere tested periodically for the viability of the \n\nbacterial cells. The result of the viability test done \n\nup to 190 days is shown in the Table 4: \n\nThe result of the viability tests shows the \n\ndiversified results. The beads prepared at 1%, 2% \n\nand 3% sucrose concentration had shown the \n\nviability up to six months. On the experiment \n\ndone on 170th day and 190th day, the Rhizobial \n\nstrain was absent and the zone clearance rings \n\nwere observed around the beads having 5% and \n\n10% sucrose concentration on the YEMA plates.  \n\nDiscussion \nPresent study was carried on Bradyrhizobium \n\njaponicum and it was based on the Rhizobium \n\npresent on the root nodules of the soybean \n\nspecies found in Nepal. Different methods and \n\nthe materials were used for the isolation, \n\nidentification, mass culture, immobilization and \n\nviability tests. The data obtained have been \n\nTable 2: Biochemical tests on Rhizobium spp \n\nS.N Biochemical tests  Result Remarks \n\n1. Catalase production test + Ve  \n2. Penicillin resistance test -Ve  \n3. pH tolerance test   \n pH 4.5 +Ve  \n pH 7 +Ve Slow growing rhizobia \n pH 9 -Ve  \n pH 9.5 -Ve  \n4. NaCl tolerance test   \n 1% NaCl Extreme   \n 2% NaCl More  \n 4% NaCl Less  \n5. Color change of BTB Yellow   \n6. Nodulation test +ve  \n\n+Ve = positive, -Ve= negative  \nTable 3: Number of beads formed from the 25ml of cultured solution in different concentration \nof sucrose. \n\nS.N % of soidum. \nalginate  \n\n% of sucrose Calcium carbonate \nCaCl2(M) \n\nBeads per \n25ml   \n\nAverage \nbeads \n\n1. 2% 10% 0.2 169  \n2. 2% 5% 0.2 127  \n3. 2% 3% 0.2 126 137 \n\n4. 2% 2% 0.2 146  \n5. 2% 1% 0.2 117  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No.", "start_char_idx": 27251, "end_char_idx": 30251, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "dc0981dd-965c-4989-b1f9-c92d50068776": {"__data__": {"id_": "dc0981dd-965c-4989-b1f9-c92d50068776", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d282287f-4c5a-49c0-ae73-4667d0fcf21a", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "5de3646b39735d0577b28de595c8469a8a80ebf302851edb6f19b8eab23fcbc9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6856efa2-204d-44ff-896c-748c4a015850", "node_type": "1", "metadata": {}, "hash": "27f963d0f5cb03e0e4e7cbb669f7f0142374549bf5858e946b9b0f0de46ba372", "class_name": "RelatedNodeInfo"}}, "text": "2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  46 Nepjol.info/index.php/njb. \n\n \n\n \n\ndiscussed with the relevant information and the \n\nsimilar works carried out by the different \n\ninvestigators. Very few works have been done in \n\nNepal but several works have been done by the \n\nforeign researcher. From the present study \n\nperformed on the Bradyhzobium japonicum, varied \n\nresponses were obtained.  \n\nFor the identification of the bacterial species \n\npresent in the root nodules of the soybean, \n\ndifferent tests have been performed. Different \n\nbiochemical tests performed for present study \n\nreveals that the strain of the Rhizobium under \n\nstudy was the slow growing species. The catalase \n\nproduction test of the Bradyrhizibium japonicum \n\nshows the positive result which is adjacent to the \n\nRhizobial isolates of the alfalfa as in the \n\nbiochemical characterization performed by \n\nShahzed et al.[36]. It means that the Rhizobial \n\nisolates of the present study contain the catalase \n\nenzyme which decomposes the hydrogen \n\nperoxide to release oxygen. This conforms that \n\nthe Bradyrhizobium japonicum is the cytochrome \n\ncontaining aerobic bacteria as described by \n\nBuchanan and Gibbons [37] that Rhizobia are \n\naerobic bacteria utilizing oxygen as the terminal \n\nelectron acceptor. Similarly the Rhizobial isolates \n\nof the present study shows the less resistance to \n\nthe penicillin disc 10\u00b5g which indicate that \n\npenicillin is effective to the Bradyrhizobium \n\njaponicum which reduced the growth of the \n\nrhizobium showing the antimicrobial activity to \n\nrhizobia. The pH tolerance test performed for the \n\npresent study shows that the rhizobial isolates \n\ncan tolerate the low pH but cannot tolerate the \n\nhigh pH. It means that Bradyrhizobium japonicum \n\nis the acid tolerant species of the rhizobium. As  \n\nThornton & Davey [38]; Richardson & Simpson \n\n[39] mentioned that  slight change in pH alone \n\ncan significantly affect the growth of root nodule \n\nbacteria ,the Bradyrhizobium shows the high \n\ngrowth in pH 4.5 and 7 whereas it can\u2019t grow in \n\npH 9 and and 9.5. The concentration of the \n\nsodium chloride also effects the growth and the \n\nsurvival of the Rhzobium species. As mentioned \n\nby Singleton et al [22]  that increasing salt \n\nconcentration may have detrimental effects on \n\nrhizobial population, the Bradyrhizobium \n\njaponicum grow well in the 1% and 2% NaCl but \n\ndo not grow well in 4% NaCl concentration and \n\nit has also vital role in the cell viability for 7 \n\nweeks in the YEMA plates. Also the nodulation \n\ntest shows the positive result of the present study \n\nconfirmed the isolates as the Bradyrhizobium \n\njaponicum since it is host specific. The color \n\nchange of BTB to yellow showed that it is the fast \n\ngrowing species but the all other results \n\nbiochemical tests points it as the slow growing \n\nbacteria.  \n\nThe mass culture of the Rhizobial isolates of the \n\npresent study shows the dense growth of the \n\nbacteria at 7-9 days of the inoculation forming the \n\ndense mass at the surface of the YEMA broth. It \n\nalso indicate that it is the slow growing species of \n\nRhizobium since the fast growing species grow \n\ndensely at 4-6 days of inoculation at the \n\ntemperature of 300C. \n\nThe cultured mass of the Rhizobium was \n\nimmobilized in the form of the encapsulated \n\nbeads by using the sodium alginate extracted \n\nfrom algae as studied by Neely & Pettitt (1973) \n\n[40]. The preparation of encapsulated beads of \n\nRhizobium was not easier and it has many \n\nlimitations in its procedure. The missing of one \n\nstep hampers severely the formation of beads. \n\nThe height of dropping, time and rotation of \n\nmagnetic stirrer are the most important factors.", "start_char_idx": 30231, "end_char_idx": 33994, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6856efa2-204d-44ff-896c-748c4a015850": {"__data__": {"id_": "6856efa2-204d-44ff-896c-748c4a015850", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "dc0981dd-965c-4989-b1f9-c92d50068776", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "bdedb948348b8c07ffdeaeb200c661fd195dc6ca70ed4386f3fe29d1408588c5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fc521750-c0aa-4a29-895d-8d205fcca75e", "node_type": "1", "metadata": {}, "hash": "6c458d7ec85064282a4c7ed3e49380b90d4f4d304743e6167bc64cba25a1809e", "class_name": "RelatedNodeInfo"}}, "text": "An average of 137 beads was prepared from 25ml \n\nof broth. Thus 548 beads per 100 ml can be \n\nprepared within the limitation of time and \n\nrotation of magnetic stirrer. The less rotation and \n\nthe over rotation results in the deformation of \n\nbeads. The large volume of the inoculant in the \n\nsmall beaker with small magnet could not \n\ndissolve the sodium alginate and hence the beads \n\nformation is effected which could not remain in \n\nthe beaded form for the longer period at room \n\ntemperature and dissolves itself. As Saiprasad \n\n(2001) [14] reported that Sodium alginate was the \n\nmost accepted hydro-gel and frequently used as \n\na  matrix for the synthetic seeds because of its low \n\ntoxicity, low cost, quick gelation and \n\nbiocompatibility characteristics, it was used as \n\nthe gelling agent along with the sucrose as the \n\nadditives for their survival on the basis of study \n\nperformed by Vincent [41] and found that 24-44% \n\nof cells suspended in a 10% sucrose solution  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  47 Nepjol.info/index.php/njb. \n\n \n\n \n\nsurvived primarily drying whereas only 0.1 % \n\nsurvived when suspended in water. 2% of \n\nsodium alginate was found to be the best for the \n\nencapsulation which are hardened by 0.1 M \n\nCaCl2 as noticed by Kierstan & Bucke (1977) [42]. \n\nWhen the beads encapsulated with sodium \n\nalginate were stored at the room temperature and \n\ntested for their viability, they showed the viable \n\ncells for six months. The air dried beads kept \n\nsealed in the culture tube have maintain their \n\nbeaded structure for several months. The \n\ndifferent sucrose concentration mixed as the \n\nadditives for their survival have played the \n\nimportant role. In 1%, 2% and 3% sucrose \n\nconcentration the cells were viable for 190 days of \n\ninoculation whereas in 5% and 10% sucrose \n\nconcentration the cells were survived only for 145 \n\ndays. Mcleod (1961) [43] had found that the \n\nincorporation of 10% sucrose in yeast Mannitol \n\nbroth improved the survival on glass beads \n\ncompared with un-amended broth cited by \n\nVincent [41] but the survival of the \n\nBradyrhizobium japonicum is less at higher sucrose \n\nconcentration and vice versa. It shows that the \n\nsucrose at low concentration maintains the \n\nmoisture content and support for the viability of \n\nthe rhizobium whereas the higher concentration of \n\nthe sucrose effects their survival after few \n\nmonths. Thus it can be said that beads of \n\nBradyrhizobium japonicum prefers the lower \n\nconcentration of the sucrose. \n\nConclusion \nFindings of the present study carried on \n\nBradyrhizobium japonicum concluded that it can be \n\nisolated, identified and encapsulated in the forms \n\nof beads which looks like chemical fertilizers \n\nfound in the market. It also shows that \n\nBradyrhizobium japonicum is slow-growing \n\nbacteria. Besides soil, peat, charcoal as the solid \n\ninoculants and the broth as a liquid inoculant, the \n\nrhizobial inoculants can be immobilized in the \n\nform of encapsulated beads by using 2% sodium \n\nalginate, 1-3% sucrose as additives and 0.1M \n\nCaCl2 as the hardening substances. This \n\nmaintains the moisture content of the beads as \n\nwell as prevents the contaminants and preserved \n\nthe cells for several months. This study concludes \n\nthat the encapsulated beads with sucrose (1-3%) \n\nas the additives can be viable for more than 190 \n\ndays whereas with the 5% and 10% sucrose cells \n\nsurvive only for five months. Also, they are easy \n\nfor handling as well as can be viable for more \n\nthan six months in the room temperature.", "start_char_idx": 33997, "end_char_idx": 37657, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fc521750-c0aa-4a29-895d-8d205fcca75e": {"__data__": {"id_": "fc521750-c0aa-4a29-895d-8d205fcca75e", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6856efa2-204d-44ff-896c-748c4a015850", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c2ce2f41d6a5fad271a4b9529fe6e4166725ead723d37be0c2fe27d13146ee26", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b8214219-64b9-4a98-9eb5-f97d74a6ff60", "node_type": "1", "metadata": {}, "hash": "d5ce37378466c04de58347078614acb89fb53981d70e497e6d2c6f2f93c87695", "class_name": "RelatedNodeInfo"}}, "text": "Thus \n\nthe rhizobial strain can be easily immobilized by \n\nusing sodium alginate and sucrose as additive. \n\nReferences  \n1. Fred EB, Baldwin LL, Mccoy E: Root nodule \n\nbacteria and leguminous plants. 1932 \nUniversity of Wisconsin press, Madison. \n\n2. Graham PH:. Ecology of root nodule bacteria \nof legumes. In M. J. Dil-worth et al. (ed.). \nNitrogen fixing symbiosis, 2008 23-58.  \n\n3. Robson AD: The role of self-regenerating \n\npasture in rotation with cereals in \n\nMediterranean areas. In: The role of legumes \n\nin the farming system of the Mediterranean \n\nareas.1990 217-236. \n\n4. Unkovich MJ, Pate JS, Armstrong EL, Sanford \nP: Nitrogen economy of annual crop and \npasture legume in southeast Austr Soil biol \nBiochem 1995 27: 585-588. \n\n5. Reeves TJ, Smith IS: Pasture management \n\nand cultural methods for the control of \n\nannual ryegrass (lolium rigidum) in wheat. \n\nAust J Expl Agric Anim husb. 1975 15: 527-530 \n\n6. O\u2019Hara GW, Howieson JG, Graham PH: \n\nNitrogen fixation and agricultural practices. \n\nIn: Leigh GJ (ed.) Nitrogen fixation in \n\nmillennium. 2002 Elsevier, Amsterdam. The \n\nNetherlands, 391-419 \n\n7. Dommergues YR, Diem HG, Divies C. \n\nPolyacrylamide entrapped Rhizobium as an \n\ninoculants for legumes. Appl Environ Microb. \n\n1979 37:779-781 \n\n8. Bashan Y: Alginate beads as synthetic \n\ncarriers for slow release of bacteria that \n\naffect plant growth. Appl Environ Microbiol. \n\n1986 51: 1089-1098 \n\n9. Cassman KG, Munns DN, Beck DP: Growth \nof Rhizobium strain  at low concentrations of \nphosphate. Soil Sci Soc Amer J. 1997 45: 520-\n523. \n\n10. Peoples MB, Herridge DF, and Lahda JK: \n\nBiological nitrogen fixation:  an efficient \n\nsource of nitrogen for sustainable \n\nagricultural production. Plant Soil. 1995 174: \n\n3-28. \n\n11. Brockwell J, Bottomley PJ: Recent advances \n\nin inoculant technology and prospects for \n\nthe future. Soil Biol Biochem. 1995 27(4): 683-\n\n697 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  48 Nepjol.info/index.php/njb. \n\n \n\n \n\n12. Graham P H, Vance C P: Nitrogen fixation in \n\nperspective: an overview of research and \n\nextension needs. Field Crop Res. 2000 65: 93-\n\n106 \n\n13. Kennedy IR. Cocking EC: Biological \nNitrogen Fixation; the global challenge and \nthe future needs. In Rockeller Foundation \nBellagio conference Proceedings, 1997 83 \n\n14. Saiprasad GVS: Artificial seed and their \n\napplication. Resonance. 2001 50: 22-32 \n\n15. Sullivan JT, Eardly BD, Van-Berkum P, \n\nRonson CW: Four unknown species of non-\n\nsymbiotic rhizobia isolated from root \n\nnodules of rhizosphere of  Lotus  \n\ncorniculatus. Appl Environ Microbiol. 1996 62: \n\n2818-2825 \n\n16. Somasegaran P, Hoben HJ: Handbook of \n\nRhizobia. Methods in Legume-Rhizobium \n\nTechnology. 1994 Springer-Verlag, New \n\nYork, NY, 450 pp \n\n17. Date RA: Advances in inoculant technology: \n\na brief review.", "start_char_idx": 37658, "end_char_idx": 40583, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b8214219-64b9-4a98-9eb5-f97d74a6ff60": {"__data__": {"id_": "b8214219-64b9-4a98-9eb5-f97d74a6ff60", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fc521750-c0aa-4a29-895d-8d205fcca75e", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "5d84f778e3a94f4bb540560cab5482fbb216439d0bd64a01e6f289725823f4f8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "809eedb6-0df5-46b6-9e32-cc51cd3263c4", "node_type": "1", "metadata": {}, "hash": "033fe5565ae6e3b6cb443ac16e8873a554bdc382cee480a0f547318bc5257515", "class_name": "RelatedNodeInfo"}}, "text": "Date RA: Advances in inoculant technology: \n\na brief review. Aus J Exp Agric. 2001 41: 321-\n\n325. \n\n18. Burlison WL, Sears OH, Hackleman J C: \n\nGrowing alfalfa in Illinois. Agric Exp Stn \n\nBull. 1930 349: 411-448 \n\n19. Smith RS: Legume inoculant formulation \n\nand application.  Can J Microbiol. 1992 38: 485-\n\n492 \n\n20. Herridge D, Gemell G, Hartley E: Legume \n\ninoculants and quality control. In Herridge \n\nD. (ed.). Inoculants and nitrogen fixation of \n\nlegumes in Vietnam, Australia 2002 p105-115.  \n\n21. Allen EK:  Biological aspects of symbiotic \n\nnitrogen fixation. Encycl Pl Physiol. 1958 8: 48-\n\n118 \n\n22. Singleton P, Keyser H, Sande E: \nDevelopment and evaluation of liquid \ninoculants. In: Herridge D. (ed). Inoculant and \nnitrogen fixation of legumes in Vietnam. \nAustralia 2002 p95-104. \n\n23. Olsen PE, Rice WA, Bordeleau LM, Demidoff \nAH, Collins MM:  Levels and identities of \nnon-rhizobial microorganisms found in  \ncommercial legume inoculant made with \nnon-sterile peat carrier.  Can J Microbiol. 1996 \n42: 72-75. \n\n24. Gyaneshwar P, Nareshkumar G, Parckh LD: \n\nEffect of Buffering on the Phosphate \n\nSolubilizing Ability of Microorganisms. \n\nWorld J Microbiol Biotechnol. 1998 14: 669-673 \n\n25. Lee J, Song SH:. Evaluation of Groundwater \n\nQuality in Coastal Areas: Implications for \n\nSustainable Agriculture. Environ Geol. 2007 \n\n52: 1231-1242 \n\n26. Elkoca E, Kantar F, Fiahin F: Influence of \nNitrogen Fixing and P Solubilizing Bacteria \non the Nodulation, Plant Growth, and Yield \nof Chickpea. J Plant Nutr. 2008 31: 157-171. \n\n27. Bohlool BB, Ladha J K, Garrity DP, George T: \n\nBiological nitrogen fixation for sustainable \n\nagriculture - a perspective. Plant and Soil. \n\n1992 141: 1-11 \n\n28. Galloway JN, Townsend AR, Erisman JW, \n\nBekunda M, Cai ZC, Freney JR, Martinelli LA, \n\nSeitzinger SP, Sutton MA:. Transformation of \n\nthe nitrogen cycle: Recent trends, questions, \n\nand potential solutions. Science. 2008 320: \n\n889-892 \n\n29. Peoples MB, Hauggaard-Nielsen H, Jensen \nES: The potential environmental benefits \nand risks derived from legumes in rotations. \nIn: D. W. Emerich and H. B. Krishnan, \neditors. Nitrogen Fixation in Crop \nProduction. Am Soc Agro. 2009 349-385 \n\n30. Dubey RC, Maheshwori DK:. Practical \n\nmicrobiology. S. Chand and co. Ltd. 2002 \n\nNew Delhi \n\n31. Lowe GH: The rapid detection of lactose \nfermentatation in paracolon organism by \ndemonstration of 6- D-galactosidase. J Med \nLab Technol. 1962 19:21-25 \n\n32. Shah PK, Dahal PR, Amatya J: Practical \n\nMicrobiology 2009 (Revised Ed.) 158-162. \n\nDelta Offset Press. Thapathali. Kathmandu \n\n33. Dubey RC: A test book of Biotechnology. S. \nChand Publication and Company Limited, \n1993 Ram Nagar New Delhi. \n\n34. Heller, G: A quantitative study of \n\nenvironmental factors involved in survival \n\nand death of bacteria in the desiccated state.", "start_char_idx": 40523, "end_char_idx": 43361, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "809eedb6-0df5-46b6-9e32-cc51cd3263c4": {"__data__": {"id_": "809eedb6-0df5-46b6-9e32-cc51cd3263c4", "embedding": null, "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-189", "node_type": "4", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "c484b04fd4170168df4659ac2e24827c77b59db219fe07ed07e74784d588256e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b8214219-64b9-4a98-9eb5-f97d74a6ff60", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "65badb49d85b00c4bf70fa2a17323789524f7eb841458385446d1451b6d5afb9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "94c93d2e-00f6-4858-b146-1de83109ff2b", "node_type": "1", "metadata": {}, "hash": "d11a9c7771571c28f4a9c42ac3c6a70c112496762c0423fe8ccf4fd96ca0c613", "class_name": "RelatedNodeInfo"}}, "text": "J Hygeine 1941 41: 109-126 \n\n35. Jung G, Mugnier J, Diem HG, Dommergues, \n\nYR: Polymer entrapped  Rhizobium as an \n\ninoculant for legumes. Plant Soil. 1982 65: \n\n219-231 \n\n36. Shahzed F, Shafee M, Abaas F. Babar S, Tariq \nMM, Ahmad Z: Isolation and biochemical \ncharacterization of Rhizobium meliloti from \nroot nodules of alfalfa (Medico sativa). Anim \nPlant Sci. 2012 22(2): 522-524. \n\n37. Buchanan RE, Gibbons NE: Bergey\u2019s manual \n\nof Determinative Bacteriology, 8th edition. \n\n1974 Baltimore Williams and Wilkins \n\n38. Thornton FC, Davey CB: Acid dolerance of \n\nRhizobium trifolii in culture media. Soil Sci \n\nSoc Am J. 1983 47: 496-501 \n\n39. Richardson EA, Simpson RJ: Acid tolerance \n\nand symbiotic effectiveness of Rhizobium \n\ntrifolii associated with a trifolium \n\nsubterraneum L. based pasture growing acid \n\nsoils. Soil Biol Biochem. 1989 21: 87-95 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 39-49                     Chhetri et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  49 Nepjol.info/index.php/njb. \n\n \n\n \n\n40. McNeeley W H, Pettitt DJ:. Algin. In \nIndustrial Gums polysaccharides and Their \nDerivatives. In Whistler RL. (ed) 1973 2nd \nedition. Academic press , New York, 49-88 \n\n41. Vincent JM: Survival of root-nodule bacteria. \n\nIn E.G. Hallsworth (ed.).  Nutrition of the \n\nLegumes (p108\u2013123). Soil Biol Biochem. 1958 \n\n36: 1275-1288 Quoted in Deaker R, Roughley \n\nRJ, Kennedy IR. (2004). Legume seed \n\ninoculation technology-a review.   \n\n42. Kierstan M, Bucke: The immobilization of \n\nmicrobial cells, subcellular organelles and \n\nenzymes in calcium alginate gels. Biotech \n\nBioengin. 1977 14: 387-397 \n\n43. McLeod RW, Roughley RJ: Freeze-dried \n\ncultures as commercial legume inoculants. J  \n\nExp Agric Annimal Husband.19611: 29-33 \n\n44. Nobbe F, Hiltner L: Inoculation of the soil for \n\ncultivating leguminous plants. 1896 USA. \n\nUS Patent No. 570813", "start_char_idx": 43364, "end_char_idx": 45270, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "94c93d2e-00f6-4858-b146-1de83109ff2b": {"__data__": {"id_": "94c93d2e-00f6-4858-b146-1de83109ff2b", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "809eedb6-0df5-46b6-9e32-cc51cd3263c4", "node_type": "1", "metadata": {"identifier": "njb-189", "author": "Chhetri, Til Kumari; Subedee, Bijay Raj; Pant, Bijaya", "title": "Isolation, Identification and Production of Encapsulated Bradyrhizobium japonicum and Study on their Viability", "date": "2019-12-29", "file": "njb-189.pdf"}, "hash": "19792e8bda68e64c917042bb1d45dcd11245f6d775859362b407c9f118f101b0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2207d609-e4ad-45f0-b313-c23c8689364a", "node_type": "1", "metadata": {}, "hash": "81d637e5eddc9d8756fb7666d8dc0c3ea6243ac47c797a8b885a3c76b96f6e57", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102   DOI: https://doi.org/10.3126/njb.v7i1.26949 \n\n\u00a9NJB, Biotechnology Society of Nepal  96 Nepjol.info/index.php/njb. \n\nREVIEW ARTICLE \n\n \n\nInfectious Sources of Histoplasmosis and Molecular \nTechniques for its Identification  \n\nSudip Bhandari1, Binod Rayamajhee2, Laxmi Dhungel1, Sami Poudel1, Bhagwati Gaire1,  \n\nSunil Shrestha1, Niranjan Parajuli3* \n\n1Department of Biotechnology, National College, Tribhuvan University, Kathmandu, Nepal \n2Department of Infectious Diseases and Immunology, Kathmandu Research Institute for Biological \n\nSciences (KRIBS), Lalitpur, Nepal \n3Central Department of Chemistry, Tribhuvan University, Kathmandu, Nepal \n\nAbstract \nHistoplasmosis, a fungal infection caused by Histoplasma capsulatum (H. capsulatum), acquired \nfrom contaminated soil with droppings of chicken or birds and found to be distributed in many \nparts of the world. The prevalence of histoplasmosis has not well studied in Nepal. The common \nsymptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and \nweight loss.  Most of the infections associated with histoplasmosis are asymptomatic. People with \ncompromised immune systems such as HIV/AIDS (PLWHA), cancer, and organ transplant \nrecipients are at risk of developing this disease. In this review, we have summarised the current \nstatus of histoplasmosis in Nepal and molecular techniques available for its identification. To date, \nthe significant outbreak is not reported in Nepal, but the risk of infection for the vulnerable \npopulation cannot be undermined. Appropriate preventive measures and treatment on time can \nreduce the burden of this fungal disease. Further, this review is also focused on molecular \nidentification of H. capsulatum. Hence, careful considerations by concerned stakeholders for \nnational surveillance programs and the treatment of patients on time after proper diagnosis is \nhighly recommended.  \nKeywords: Histoplasmosis, asymptomatic, vulnerable, treatment \n\n*Corresponding author: \n\nEmail:  nparajuli@cdctu.edu.np \n\nIntroduction  \nHistoplasmosis is acquired by inhalation of \n\nspores of H. capsulatum, which is usually found \n\nin the soil contaminated by bird droppings, \n\nwhich reveals the higher risk of disease in \n\nfarmers, gardeners, poultry keepers, \n\nconstruction workers, pest control workers and \n\nin some instances travelers visiting caves and \n\ntunnels[1]. Histoplasma is a dimorphic fungus, \n\nwhich produces mycelial form in the soil \n\nenvironment and converts to the yeast form at \n\nhost body temperature (37 \u00b0C), and it usually \n\ndoes not develop the symptomatic infection. So \n\npeople need not concern about the infection [2]. \n\nHowever, for immune-compromised people, \n\nthe fungus can result in severe infection. Very \n\nyoung children and elderly people who have a \n\nweak immune system; are more likely to get \n\nhistoplasmosis disease [3]. \n\nThe development of infection and the \n\ndissemination of H. capsulatum are initially \n\ndependent on the condition of the host body[4]. \n\nThe majority of infected persons could have \n\neither no symptoms or a very less mild sickness, \n\nwhich is hard to recognize as the cause of \n\nhistoplasmosis [5]. The clinical symptoms \n\ngenerally occur only in a small number (around \n\n1%) of the population when exposed with H. \n\ncapsulatum spores [6].", "start_char_idx": 47, "end_char_idx": 3433, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2207d609-e4ad-45f0-b313-c23c8689364a": {"__data__": {"id_": "2207d609-e4ad-45f0-b313-c23c8689364a", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "94c93d2e-00f6-4858-b146-1de83109ff2b", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "b54dfe61762b8d18ca8bd348c4954c4d6d5b5c956f12a11b087cc0014a30fd21", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d0f516d1-dd81-41de-b138-6533a27d4ea5", "node_type": "1", "metadata": {}, "hash": "7cf597aec7448ae2d5ef91331ff413557e55782bf53518c7ee6de0a219792b94", "class_name": "RelatedNodeInfo"}}, "text": "Persons who are usually \n\nimmuno-compromised and are unable to \n\ndevelop effective cell-mediated response are \n\nlikely to develop symptomatic disease during \n\nthe period of acute dissemination in body [5], \n\nwhich includes infant child, patients with \n\nHIV/AIDS, organ transplant recipients, and \n\nthose with hematologic deficiencies, and also \n\nthose patients, who are on corticosteroids drugs \n\ntreatment [7]. An individual is under the risk of \n\ndeveloping symptoms even after years leaving \n\nthe endemic vicinity of histoplasmosis if the \n\nman or woman was in an immuno-suppressive \n\nsituation at that time [8]. Histoplasmosis is of \n\nfour major types: pulmonary histoplasmosis, \n\nprogressive disseminated histoplasmosis, \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  97 Nepjol.info/index.php/njb. \n\n \n\n \n\ncutaneous histoplasmosis, and African \n\nhistoplasmosis [9, 10]. \n\nPathogenesis of Histoplasmosis \nThe fungal infection starts after the inhalation of \n\nspores produced by the mycelial form of H. \n\ncapsulatum and the spores deposited inside the \n\nalveoli of the lungs [11]. After some time, the \n\nspores start to germinate inside the alveoli at \n\nnormal body temperature to become dimorphic \n\nform. Then pulmonary macrophages engulf that \n\nyeast form of dimorphic fungus [12]. After the \n\nengulfment, the yeasts become a parasite and \n\nmultiply within the alveolar cells, then travel to \n\nhilar and mediastinal lymph nodes [13]. When \n\nyeast form fungus gets access to the blood \n\ncirculation system, then disseminates more \n\nrapidly and swiftly across various organs of the \n\nbody. About after two weeks of exposure, the \n\nmacrophages become fully fungicidal, then the \n\ncellular immunity starts a defense against the \n\nfungal particles [14]. It leads to necrosis at the \n\nsite of infection like in the lungs, liver, spleen, \n\nlymph nodes, and on bone marrow, adrenal \n\nglands, and mucocutaneous membranes, which \n\nresults in progressive disseminated \n\nhistoplasmosis [4] as shown in Figure 1. The \n\nprogressive type of histoplasmosis is much \n\nmore lethal and severe as compared to other \n\nkinds of histoplasmosis [15]. \n\nEpidemiology \nHistoplasmosis is distributed worldwide, but \n\nincidence cases are often reported around the \n\nperiphery of river valleys [16]. The majority of \n\ninfections in humans are reported from the \n\ncentral United States, whereas the small number \n\nof cases are also reported from  Brazil, \n\nArgentina, India, and South Africa [17]. \n\nCurrently, due to an increase in the occurrence \n\nof histoplasmosis worldwide, scientists have \n\nbeen developing several diagnostic strategies. \n\nHistoplasmosis endemicity can be evaluated by \n\npopulation-based use of a histoplasmosis skin \n\ntest [18]. The skin test was found to be useful \n\nwhen a major endemic area of histoplasmosis \n\nidentified in the north-eastern and Midwestern \n\nUnited States [19]. \n\nIncreased number of infectious diseases like \n\nhistoplasmosis is being reported in areas where \n\nit was previously not thought to be prevalent, \n\nand the changing distribution of infectious \n\ndiseases can become an important step to guide \n\ndiagnostic workup and direct public health \n\nawareness [17]. Notably, in tuberculosis-\n\nendemic regions, disseminated histoplasmosis \n\ncan easily be misdiagnosed as tuberculosis \n\nbecause of its similar clinical symptoms and \n\nmust be considered as histoplasmosis infection \n\nin patients who do not respond to empiric anti-\n\ntubercular therapy [20]. \n\nAn estimated discern indicates that nearly 3,000 \n\npeople develop kidney failure yearly in Nepal \n\n[21].", "start_char_idx": 3434, "end_char_idx": 7123, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d0f516d1-dd81-41de-b138-6533a27d4ea5": {"__data__": {"id_": "d0f516d1-dd81-41de-b138-6533a27d4ea5", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2207d609-e4ad-45f0-b313-c23c8689364a", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "ed75ffc31f9ee1825274a767469c24e811eccc98f192a4821f5b2eab75f111e2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8f7729e6-77c4-4d90-9f8c-9b4292577d08", "node_type": "1", "metadata": {}, "hash": "e492ac85ecd63f65091040e5600d03c0568804d1709d638967c931e777fd6cd6", "class_name": "RelatedNodeInfo"}}, "text": "Similarly, there are additionally a high \n\nnumber of the population in Nepal with a \n\ndiabetic condition; the 2016 Diabetes Profile has \n\nshown that 9.1 %of the Nepali people are \n\ndwellings with diabetes [22]. Likewise, the cases \n\nof HIV/AIDS is also in excessive number inside \n\nthe country [23]. According to National Centre \n\nfor AIDS and STD Control (Nepal), there \n\nare around 31,020 HIV/AIDS sufferers in the \n\ncountry, and it is estimated to upward thrust at \n\na rate of 2 patients per day. Those persons \n\nhaving conditions like a diabetic, organ \n\ntransplant, and the person with HIV/AIDS have \n\na weak immune system, which means that they \n\nare at high risk of developing other secondary \n\ninfections like histoplasmosis during their \n\nlifetime.  \n\nAccording to the published reports,  the \n\ngeographical distribution of histoplasmosis has \n\nnot been clearly understood [4]. The occurrence \n\nof histoplasmosis in Asia has not fully \n\nappreciated until recently. Malaysia is the first \n\ncountry in Asia, where H. capsulatum was \n\nisolated from soil samples [24]. India, a close \n\nneighboring of Nepal, has also reported a high \n\nnumber of histoplasmosis cases in West Bengal \n\nand Assam till 2018[24]. From, 1995 to 2018, \n\nabout 388 cases of H. capsulatum has been \n\nreported, and the number is in increasing order \n\nin India [24]. In recent years, an increasing \n\nnumber of histoplasmosis cases have been \n\nrecognized among HIV-positive and/or \n\ndiabetic patients in India [1]. In case of Nepal, \n\nhistoplasmosis has only been described four \n\ntimes, Amatya et al., (2014) reported that the \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  98 Nepjol.info/index.php/njb. \n\n \n\n \n\ncases of histoplasmosis in an Indian national \n\nwho traveled to Nepal for medical care and once \n\nin a Nepali citizen, making this the second case \n\ndescribed in the literature by Subedi et al., (2016) \n\n[25]. Recently, another case has been published \n\nby Sharma and Adhikari (2019). According to \n\nthem, a 52 years old man with diabetes shows \n\nadrenal involvement in histoplasmosis [26]. \n\nAdditionally, one case has reported in a \n\nNepalese migrant to the USA with evidence of \n\ninfection being acquired in Nepal by Gandi et \n\nal., in 2015 [27]. Nepal and India have an open \n\nborder, and a large number of Nepalese \n\nmigrants is directly dependent on Indian \n\neconomic and cultural aspects such as \n\nemployment, education, social relationship, and \n\ntrade among other elements. Such socio-\n\neconomic relationship directly has an impact on \n\nthe dissemination of the diseases, including \n\nhistoplasmosis. \n\nIdentification of H. Capsulatum and diagnosis \n\nof histoplasmosis \n\nTraditionally, identification of causative agent \n\nof histoplasmosis is carried out through tissue \n\nculture or body fluids at 25\u00b0C on Sabouraud\u2019s \n\ndextrose agar to allow the growth of mycelial \n\nphase of H. capsulatum. For the proper growth of \n\nmold, it takes about six weeks. After the \n\nincubation period, two different types of conidia \n\nare formed. The tuberculate or macroconidia are \n\nof about 8 \u2013 15 \u00b5m in diameter and have a thick \n\nwall. Similarly, the microconidia are tiny about \n\n2 -4 \u00b5m in diameter and are more infectious \n\nwhen compared to the more abundant conidia. \n\nHowever, the procedure of culturing is time-\n\nconsuming [28]. \n\nLikewise, histopathological examination is \n\nanother technique for detection of the \n\nHistoplasma. It is generally done for severely ill \n\npatients [29]. The method includes tissue \n\nbiopsy.", "start_char_idx": 7124, "end_char_idx": 10741, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8f7729e6-77c4-4d90-9f8c-9b4292577d08": {"__data__": {"id_": "8f7729e6-77c4-4d90-9f8c-9b4292577d08", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d0f516d1-dd81-41de-b138-6533a27d4ea5", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "44eef71f3b5dce467cd5358e431f2a44ad79ccc7772c4d7c3f49317a5c0825d8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e97489c7-3090-4190-a8e0-411d5b527e26", "node_type": "1", "metadata": {}, "hash": "f2f8dcd2826280c85711a798668ac40e0782ab989b6f99449304c0e02a6c2944", "class_name": "RelatedNodeInfo"}}, "text": "The method includes tissue \n\nbiopsy. During the histopathological \n\nobservation, other organisms also could mimic \n\nthe appearance like that of H. capsulatum. So, this \n\ncould be eliminated by using another stain like \n\nmethenamine silver or periodic acid- Schiff \n\nstains, which visualize H. capsulatum [30]. \n\nDetection of circulating Histoplasma associated \n\nantigen in urine and serum is another diagnostic \n\noption to detect the presence of the fungi using \n\nan immunological technique like sandwich \n\nenzyme immunoassay (EIA), and it was first \n\ndescribed in 1986 [31]. This technique generally \n\nuses H. capsulatum antigen, which is a \n\npolysaccharide, a polyclonal rabbit anti-\n\nHistoplasma immunoglobulin G linked to biotin, \n\nand horseradish peroxidase [4, 18]. The assay \n\nalso shows the highest sensitivity against AIDS \n\npatients who had disseminated histoplasmosis \n\n[9].  \n\nAntibody tests are also performed to detect \n\nseveral forms of Histoplasma cases. There are \n\ntwo standard assays for antibody detection they \n\nare, (i) complement fixation (CF) test, which is \n\nbased on the use of two separate antigens- yeast \n\nand mycelial (or histoplasmosis)- and (ii) \n\nimmunodiffusion (ID) assay [32]. The ID assay \n\ntests generally detect the presence of M and H \n\nprecipitin bands. An M \n\nband often existing in persistent types of \n\nhistoplasmosis and lasts for many months to \n\nyears after the infection. An H band is also \n\nindicative of the severe form of histoplasmosis \n\n[17]. The ID assay is approximately only 80% \n\nsensitive but is more specific than CF assay \n\nbecause in CF assay, cross-reaction may occur \n\nwith other fungal infections like sarcoidosis [33]. \n\nMolecular identification of Histo-\nplasmosis \nIn recent days, the molecular approach is widely \n\nused as a reliable and rapid technique for the \n\ndetection of diseases with higher sensitivity and \n\nspecificity. At present, the use of chemo-\n\nluminescence labeled DNA probe for the \n\ndetection of specific sequences of ribosomal \n\nRNA (rRNA) has been able to detect and \n\nconfirm all the H. capsulatum isolates from the \n\nculture [32]. AccuProbe is one of them, which is \n\nbased on the principle of nucleic acid \n\nhybridization. A single-stranded \n\nchemiluminescent labeled DNA probe is \n\ncomplementary to the sequence of ribosomal \n\nRNA of fungus.  After the rRNA released from \n\nfungi, the steady DNA-RNA hybrid formed and \n\nthat hybrids are detected and measured by \n\nusing Hologicluminometer. A positive result is \n\ngiven by reading equal to or higher than the \n\npredetermined cut-off values, and an adverse \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  99 Nepjol.info/index.php/njb. \n\n \n\n \n\neffect is the cost less than that of cut-off values \n\n[39].  \n\nPolymerase chain reaction (PCR) \nAssay \nPCR is based on the amplification of the fungal \n\ngene for identifying mycoses. In2003, Guedes et \n\nal. designed a nested PCR based technique for \n\nthe rapid detection of fungi. The \n\noligonucleotides used in this method is based on \n\nthe M - antigens of H. capsulatum var. capsulatum.", "start_char_idx": 10705, "end_char_idx": 13883, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e97489c7-3090-4190-a8e0-411d5b527e26": {"__data__": {"id_": "e97489c7-3090-4190-a8e0-411d5b527e26", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8f7729e6-77c4-4d90-9f8c-9b4292577d08", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "217f23126f48dc3750a84cd91ef1eccb905c44e036334c56d0bd48a87fbe8538", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3263bb80-d5f3-4b95-8e16-226baee3eaad", "node_type": "1", "metadata": {}, "hash": "e6865aefcbec9dc243fdf10c5955e83eec5e06b3b293fcb6c2dfe3c69488a731", "class_name": "RelatedNodeInfo"}}, "text": "capsulatum. \n\nThe oligonucleotidesMsp1F - Msp1R (pair 1) and \n\nMsp2F - Msp2R (pair 2) results amplicon of size \n\n111 bp and 279 bp, respectively[34]; (Msp1F: 5\u2032 - \n\nACA AGA GAC GAC GGT AGC TTC ACG - 3\u2032 \n\nandMsp1R: 5\u2032 - GCG TTG GGG ATC AAG CGA \n\nTGA GCC - 3\u2032), (Msp2F: 5\u2032 - CGG GCC GCG TTT \n\nAAC AGC GCC - 3\u2032 and Msp2R: 5\u2032 - ACC AGC \n\nGGC CAT AAG GAC GTC - 3\u2032). The PCR \n\nreaction was performed with 100ng DNA \n\namplified in a 25 \u00b5l reaction using 20 pmol of \n\noligonucleotides(35 cycles of denaturation, \n\nannealing, and chain extension) [34].  \n\nBy using this protocol,100% specificity and \n\naccuracy were mentioned in the 31 examined \n\nstrains from the animal, soil, and human [34]. \n\nThe results also confirmed the absence of other \n\nfungal strains such as H. capsulatum var. \n\nfarciminosum, Paracoccidioides brasiliensis, \n\nCandida spp., Sporothrix schenckii, Cryptococcus \n\nneoformans,etc in the amplified products[34]. \n\nReal-time PCR (RT- PCR) \nThe real-time RT-PCR or qPCR shows \n\npromising results in the diagnosis of \n\nhistoplasmosis infection, which currently \n\nidentified the H. capsulatum among several other \n\nfungi [35]. Simon et al., (2010) carried out \n\nresearch for the detection of H. capsulatum based \n\non real-time PCR using the TaqMan probe [36]. \n\nFor this study, specific primers and probe \n\n(TaqManlabeled with fluorescent dye 6-\n\ncarboxyfluorescein at 5\u2032 end and a \n\nnonfluorescent quencher at the 3\u2032 end) were \n\nused for the analysis of the internal transcribed \n\nspacer (ITS) region of the rRNA. The \n\noligonucleotides HcITS-167F (5\u2032- \n\nAACGATTGGCGTCTGAGCAT-3\u2032) and HcITS-\n\n229R (5\u2032-GAGATCCGTTGTTGAAAGTTTTGA-\n\n3\u2032) were used. The following probeHcITS-188P \n\n5\u2032-6- FAM- AGAGCGATAATAATCC- MGB -3\u2032) \n\nwas used. The specificity of RT- PCR was \n\ncalculated to be 95.4% in comparison to the \n\nculture method. During the process, there is no \n\nPCR inhibitor detected. Among the 275 samples \n\nwhich were previously reported to be negative \n\nin culture method, 11 samples were reported as \n\npositive by RT-PCR with a specificity of 96.0%. \n\nSimilarly, among the 341 samples tested, zero \n\nnonspecific signals were recorded [36].  \n\nDespite this promising research result, for the \n\ndetection of H. capsulatum in clinical samples, \n\nthere are no currently FDA-approved molecular \n\nassays available. Moreover, to date, the \n\nmolecular approach for the detection of \n\nhistoplasmosis is in a developing stage. \n\nSimilarly, an improved and reliable technique \n\nhas to be designed as a dependable approach for \n\nthe detection of the disease. Regarding the \n\nfuture accessibility of molecular techniques for \n\nthe detection of histoplasmosis is not unclouded \n\nin resource-limited countries like Nepal [37]. \n\nTreatment of Histoplasmosis \nBased on the current status, most patients with \n\nmild acute histoplasmosis limited to lungs will \n\nbe resolved after a month without specific \n\ntreatment. However, the disseminated or the \n\nsevere form of histoplasmosis should be treated \n\nwith antifungal agents[17].", "start_char_idx": 13872, "end_char_idx": 16890, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3263bb80-d5f3-4b95-8e16-226baee3eaad": {"__data__": {"id_": "3263bb80-d5f3-4b95-8e16-226baee3eaad", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e97489c7-3090-4190-a8e0-411d5b527e26", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "bed02f09515e166245686d066dc2f7108ff53bf109dea4931b455bbd0ff47bc3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "559a7e14-ef22-4a12-b9fb-f7b8c743b5bc", "node_type": "1", "metadata": {}, "hash": "1bcc5b4332af22ec385200105906af87b31152437c189d70589c2abf86048db0", "class_name": "RelatedNodeInfo"}}, "text": "The treatment of chronic and severe form of \n\npulmonary histoplasmosis is generally done by \n\nprescribing amphotericin B (3.0\u20135.0 mg/kg \n\ndaily for 1\u20132 weeks, intravenously), followed by \n\nitraconazole (200 mg 3 times per day for 3 days \n\nand after that 200 mg for two times a day, for a \n\ntotal of 3 months) is required and \n\nrecommended[38]. Similarly, for progressive \n\ndisseminated histoplasmosis amphotericin B \n\n(3.0 mg/kg daily) is recommended for about 7-\n\n15days, which is followed by oral drug \n\nitraconazole having 200 mg concentration for \n\ndaily three times for three days and after that \n\n200 mg daily for two times for a total of at least \n\na year). Likewise, for the patients with the \n\nimmunosuppressed condition, itraconazole \n\ndaily (200 mg) is recommended as \n\nprophylaxis[19,38]. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  100 Nepjol.info/index.php/njb. \n\n \n\n \n\nResearch status of histoplasmosis in \nNepal \nIn Nepal, there is poor hygienic practices and \n\nsanitation in major parts of the country and is \n\noffering the home for the continuous emergence \n\nand re-emergence of several life-threatening \n\ninfectious diseases. So, Histoplasmosis could be \n\na serious issue of public health in days to come. \n\nIn Nepal, the majority of the population lives in \n\nrural areas with minimal health care facilities. \n\nThe burden of the disease is much higher in \n\nrural settings compared to the urban areas. \n\nAlthough the pattern of diseases might change \n\nregularly, infectious diseases remain the leading \n\ncause of mortality and morbidity in Nepal. In \n\nmany rural settings, cases of some diseases are \n\noften identified only through their clinical signs \n\nand symptoms. Lack of proper public health \n\nawareness and treatment of infectious diseases \n\nmade people prone to several fungal infections, \n\nincluding Histoplasmosis, which may be a \n\nsevere issue [40]. As the pervasiveness of \n\nHIV/AIDS and Tuberculosis are budding \n\nswiftly, the infection from the Histoplasma may \n\nlead to a significant effect [41]. Thus, careful \n\ndiagnosis and treatment on time are paramount \n\nto control the future outbreaks of \n\nhistoplasmosis. Moreover, the probability of \n\nundiagnosed histoplasmosis dissemination also \n\nurges for the need of proper diagnostic \n\nstrategies of Histoplasma spp. infection in Nepal. \n\nAs per institutional information, there is no \n\nroutine examination and treatment of \n\nhistoplasmosis infection in Tribhuvan \n\nUniversityTeaching Hospital and National \n\nPublic Health Laboratory (NPHL-Nepal), while \n\nboth of them are considered as reference health \n\nservice centres of the country.  \n\nChallenges in Nepal \nAs Nepal is under the way of development, the \n\nbudgeting for the health and its related sector is \n\nvery less. Lack of proper investment in the \n\nhealth sector for the diagnosis and treatment of \n\ndiseases is a significant problem in Nepal. Death \n\nof hundreds of people by unknown disease is \n\nalso prevalent. Lack of proper government \n\nstrategies and operative ventures in the health \n\nsector has been a cardinal cause of those strange \n\ndeaths. Allocation of the budget on the health \n\nsector is only attentive to the treatment of some \n\nsevere disease, which shows that the country \n\nhas less interest in disease diagnosis. It is one of \n\nthe significant consequences of the death of \n\npeople from unknown conditions. Therefore, \n\nearly diagnosis of histoplasmosis is mandatory \n\nto reduce the burden of illness.  \n\nConclusion \nThe clear and accurate picture of the \n\ndissemination of histoplasmosis is complicated \n\nto understand in the resource-limited clinical \n\nsector of Nepal. Many factors could contribute \n\nto this, like lack of proper lab facilities, lack of \n\nexpertise on fungal infections, etc.", "start_char_idx": 16893, "end_char_idx": 20760, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "559a7e14-ef22-4a12-b9fb-f7b8c743b5bc": {"__data__": {"id_": "559a7e14-ef22-4a12-b9fb-f7b8c743b5bc", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3263bb80-d5f3-4b95-8e16-226baee3eaad", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "397753a604726aea4d64ed65d4bcb5eaf78e9697b030424803e52d2bba595749", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4c4e8ce3-1a91-4905-b0c5-0d8edb8df63e", "node_type": "1", "metadata": {}, "hash": "61ec70bc8aeb3ff2e07679184a96093b501413774d17102fbce257c409199804", "class_name": "RelatedNodeInfo"}}, "text": "Additionally, \n\nnot much reliable data are available from the \n\ngovernment and academia due to incompetence \n\nin the diagnosis strategies. The published data \n\nare also in the form of case reports based on a \n\nhospital visit. It is already emphasized that the \n\npockets of endemicity do exist in Nepal, which \n\ncould lead to a significant impact on public \n\nhealth. Hence, careful considerations and the \n\nmolecular approach of disease identification \n\nmay be a suitable alternative. To address this \n\nproblem, further research should be done \n\nimmediately in Nepal \n\nConflict of Interest  \nThe authors declare no conflict of interest. \n\nReferences \n1. Chakrabarti A, Slavin MA :Endemic fungal \n\ninfections in the Asia-Pacific region. Med \nMycol 2011 49: 337\u2013344. \n\n2. Stobierski MG, Hospedales CJ, Hall WN, \nRobinson-Dunn B, Hoch D,  Sheill DA: \nOutbreak of histoplasmosis among \nemployees in a paper factory - Michigan. J \nClin Microbiol 1993 34:1220\u20131223. \n\n3. Gajurel K, Dhakal R, Deresinski S: \nHistoplasmosis in transplant recipients. \nClinical Transplantatfile 2017 13:1-7. \n\n4. Wheat LJ, Azar MM, Bahr NC, Spec A: \nHistoplasmosis. Chest. 2016 30: 207\u2013227. \n\n5. Kilburn CD, McKinsey D S: Recurrent \nmassive pleural effusion due to pleural, \npericardial, and epicardial fibrosis in \nhistoplasmosis. Chest. 1991 100: 1715\u20131717. \n\n6. Holbrook ED, Kemski MM, Richer SM, Wheat \nLJ, Rappleye CA: Glycosylation and \nimmunoreactivity of the Histoplasma \ncapsulatum Cfp4 yeast-phase exoantigen. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  101 Nepjol.info/index.php/njb. \n\n \n\n \n\nInfect Immun. 2014 82:4414\u20134425. \n7. Assi M, Martin S, Wheat LJ, Hage C, Freifeld \n\nA, Avery R,  et al.: Histoplasmosis after solid \norgan transplant. Clin Infect Dis. 2013 \n57:1542\u20131549. \n\n8. Kauffman CA, Israel KS, Smith JW, White \nAC, Schwarz J, Brooks GF: Histoplasmosis in \nimmunosuppressed patients. Am J Med. 1978 \n64: 923\u2013932. \n\n9. Guimar\u00e3es AJ, Nosanch JD, Zancop\u00e9-Oliveira \nRM: Diagnosis of histoplasmosis. Brazilian J \nMicrobiol. 2006  37:1\u201313. \n\n10. Gugnani HC: Histoplasmosis in Africa: a \nreview. Indian J Chest Dis Allied Sci. 2000 42: \n271\u2013277. \n\n11. Faiolla RCL, Coelho MC, Santana RdeC, \nMartinez R: Histoplasmosis in \nimmunocompetent individuals living in an \nendemic area in the Brazilian Southeast. Rev  \nSoc Bras Med Trop. 2013 46: 461\u2013465. \n\n12. Eissenberg LG, Goldman WE: Histoplasma \nvariation and adaptive strategies for \nparasitism: New perspectives on \nhistoplasmosis. Clin Microbiol Rev. 1991 4: \n411\u2013421. \n\n13.", "start_char_idx": 20761, "end_char_idx": 23388, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4c4e8ce3-1a91-4905-b0c5-0d8edb8df63e": {"__data__": {"id_": "4c4e8ce3-1a91-4905-b0c5-0d8edb8df63e", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "559a7e14-ef22-4a12-b9fb-f7b8c743b5bc", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "6afbcd4345d443376e3a737482dd591ae41a5aa6a76446dadb8b297aa1b189bd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "39a36414-49ab-4bd6-9743-5169a03c0cf4", "node_type": "1", "metadata": {}, "hash": "f05c54e460c60d0a9543fd19eec0a8491bee0f8947aa4be2d2a2dd6a86d63904", "class_name": "RelatedNodeInfo"}}, "text": "1991 4: \n411\u2013421. \n\n13. Pervez MM, Cobb B, Matin N, Shahrin L, \nFord ER, Pietroni M: Disseminated \nhistoplasmosis in a patient with advanced \nHIV disease-lessons learnt from \nBangladesh. J Heal Popul Nutr. 2010 28:305\u2013\n307. \n\n14. Guha A, Kulkarni HS: Histoplasmosis - \nPatient Information Series. Am J Respir Crit \nCare Med. 2012 185: 1\u20132. \n\n15. Silverman FN, Schwarz J, Lahey E: \nHistoplasmosis. Am J Med 1945 6: 410\u2013460. \n\n16. De D, Nath UK: Disseminated \nHistoplasmosis in Immunocompetent \nIndividuals- not a so Rare Entity, in India. \nMed J of Hem and Infe Dis. 2015 1: 3\u20137. \n\n17. Kauffman CA: Histoplasmosis: A clinical \nand laboratory update. Clin Microbiol Rev.  \n2006 19: 110\u2013122. \n\n18. Kauffman CA: Histoplasmosis: A clinical \nand laboratory update. Clin Microbiol Rev. \n2007 20:115\u2013132. \n\n19. Bahr NC, Antinori S, Wheat LJ, Sarosi G A: \nHistoplasmosis Infections Worldwide: \nThinking Outside of the Ohio River Valley. \nCurr Trop Med Reports. 2015 2:70\u201380. \n\n20. Thapa S, Jha SC, Trotter AB: Images in \nclinical tropical medicine persistent fever \nand skin lesions due to histoplasmosis in a \nboy from rural Nepal. Am J Trop Med Hyg. \n2016 94: 70-80. \n\n21. Karki KB, Maskey J, Giri M, Pandey AR, \nMakai P, Subedi R, et al: Assessment of \nChronic Kidney Disease Support Program \nof Government of Nepal. Nepal Health \nResearch Council, Nepal 2016. \n\n22. Acharya RP: Epidemic of Diabetes in Urban \nNepal - Time To Act. J Nepal Med Assoc.  2003 \n\n42:1-2. \n23. NCASC: Fact sheet 1: HIV Epidemic Update \n\nof Nepal Facts about HIV Epidemic in Nepal \nHIV Estimates in Nepal. 2018. \n\n24. Randhawa HS, Gugnani HC: Occurrence of \nHistoplasmosis in the Indian Sub-\nContinent: An Overview and Update. J Med \nRes Pr. 2018 7: 3\u201371. \n\n25. Amatya R, Koirala R, Khanal B, Gurung R, \nRijal A, Dhakal K: Case Report \nhistoplasmosis\u202f: first case from nepal, 2010 \nDepartment of Microbiology. Nepal 60\u201363. \n\n26. Sharma N, Adhikari R: Adrenal involvement \nin histoplasmosis. J Pathol Nep. 2019 9: 1502\u2013\n1504. \n\n27. Gandhi V, Singh A, Woods GL, Epelbaum O: \nA 66-year-old woman with fever, cough, and \na tongue lesion, Chest. 2015 147: 140\u2013147. \n\n28. Mansoor CA, Bhargavan PV, Rajanish R, and \nNair LR: Disseminated histoplasmosis. \nIndian J Orthop. 2013 47:  639\u2013642. \n\n29. Hage CA, Connolly P, Horan D, Durkin M, \nSmedema M, Zarnowski R, et al: \nInvestigation of the efficacy of micafungin \nin the treatment of histoplasmosis using two \nNorth American strains of Histoplasma \ncapsulatum. Antimicrob Agents Chemother. \n2011 55: 4447\u20134450.", "start_char_idx": 23365, "end_char_idx": 25889, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "39a36414-49ab-4bd6-9743-5169a03c0cf4": {"__data__": {"id_": "39a36414-49ab-4bd6-9743-5169a03c0cf4", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4c4e8ce3-1a91-4905-b0c5-0d8edb8df63e", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "f8e97705c59eced9727585f72c59387f89878db705d0015bb2239d95687e68c8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fb414694-5276-49d2-a430-eac4c526e6d8", "node_type": "1", "metadata": {}, "hash": "cf79bc622f4ab7ad71765830e7cc5616121205c39b99861b28df014aa95147da", "class_name": "RelatedNodeInfo"}}, "text": "2011 55: 4447\u20134450. \n\n30. Gudala M: Delayed Diagnosis of Pulmonary \nHistoplasmosis in an Immunocompetent \nYoung Asthmatic Female from Florida, a \nNon-Endemic Area for Histoplasma \nCapsulatum. Med Mycol Open Access. 2015 \n1:1\u20136. \n\n31. Rajeshwari M, Xess I, Sharma MC, Jain D: \nAcid-fastness of Histoplasma in surgical \npathology practice. J Pathol Transl Med. 2017 \n51, 482\u2013487. \n\n32. Azar MM, Hage CA: Laboratory diagnostics \nfor histoplasmosis. J Clin Microbio.  2017 55: \n1612\u20131620. \n\n33. Picardi JL, Kauffman CA, Schwarz J, Phair JP: \nDetection of precipitating antibodies to \nHistoplasma capsulatum by \ncounterimmunoelectrophoresis. Am Rev \nRespir Dis. 1976 114: 171\u2013176. \n\n34. Guedes HL, Guimar\u00e3es AJ, Muniz Mde M, \nPizzini CV, Hamilton AJ, Peralta JM et al: \nPCR assay for identification of Histoplasma \ncapsulatum based on the nucleotide \nsequence of the M antigen. J Clin Microbiol. \n2003 41(2): 535\u2013539. \n\n35. Muraosa Y, Toyotome T, Yahiro M, Watanabe \nA, Shikanai-Yasuda MA, Kamei K: Detection \nof Histoplasma capsulatum from clinical \nspecimens by cycling probe-based real-time \nPCR and nested real-time PCR. Med Mycol. \n2016 54: 433\u2013438. \n\n36. Simon S, Veron V, Boukhari R, Blanchet D, \nand Aznar C: Detection of Histoplasma \ncapsulatum DNA in human samples by real-\ntime polymerase chain reaction. Diagn", "start_char_idx": 25870, "end_char_idx": 27186, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fb414694-5276-49d2-a430-eac4c526e6d8": {"__data__": {"id_": "fb414694-5276-49d2-a430-eac4c526e6d8", "embedding": null, "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-190", "node_type": "4", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "8a7572c22cc6d70d213212ac2216f1b66225fd9c103a5c7c75704e5b7c5cd685", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "39a36414-49ab-4bd6-9743-5169a03c0cf4", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "4615761ee2a1d9e14f026871fc88bb3490359c0ae6bff69eff4bbd07bca3dfcb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "78090a98-f3fd-4ed0-9fbd-bb0d45d31293", "node_type": "1", "metadata": {}, "hash": "a5a6c07972b3428e2bbd2bdcabc482e7d0bb78cb923dba2cbdc7fa55db258659", "class_name": "RelatedNodeInfo"}}, "text": "Diagn \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 96-102                Bhandari et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  102 Nepjol.info/index.php/njb. \n\n \n\n \n\nMicrobiol Infect Dis. 2010 66: 268\u2013273. \n37. Ha HTT, Ohnoc H , Tram NT, My TN, Hai PT, \n\nHoa LM, et al: Histoplasmosis in Pulmonary \nInfection Patients from Hospitals in Hanoi, \nVietnam. Appl Microbiol. 2017 3: 3\u20135. \n\n38. Wheat LJ, Freifeld AG, Kleiman MB, Baddley \nJW, McKinsey DS, Loyd JE et al: Clinical \nPractice Guidelines for the Management of \nPatients with Histoplasmosis: 2007 Update \nby the Infectious Diseases Society of \nAmerica. Clin Infect Dis. 2007 45: 807\u2013825. \n\n39. Wu XS, Yang YY, Zhang LJ, Lu GM: \nDiagnosis of Histoplasmosis. Chinese J \nRadiol. 2008 42: 771\u2013773. \n\n40. Baker J, Setianingrum F, Wahyuningsih R, \nDenning DW: Mapping histoplasmosis in \nSouth East Asia \u2013 implications for diagnosis \nin AIDS. Emerg Microbes Infect. 2019 8:1139\u2013\n1145.  \n\n41. Nacher M, Blanchet D, Bongomin F, \nChakrabarti A, Couppi\u00e9 P, Demar M, et al: \nHistoplasma capsulatum Antigen Detection \nTests As An Essential Diagnostic Tool For \nPatients With Advanced HIV Disease In \nLow And Middle Income Countries: A \nSystematic Review Of Diagnostic Accuracy \nStudies. PLoS Negl Trop Dis. 2018 12:1-12.", "start_char_idx": 27181, "end_char_idx": 28477, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "78090a98-f3fd-4ed0-9fbd-bb0d45d31293": {"__data__": {"id_": "78090a98-f3fd-4ed0-9fbd-bb0d45d31293", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fb414694-5276-49d2-a430-eac4c526e6d8", "node_type": "1", "metadata": {"identifier": "njb-190", "author": "Bhandari, Sudip; Rayamajhee, Binod; Dhungel, Laxmi; Poudel, Sami; Gaire, Bhagwati; Shrestha, Sunil; Parajuli, Niranjan", "title": "Infectious sources of Histoplasmosis and molecular techniques for its identification", "date": "2019-12-29", "file": "njb-190.pdf"}, "hash": "394cf9b90b16a34f909322758461e27dde589de195457b776d272c77524c194b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b3e50d60-4e20-4177-8880-6bd8f19a6d5d", "node_type": "1", "metadata": {}, "hash": "deb688cc6840b300e5503aadd7b9cb985beaf3afc725a311952a0dd66a34880c", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7   DOI: https://doi.org/10.3126/njb.v7i1.26944 \n\n\u00a9NJB, Biotechnology Society of Nepal  1 Nepjol.info/index.php/njb. \n\nORIGINAL RESEARCH ARTICLE \n\n \n\nAntibiotic Susceptibility Profile of Respiratory Pathogens \nObtained at Tertiary Care Hospital from Western Nepal \n\nDeepak Subedi1, Surya Prasad Devkota1,2,3*, Dharm Raj Bhatta4, Binita Koirala Sharma1,  \nAshmita Paudel2, Krishna Gurung1,5, Damodar Gautam1 \n\n1Pokhara Bigyan Tatha Prabidhi Campus, Nayabazar, Pokhara \n2Regional College of Health Science and Technology, Nayabazar, Pokhara \n\n3 School of Health and Allied Sciences, Pokhara University, Pokhara \n4Manipal College of Medical Sciences, Pokhara \n\n5Prithvi Narayan Campus, Pokhara \n\nABSTRACT \nThe prevalence and drug resistance of the respiratory pathogens is increasing gradually in \nNepal. However, their detail study is rare in the western region of Nepal. Hence, this study was \ncarried out to know the incidence and antibiotic susceptibility profile of the respiratory \npathogens obtained at a tertiary care center located at Pokhara. 139 pathogens were isolated \nfrom 460 clinical samples included.  Significant pathogens were Gram-negative bacteria 94 \n(67.62%), followed by 28 (20.15%) Candida, and Gram-positive isolates 17 (12.23%). The growth \nrate was significantly higher for sputum samples in comparison to throat swabs. Pseudomonas \naeruginosa, Klebsiella pneumoniae, and Acinetobacter spp were significant Gram-negative isolates \nwhile Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes were Gram-\npositive pathogens. Sensitivity rate was higher for colistin and imipenem among Gram-negative \nisolates while lower sensitivity was for cefepime. Vancomycin was effective against all tested \nGram-positive isolates while erythromycin and ciprofloxacin were less effective. \nKeywords: respiratory pathogens, western Nepal, Pseudomonas aeruginosa, Acinetobacter spp, \n\nKlebsiella pneumonia, S. aureus. \n\n*Corresponding Author \n\nEmail: devkotasp1@gmail.com \n\nIntroduction \nInfection of the respiratory tract is a significant \n\ncause of mortality as well as morbidity among the \n\nelderly and young people of Nepal [1]. \n\nManagement of these infections is more complex \n\nin developing countries due to the lack of the \n\nproper identification of pathogens and their \n\nsuitable treatment [2]. The causative agents of the \n\nrespiratory infections are not detected in many \n\ncases hence physicians depend on clinical \n\nmanifestations for the diagnosis [3]. Klebsiella \n\npneumaniae, Streptococcus pneumoniae, and \n\nHaemophilus influenzae are the significant \n\npathogens causing respiratory infections [4, 5].   \n\nIn addition to this, pathogens like Staphylococcus \n\naureus, Acinetobacter, and Pseudomonas aeruginosa \n\nare also frequently isolated from respiratory \n\nspecimens [6, 7].   \n\nPolymicrobial respiratory infections by two or \n\nmore bacteria, two or more viral pathogens, and \n\nmixed viral and bacterial pathogens also have \n\nbeen reported [4, 5]. Pneumonia and infection of \n\nlower respiratory tract are the causes of more \n\nthan 4 million deaths per year and this problem \n\nis more frequent in middle-and low-income \n\nnations [8]. Drug resistance among these \n\npathogens has been increasing as there are \n\nmultiple reports of multidrug resistance among \n\nvarious respiratory pathogens and many of them \n\nare not susceptible to several routine antibiotics \n\n[4, 9].", "start_char_idx": 47, "end_char_idx": 3527, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b3e50d60-4e20-4177-8880-6bd8f19a6d5d": {"__data__": {"id_": "b3e50d60-4e20-4177-8880-6bd8f19a6d5d", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "78090a98-f3fd-4ed0-9fbd-bb0d45d31293", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "407f002b20c8dea1c6a89612fb76464cc126233624a62691f073dcd374a5a525", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0f840eb5-c167-43c5-864d-d8ef137bbd5a", "node_type": "1", "metadata": {}, "hash": "821c053216cc4ffd47dc3519bb7f2ec8edda31f33668dffe5a3bbdfe3b56db42", "class_name": "RelatedNodeInfo"}}, "text": "Though most of the bacterial respiratory \n\ninfections are treatable, the huge death is due to \n\na lack of proper preventive measures and \n\nunavailability of healthcare facilities [8]. Several \n\nreports are indicating a gradual increase in \n\nantibiotic resistance among many bacterial \n\npathogens responsible for respiratory diseases \n\n[10, 11]. \n\nThe imprudent use of antibiotics for treating \n\nthese infections has resulted in a very rapid \n\nincrease in drug resistance of the respiratory \n\npathogens [12]. Though various pathogens [13] \n\nand risk factors [14] are associated with \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  2 Nepjol.info/index.php/njb. \n\n \n\n \n\nrespiratory infections, their detail study is very \n\nlimited in this region. Surveillance study on \n\nrespiratory pathogens was imminent in this part \n\nof the nation. Hence this study was done to access \n\nthe prevalence, distribution and drug resistance \n\nprofile of the respiratory pathogens isolated at a \n\ntertiary care center of western Nepal. \n\nMaterials and Methods \nStudy site and duration \n\nThe study site was Manipal Teaching Hospital, a \n\n750-bed multi-specialty tertiary care hospital \n\nlocated at Pokhara, Nepal. The study was \n\nconducted for a period of six months (July 2016 \n\nto January 2017) at the Microbiology laboratory \n\nof the hospital.  \n\nSample collection \n\nSamples were collected from patients with \n\nclinical symptoms of respiratory tract infection as \n\nindicated by physicians. For the collection of \n\nsputum samples, patients were given various \n\ninstructions on how to collect a sputum sample \n\ncorrectly. Clean, well-labeled, wide-necked, dry \n\nand leak-proof screw-cap container was \n\nprovided to the patients for sputum collection. \n\nThe sample was accepted only if it was sputum \n\nbut samples containing saliva, nasal secretions, \n\nmucus, etc. were not analyzed. In the case of \n\nunacceptable samples, repeated samples were \n\nrequested. Throat sample was collected by \n\ntrained personals. The patient was first allowed \n\nto sit comfortably in a good light and using a \n\ntongue depressor the throat was observed for any \n\nswelling, redness, pus, ulcerations, exudates, and \n\npresence of the membrane. A sterile cotton swab \n\nwas used to collect a sample of the infected \n\nthroat. Special care was taken not to contaminate \n\nthe swab with saliva and placed it into a sterile \n\ncontainer.  \n\nMacroscopic and microscopic \nexamination of the sample \n\nAppearance, as well as presence or absence of \n\nblood in the given sputum, was observed and \n\nnoted. Similarly, gram staining was performed \n\nfrom both sputum and throat swabs to observe \n\npus cells and bacteria. \n\nIsolation and identification \n\nRespiratory tract samples obtained from both \n\nadmitted and outpatients visiting the hospital \n\nwere included in this study. Soon after collection, \n\nall the specimens were cultured on chocolate agar \n\nand 5% sheep blood agar. The inoculated plates \n\nwere then incubated on candle jar for 18-24 \n\nhours. Optochin and bacitracin discs were placed \n\non the primary inoculation for the presumptive \n\nscreening of S. pneumoniae and H. influenzae as \n\nwell as S. pyogens respectively. Identification of \n\nthe pure culture was carried out by observing \n\ncolony characteristics and gram staining \n\nfollowed by oxidase test, catalase test, coagulase \n\ntest, urease test, TSI test, and IMViC tests. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  3 Nepjol.info/index.php/njb. \n\n \n\n \n\nAntibiotic susceptibility test \n\nKirby-Bauer disc diffusion method was used \n\nusing Muller Hinton agar (Hi-Media, India). \n\nClinical and Laboratory Standards Institute \n\n(CLSI) guidelines were used for the \n\ninterpretation of the results [15].", "start_char_idx": 3528, "end_char_idx": 7448, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0f840eb5-c167-43c5-864d-d8ef137bbd5a": {"__data__": {"id_": "0f840eb5-c167-43c5-864d-d8ef137bbd5a", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b3e50d60-4e20-4177-8880-6bd8f19a6d5d", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "ce168610575b19a70fb466df92c81e96d9f6af3b8bdd356a10131ab41e196885", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "995bb380-8bad-49ba-b8c5-b9ccddb7bf6e", "node_type": "1", "metadata": {}, "hash": "6c3fc8d187063e383253f5118eda49fcbaab647dfbec17ebad9d0556be87ce89", "class_name": "RelatedNodeInfo"}}, "text": "0.5 Mac Farland \n\nsuspension of the isolates were used for \n\ninoculation. Amikacin (30\u00b5g), Azithromycin \n\n(15\u00b5g), Cefepime (30\u00b5g), Ceftriaxone (30\u00b5g), \n\nCiprofloxacin (5\u00b5g), Colistin, (10\u00b5g) Co-\n\nTrimoxazole (25\u00b5g), Imipenem (10\u00b5g), \n\nPiperacillin/ Tazobactam (100\u00b5g/10), \n\nVancomycin (30\u00b5g), Levofloxacin (5\u00b5g), \n\nTeicoplanin (30\u00b5g), and Erythromycin (15\u00b5g) \n\nwere used for the test. E. coli ATCC 25922 was \n\nused as a control organism for Gram-negative \n\nisolates while Staphylococcus aureus ATCC 25923 \n\nwas used as a control organism for Gram-positive \n\nisolates during this test.  \n\nResults \nAmong 460 samples, 139 (30.2%) showed the \n\ngrowth of respiratory pathogens. The incidence \n\nof the pathogens was higher from the sputum \n\nsample in relation to the throat sample (Table 1). \n\nPseudomonas aeruginosa, Acinetobacter spp., \n\nKlebsiella pneumoniae, Staphylococcus aureus, \n\nEscherichia coli, and Candida spp. were the \n\ncommon pathogens isolated followed by \n\nHaemophilus influenzae, Streptococcus pneumoniae, \n\nStreptococcus pyogenes, and Enterobacter spp. \n\nStreptococcus pyogenes were isolated only from \n\nthroat swab while all gram-negative isolates were \n\nonly from the sputum sample. Only Candida was \n\nisolated from both of the samples (Table 2). \n\nTable 1: Total cases and types of samples \n\nSamples \nGrowth \n\npositive (%) \n\nGrowth \n\nnegative (%) \nTotal (%) \n\nSputum 134 (29.13%) 299 (65%) 433 (94.13%) \n\nThroat \n\nswab \n5 (1.07%) 22 (4.8%) 27 (5.87%) \n\nTotal 139 (30.2%) 321 (69.8%) 460 (100%) \n\nVancomycin was the most effective drug for all \n\nthree Gram-positive pathogens as all of these \n\nisolates were sensitive to vancomycin. \n\nTeicoplanin and clindamycin were also highly  \n\n \n\nTable 2: Distribution of Gram-positive cocci, Gram-\nnegative bacilli and Candida spp. in throat swab and \nsputum samples \n\nMicroorganisms \n\nSamples \n\nTotal (%) \nThroat \nswab \n(%) \n\nSputum \n(%) \n\nPseudomonas \naeruginosa \n\n0 31(22.3) 31 (22.3) \n\nAcinetobacter spp 0 26 (18.7) 26 (18.7) \n\nKlebsiella \npneumoniae \n\n0 23 (16.55) 23 (16.55) \n\nStaphylococcus \naureus \n\n0 9 (6.47) 9 (6.47) \n\nEscherichia coli 0 8 (5.76) 8 (5.76) \n\nHaemophilus \ninfluenza \n\n0 4 (2.88) 4 (2.88) \n\nStreptococcus \npneumoniae \n\n0 4 (2.88) 4 (2.88) \n\nStreptococcus \npyogenes \n\n4 (2.88) 0 4 (2.88) \n\nEnterobacter spp 0 2 (1.44) 2 (1.44) \n\nCandida spp.", "start_char_idx": 7449, "end_char_idx": 9770, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "995bb380-8bad-49ba-b8c5-b9ccddb7bf6e": {"__data__": {"id_": "995bb380-8bad-49ba-b8c5-b9ccddb7bf6e", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0f840eb5-c167-43c5-864d-d8ef137bbd5a", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "76b55bd9c1a0eb0c1d778258e9c687e334ae2669fa5ce94e9444bb96dc84cb51", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2db0b8bd-4ad0-4d6d-91b9-b2721b5a2958", "node_type": "1", "metadata": {}, "hash": "9e8caf0b48ef51768f606e021feb5195c2c8c58b65d86835f1f4ca323a4c5f79", "class_name": "RelatedNodeInfo"}}, "text": "1 (0.72) 27 (19.42) 28 (20.14) \n\nTotal 5 (3.6) 134 (96.4) 139 (100) \n\nactive against S. aureus and Streptococcus pyogenes \n\nrespectively while other drugs were less effective \n\nas there was resistance ranging from 22 to 100% \n\nfor antibiotics other than vancomycin, \n\nteicoplanin, and clindamycin (Table 3). Colistin \n\nwas the most effective drug for treating \n\nPseudomonas aeruginosa and Acinetobacter. Least \n\ndrug resistance was noted on Enterobacter while \n\nthe highest resistance was observed on \n\nAcinetobacter. Resistance towards all common \n\nantibiotics was observed in all Gram-negative \n\npathogens excluding Enterobacter with varying \n\npercentages (Table 4). \n\nDiscussion \nNearly one-third of the samples were positive for \n\nthe respiratory pathogens. Findings of the \n\nShrestha et al (2005) also reported that 31% of the \n\nsputum samples were positive for pathogens \n\n[16]. Among the isolated pathogens Gram-\n\nnegative isolates were more prevalent than \n\nGram-positive pathogens.  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  4 Nepjol.info/index.php/njb. \n\n \n\n \n\nTable 3: Antimicrobial susceptibility profiles of Gram-positive isolates \n\n \n\n \n\nAntibiotics \n\nused \n\nPathogens \n\nStaphylococcus aureus \n\n(n=9) \n\nStreptococcus pyogenes \n\n(n=4) \n\nStreptococcus pneumoniae \n\n(n=4) \n\nSensitive \n\nNo. (%) \n\nResistant \n\nNo. (%) \n\nSensitive \n\nNo. (%) \n\nResistant \n\nNo. (%) \n\nSensitive No. \n\n(%) \n\nResistant \n\nNo. (%) \n\nAmikacin 7 (77.8) 2 (22.2) 3 (75) 1 (25) - - \n\nCiprofloxacin 3 (33.3) 6 (66.7) - - - - \n\nClindamycin 2 (22.2) 7 (77.8) 4 (100) 0 2 (50) 2 (50) \n\nCo-Trimoxazole - - - - 1 (25) 3 (75) \n\nErythromycin 2 (22.2) 7 (77.8) 0 4 (100) 2 (50) 2 (50) \n\nLevofloxacin - - 0 4 (100) 3 (75) 1 (25) \n\nTeicoplanin 9 (100) 0 - - - - \n\nVancomycin 9 (100) 0 4 (100) 0 4 (100) 0 \n\nA very high incidence of Gram-negative \n\npathogens among respiratory samples are also \n\nreported earlier [2, 12, 16]. Gram-negative \n\nisolates like Pseudomonas aeruginosa, Acinetobacter \n\nspp., Klebsiella pneumoniae were predominant. \n\nThese findings were in accordance with the \n\nprevious studies [13, 17]. Similarly, Staphylococcus \n\naureus was the major Gram-positive pathogens. \n\nPathogens like Streptococcus pneumoniae, \n\nStreptococcus pyogenes were also isolated from the \n\nrespiratory samples. The predominance of \n\nStaphylococcus aureus among Gram-positive \n\npathogens followed by Streptococcus spp. is \n\ncommon from respiratory samples [1, 3, 18].", "start_char_idx": 9771, "end_char_idx": 12315, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2db0b8bd-4ad0-4d6d-91b9-b2721b5a2958": {"__data__": {"id_": "2db0b8bd-4ad0-4d6d-91b9-b2721b5a2958", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "995bb380-8bad-49ba-b8c5-b9ccddb7bf6e", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "932c65e46bb74452b745d8820141b88de52b5c8f8e9cb0b28d3da473ab0d4040", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ea246ce2-22d1-43e1-9bbe-57241d2614ae", "node_type": "1", "metadata": {}, "hash": "156c167ddc9cdc5400b9658e9f4c41194bc1a6fea58f0e2caa8f4642b8b029d6", "class_name": "RelatedNodeInfo"}}, "text": "is \n\ncommon from respiratory samples [1, 3, 18]. \n\nCandida isolates were also the major cause of \n\nTable 4: Antimicrobial susceptibility profiles of Gram-negative bacilli \n\nAntibiotics \n\nUsed \n\nPathogens \n\nPseudomonas \n\naeruginosa \n\n(n=31) \n\nAcinetobacter \n\nspp \n\n(n=26) \n\nKlebsiella \n\npneumoniae \n\n(n=23) \n\nE. coli \n\n \n\n(n=8) \n\nHaemophilus \n\ninfluenzae \n\n(n=4) \n\nEnteroba\n\ncter \n\nspp \n\n(n=2) \n\nS (%) S (%) S (%) S (%) S (%) S (%) \n\nAmikacin 26 (83.9) 8 (30.8) 15 (65.2) 8(100) - 2 (100) \n\nAzithromycin - - - - 2 (50) - \n\nCefepime 7 (22.6) 6 (23.1) 6 (26.1) 0 - 2 (100) \n\nCeftriaxone - - - - 3 (75) - \n\nCiprofloxacin 22 (71) 8 (30.8) 10 (43.5) 2 (25) 4 (100) 2 (100) \n\nColistin 31 (100) 26 (100) - - - - \n\nCo-Trimoxazole - - 8 (34.8) 5 (62.5) 2 (50) 2 (100) \n\nImipenem 23 (74.2) 9 (34.6) 21 (91.3) 7(87.5) - 2 (100) \n\nPiperacillin/ \n\nTazobactam \n13 (42) 5 (19.2) 7 (30.4) 3 (37.5) - 2 (100) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  5 Nepjol.info/index.php/njb. \n\n \n\n \n\nrespiratory infections as indicated by previous \n\nauthors [3, 19]. \n\nVancomycin was the most reliable treatment \n\noption for all of the gram-positive pathogens \n\nisolated. The high sensitivity of vancomycin \n\nagainst S. aureus and Streptococcus spp isolated \n\nfrom respiratory tract infection is found \n\nelsewhere [18, 19, 20]. More than 66% of S. aureus \n\nwere resistant to Ciprofloxacin, Clindamycin, \n\nand Erythromycin. Increasing resistance of S. \n\naureus towards these drugs is common globally \n\n[18, 20]. All Streptococcus pyogenes isolated in this \n\nstudy were sensitive to Clindamycin and \n\nVancomycin while they were absolutely resistant \n\nto Levofloxacin and Erythromycin. Very similar \n\nantibiotic sensitivity patterns of Streptococcus \n\npyogenes are reported earlier [21, 22]. The majority \n\nof Streptococcus pneumoniae were resistant against \n\nCo-Trimoxazole, Clindamycin, and \n\nErythromycin. An elevated level of resistance \n\namong Streptococcus pneumoniae obtained from \n\nsputum and other samples are reported from \n\ndifferent countries [23]. \n\nColistin resistance was not observed in \n\nPseudomonas aeruginosa and Acinetobacter spp. \n\nindicating that this drug is still effective as a last \n\nresort. There are many reports of absolute colistin \n\nsensitivity among gram-negative respiratory \n\npathogens [24, 25]. However, there is a report of \n\ncolistin resistance among multidrug-resistant \n\nAcinetobacter spp isolates obtained from ICU \n\npatients suffering from respiratory tract infection \n\nin Nepal [26]. Imipenem and amikacin resistance \n\nwas less in gram-negative isolates in comparison \n\nto other antibiotics like Cefepime, Ciprofloxacin, \n\nPiperacillin/ Tazobactam.", "start_char_idx": 12267, "end_char_idx": 15036, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ea246ce2-22d1-43e1-9bbe-57241d2614ae": {"__data__": {"id_": "ea246ce2-22d1-43e1-9bbe-57241d2614ae", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2db0b8bd-4ad0-4d6d-91b9-b2721b5a2958", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "9d0b5f9b5d98b694bdeda427d84b1256b796c09f9598e5e52406ff9120e8a695", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5916b492-ba6d-4c93-8693-3d5ceb5761c5", "node_type": "1", "metadata": {}, "hash": "d9ca294482e76b116f7bfbc0d6cd0b55a558ec257fb99c8481bcdfd40fc749aa", "class_name": "RelatedNodeInfo"}}, "text": "Less resistance is \n\ndetected in common respiratory gram-negative \n\npathogens against Imipenem and amikacin [24, \n\n27]. Drug resistance was higher in Acinetobacter \n\nspp among Gram-negative pathogens. Highly \n\nelevated antibiotic resistance among Acinetobacter \n\nisolated from respiratory tract infection is \n\nreported previously in Nepal [17, 26]. We also \n\nnoticed drug resistance among Haemophilus \n\ninfluenzae as some isolates were not sensitive to \n\nAzithromycin, Ceftriaxone, and Co-Trimoxazole. \n\nAntibiotic-resistant Haemophilus influenzae \n\ncausing respiratory tract infection is not new [2]. \n\nConclusion \nPseudomonas aeruginosa, Acinetobacter spp., \n\nKlebsiella pneumoniae, Candida, and S. aureus were \n\nthe major isolates causing respiratory infection. \n\nMany of these isolates were resistant to various \n\nroutine drugs. To prevent the further increase of \n\nantimicrobial resistance among the respiratory \n\nisolates antibiotic susceptibility testing must be \n\ndone before therapy. \n\nAuthor Contributions \nDS and SPD designed the research proposal, DS \n\nperformed the lab works, DRB, BKS, and KG \n\nsupervised the work, AP, SPD and DG wrote the \n\narticle. All the authors read, revised, and \n\nfinalized the manuscript. \n\nAcknowledgment \nThe authors are thankful to all the members of the \n\nMicrobiology laboratory of Manipal Teaching \n\nHospital for their continuous support during this \n\nwork as well as to the patients for providing \n\nsamples for this research.  \n\nReferences  \n1. Shrestha S, Acharya A, Gautam A, Nepal H, \n\nGautam R, Ansari S, et al: Lower respiratory \ntract pathogens and their antimicrobial \nsusceptibility pattern in a medical hospital of \ncentral Nepal. Int J BiomedAdv Res. 2013 4(5): \n335-340.  [https://www.researchgate.net/ \npublication/272566188] \n\n2. Khan S, Priti S, Ankit S: Bacteria Etiological \nAgents Causing Lower Respiratory Tract \nInfections and Their Resistance Patterns. Iran \nbiomed J. 2015 19(4): 240-46.[https://www.ncbi. \nnlm.nih.gov/pmc/articles/PMC4649860/] \n\n3. Ahmed SMA, Abdelrahman SS, Saad DM, \nOsman IS, Osman MG, Khalil EAG: Etiological \ntrends and patterns of antimicrobial \nresistance in respiratory infections. Open \nMicrobiol J. 2018 12: 34-40. [https://www.ncbi. \nnlm.nih.gov/pmc/articles/PMC5897982/] \n\n4. Bhuyan GS, Hossain MA, Sarker SK, Rahat A, \nIslam MT, Haque TN, et al: Bacterial and viral \npathogen spectra of acute respiratory \ninfections in under-5 children in hospital \nsettings in Dhaka city. PLoS ONE. 2017 12 (3): \n448-454. [https://journals.plos.org/plosone \n/article?id=10.1371/journal.pone.0174488] \n\n5. Chen J, Li X, Wang W, Jia Y, Lin F, Xu J: The \nprevalence of respiratory pathogens in adults \nwith community-acquired pneumonia in an \noutpatient cohort. Inf Drug Resist. 2019 12:2335-\n2341. [https://www.ncbi.nlm.nih.gov/ \npmc/articles/PMC6679678/] \n\n6. Chung DR, Song JH, Kim SH, Thamlikitkul V, \nHuang SG, Wang H, et al: High prevalence of \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  6 Nepjol.info/index.php/njb. \n\n \n\n \n\nmultidrug-resistant nonfermenters in \nhospital-acquired pneumonia in Asia.", "start_char_idx": 15037, "end_char_idx": 18233, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5916b492-ba6d-4c93-8693-3d5ceb5761c5": {"__data__": {"id_": "5916b492-ba6d-4c93-8693-3d5ceb5761c5", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ea246ce2-22d1-43e1-9bbe-57241d2614ae", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "6a491948674c91fa1896f5219d9e7ecdee023680cf5586cdb311e834ccd68829", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6a55a911-ca25-46e7-b879-e7309df0f43c", "node_type": "1", "metadata": {}, "hash": "a129c11ffa91f6f4bcdf3efc2a69fade00e38d9edf2e62d6cefb1df9165fabfc", "class_name": "RelatedNodeInfo"}}, "text": "multidrug-resistant nonfermenters in \nhospital-acquired pneumonia in Asia. Am J \nRespir Crit Care Med. 2011 184:1409-17.  \n[https://www.ncbi.nlm.nih.gov/pubmed/219\n20919] \n\n7. Liu F, Wen Z, Wei J, Xue H, Chen Y, Gao W, et \nal: Epidemiology, microbiology and treatment \nimplications in adult patients hospitalized \nwith pneumonia in different regions of China \na retrospective study. J thorac dis. 2017 9(10): \n3875-87. [https://www.ncbi.nlm.nih.gov/ \npmc/articles/PMC5723798/] \n\n8. Forum of International Respiratory Societies. \nThe Global Impact of Respiratory Disease - \nSecond Edition. Sheffield, European \nRespiratory Society, 2017. \n[https://www.who.int/gard/publications/Th\ne_Global_Impact_of_Respiratory_Disease.pdf] \n\n9. Odrigo-Troyano A, Sibila O. The respiratory \nthreat posed by multidrug resistant Gram-\nnegative bacteria. Respirology. 2017 22:1288-\n1299. [onlinelibrary.wiley.com/doi/full/10. \n1111 /resp.13115] \n\n10. Karchmer AW: Increased Antibiotic Resistance \nin Respiratory Tract Pathogens: PROTEKT US\u2013\nAn Update. Clin Infect Dis. 2004 39 (3): S142\u201350. \n[https://academic.oup.com/cid/article/39/Su\npplement_3/S142/288433] \n\n11. Xia W, Chen Y, Mei Y, Wang T, Liu G, Gu B et \nal.: Changing trend of antimicrobial resistance \namong pathogens isolated from lower \nrespiratory tract at a university-affiliated \nhospital of China, 2006-2010. J Thorac Dis. 2012  \n4: 284-291. [https://www.ncbi.nlm.nih.gov/ \npmc/articles/PMC3378186/] \n\n12. Thomas AM, Jayaprakash C, Amma GMR: The \npattern of bacterial pathogens and their \nantibiotic susceptibility profile from lower \nrespiratory tract specimens in a rural tertiary \ncare centre. J Evolution Med Dent Sci. 2016 5(40): \n2470-2476. [https://www.jemds.com/data_pdf \n/2_Chitra.pdf] \n\n13. Mishra SK, Kattel HP, Acharya J, Shah NP, Shah \nAS, Sherchand JB et al: Recent trend of bacterial \naetiology of lower respiratory tract infections \nin a tertiary care centre of Nepal. Int J Infect \nMicrobiol. 2012 1(1): 3\u20108. [https://doi.org \n/10.3126/ijim.v1i1.6639] \n\n14. Pant S, Bhusal KR, Manandhar S: Microbiology \nof lower respiratory tract infection in \nworkers of garment industry of Kathmandu. \nJCMS-Nepal. 2014 10(3): 14-22. [https:// \ndoi.org/10.3126/jcmsn.v10i3.12772] \n\n15. Clinical and Laboratory Standards Institute: \nPerformance standards for antimicrobial \nsusceptibility testing, twenty-second \ninformational supplement, CLSI, Document \nM100 S27, CLSI, Wayne, Pa, USA, 2017. \n\n16. Shrestha U, Singh A, Pokhrel B: Cross-sectional \nstudy of respiratory pathogens and their \nantibiotic susceptibility pattern in Tribhuvan \nUniversity Teaching Hospital. JIOM. 2006 \n28(2):59. [https://www.nepjol.info/index.php\n/JIOM/article/view/590] \n\n17. Nepal R, Shrestha B, Joshi DM, Joshi \nRD, Shrestha S, Singh A: Antibiotic \nSusceptibility Pattern of Gram-negative \nIsolates of Lower Respiratory Tract Infection. \nJ Nepal Health Res Counc.", "start_char_idx": 18159, "end_char_idx": 21049, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6a55a911-ca25-46e7-b879-e7309df0f43c": {"__data__": {"id_": "6a55a911-ca25-46e7-b879-e7309df0f43c", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5916b492-ba6d-4c93-8693-3d5ceb5761c5", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "691f353efda0b1ba8b276d3711aa90b66605a622edfd2039f49937166ad13107", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0444315c-2a8b-4255-8c24-bdb5475e7f32", "node_type": "1", "metadata": {}, "hash": "8793cdc448d1407818189c8abdeb53e7ec97891fa4e76eb3bc2550960ac08690", "class_name": "RelatedNodeInfo"}}, "text": "J Nepal Health Res Counc. 2018 16(1): 22-26. \n[http://jnhrc.com.np/index.php/jnhrc/article\n/view/1117/671] \n\n18. Bajpai T, Shrivastava G, Bhatambare GS, \nDeshmukh AB, Chitnis V: Microbiological \nprofile of lower respiratory tract infections in \nneurological intensive care unit of a tertiary \ncare center from Central India. J Basic Clin \nPharm. 2013 4(3):51-55. [https://www.ncbi. \nnlm.nih.gov/pmc/articles/PMC3979271/] \n\n19. Kumar SV, Kumar GPV, Kandati J, Pathapati \nRM, Buchineni M: A Surveillance Study of \nMicrobial Pathogens and their Antibiotic \nSensitivity of Respiratory Tract Infections in a \nTertiary Care Hospital. Int J Curr Microbiol App \nSci. 2015; 4(11):35-44. [https://www.ijcmas. \ncom/vol-4-11/S.Vijaya%20Kumar,%20et \n%20al.pdf] \n\n20. Anvari MS, Naderan M, Boroumand MA, Shoar \nS, Bakhshi R, Naderan M: Microbiologic \nspectrum and antibiotic susceptibility pattern \namong patients with urinary and respiratory \ntract infection. Int J Microbiol. 2014 2014: 682304. \n[https://www.hindawi.com/journals/ijmicro\n/2014/682304/] \n\n21. Camara M, Dieng A, boye CSB: Antibiotic \nsusceptibility of Streptococcus Pyogenes \nisolated from respiratory tract infections in \nDakar, Senegal. Microbiol. Insights. 2013 6: 71-\n75.[https://www.ncbi.nlm.nih.gov/pmc/articl\nes/PMC3987753/] \n\n22. Dhanda V, Chaudhary P, Toor D, Kumar R, \nChakraborti A: Antimicrobial susceptibility \npattern of \u00df-haemolytic group A, C and G \nstreptococci isolated from North India. J Med \nMicrobiol. 2013 62: 386-93.[https://jmm. \nmicrobiologyresearch.org/content/journal/jm\nm/10.1099/jmm.0.046672-0#tab2] \n\n23. Kim L, McGee L, Tomczyk S, Beall B: Biological \nand Epidemiological Features of Antibiotic-\nResistant Streptococcus pneumoniae in Pre- \nand Post-Conjugate Vaccine Eras: a United \nStates Perspective. Clin Microbiol Rev. 2016 \n29(3):525-52.[https://www.ncbi.nlm.nih.gov/ \npmc/articles/PMC4861989/] \n\n24. Thomas SS, Sreenath K, Sebastian S: \nCharacterization of the antibiotic profile of \nPseudomonas aeruginosa isolates from a \ntertiary care center. Int J Res Med Sci. 2016 4: 571-\n4. [https://www.msjonline.org /index.php \n/ijrms/article/view/542] \n\n25. Guzek A, Rybicki Z, Korziniewski K, \nMackiewicz K, Saks E, Chcialowski A: \nAetiological factor causing lower respiratory \ntract infections isolated from hospitalized \npatients. Adv Exp med Biol. 2015 835: 37-44. \n[https://link.springer.com/chapter/10.1007%2\nF5584_2014_23] \n\n26.", "start_char_idx": 21024, "end_char_idx": 23445, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0444315c-2a8b-4255-8c24-bdb5475e7f32": {"__data__": {"id_": "0444315c-2a8b-4255-8c24-bdb5475e7f32", "embedding": null, "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-195", "node_type": "4", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "55d65e5af5a74c627311c4c82a0ec9d2ae74167466a5f651c391b87dd5f6d803", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6a55a911-ca25-46e7-b879-e7309df0f43c", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "37064f65351c52e4f52ab1c2e795333898fef3aa0f1e57a444e63ab0872c8826", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "031c20b8-1b75-4e84-b63c-3b43b2c4d3d2", "node_type": "1", "metadata": {}, "hash": "f831f19b5179f0fb96de20fb72690f19db601d95dde98668678418e0e0b8f0a3", "class_name": "RelatedNodeInfo"}}, "text": "Bhatta DR, Hamal D, Shrestha R, Supram HS, \nJoshi P, Nayak N, et al: Burden of multidrug \n\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Nepal%20R%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Shrestha%20B%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Joshi%20DM%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Joshi%20RD%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Joshi%20RD%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Shrestha%20S%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=Singh%20A%5BAuthor%5D&cauthor=true&cauthor_uid=29717284\nhttps://www.ncbi.nlm.nih.gov/pubmed/29717284\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 9  Vol. 7, No. 1: 1-7                    Subedi et al. 2019 \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal  7 Nepjol.info/index.php/njb. \n\n \n\n \n\nresistant respiratory pathogens in intensive \ncare units of tertiary care hospital. Asian J Med \nSci. 2019 10(2):14-19. [https://www.nepjol. \ninfo/index.php/AJMS/article/view/21098] \n\n27. Regha R, Sulekha B: Bacteriological profile and \nantibiotic susceptibility patterns of lower \nrespiratory tract infections in a tertiary care \nhospital, Central Kerala. Int J Med Microbiol \nTrop Dis. 2018 4(4): 186-90. \n[http://snims.org/files/Microbiology/2.pdf]", "start_char_idx": 23446, "end_char_idx": 24917, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "031c20b8-1b75-4e84-b63c-3b43b2c4d3d2": {"__data__": {"id_": "031c20b8-1b75-4e84-b63c-3b43b2c4d3d2", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0444315c-2a8b-4255-8c24-bdb5475e7f32", "node_type": "1", "metadata": {"identifier": "njb-195", "author": "Subedi, Deepak; Devkota, Surya Prasad; Bhatta, Dharm Raj; Sharma, Binita Koirala; Paudel, Ashmita; Gurung, Krishna; Gautam, Damodar", "title": "Antibiotic Susceptibility Profile of respiratory pathogens obtained at tertiary care hospital from western Nepal", "date": "2019-12-29", "file": "njb-195.pdf"}, "hash": "8f03009046ed0f511bf6cc7b17650ce123a234ddf8a2c2548cc6a630d7e12b51", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e56a9f4e-b03d-4277-b41f-30c4222edd0f", "node_type": "1", "metadata": {}, "hash": "e0e241c38f18123024173c032d9ddc0bd55f632dd2e45dd360e1eec1e92e46aa", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1 D e c . 9 (2): 33-38 Research article DOI: https://www.doi.org/10.54796/njb.v9i2.41913  \n\n\u00a9NJB, BSN 33 \n\nSimple method devised for rapid isolation and identification of Vibrio \ncholerae from water resources of Sunsari District, Nepal \nBijay Kumar Shrestha  and Jenish Shakya\n\nDepartment of Microbiology, Central Campus of Technology, Tribhuvan University, Hattisar, Dharan, Nepal.\n\nReceived: 16th Nov 2020; Revised: 24th Jun 2021; Accepted: 26th Dec 2021; Published online: 31st Dec 2021 \n\nAbstract \nCholera is a gastrointestinal disease caused by pathogenic strain of Vibrio cholerae, the disease clinically manifested by rice-\nwater diarrhea, nausea and vomiting. This study aimed to study the incidence of Vibrio species and employ simple method \nfor rapid detection of Vibrio cholerae from water samples of Sunsari, Nepal. Identification of V. cholerae through biochemical \ntests requires extensive labor and costs. In resource limited laboratories, isolation and identification of V. cholerae often \nbecomes difficult. Therefore, this study also aimed for selecting scope of this methodology as a scientific outcome for rapid \nisolation and identification of Vibrio cholerae.  A total of 100 water samples were collected from Sunsari district in which 25 \nsamples were collected from sewage, 25 from pond, 25 from tap and 25 from tube well. The samples of collected water were \nsent to the microbiology laboratory of Central Campus of Technology maintained in ice cold box and were enriched in \nAlkaline Peptone water and selectively isolated from TCBS agar and NA agar without NaCl. Pathogens were isolated and \nidentified by conventional microbiological techniques. Out of 100 water samples collected, sucrose fermenting Vibrio species \nwere isolated only from 16 water samples. Further the selective isolation of V. cholerae from nutrient agar without NaCl \nisolated 6 isolates from sewage samples and 3 isolates from pond samples. The distribution of Vibrio cholera in the water \nsample was found to be 9%, distribution of V. alginolyticus was found to be 4% and distribution of V. fluvialis was found to \nbe 3%. In this study, non-sucrose fermenting Vibrio species were not isolated from the water samples. However, sucrose \nfermenting Vibrio species was obtained with yellow pigmentation in TCBS agar medium. The yellow pigmented colonies of \nVibrio isolates recovered from TCBS and even from Nutrient Agar devoid of sodium chloride provided sufficient evidence \nof V. cholerae  after series of other biochemical tests. This study concludes that yellow colonies (sucrose-fermenting) of Vibrio \nfrom TCBS agar medium that can grow on nutrient agar without added NaCl and which exhibit a positive oxidase reaction \ncan be confidently identified as presumptive V. cholerae. In resource-constrained environments, this simple method can \nreduce the labor cost, chemicals and time-consuming procedure of performing multiple biochemical and molecular assays \nfor identification. \n\nKeywords: Cholera, lab diagnosis, Sunsari, TCBS, Vibrio cholerae, Water \n\n Corresponding author, email: interfacebj@gmail.com \n\nIntroduction \nVibrio is a gram-negative rod-shaped bacterium that is \n\nan inhabitant of water sources. The species distribution \n\nof this bacterium is dependent on salt concentration and \n\ntemperature of water [1]. Among several species, V. \n\ncholerae , V. parahaemolyticus and V. vulnificus are known \n\nto be pathogenic. Strains of V. cholerae  of serogroups \n\nO1 and O139 are known to be associated with epidemic \n\npotential whereas non-O1 and non-O139 strains are \n\nidentified with mild diarrheal cases [2]. Cholerae \n\nsymptoms include abrupt onset of watery diarrhea (rice \n\nwatery stool), nausea, vomiting, and intestinal disorders \n\nwith abdominal pain [3].", "start_char_idx": 48, "end_char_idx": 3852, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e56a9f4e-b03d-4277-b41f-30c4222edd0f": {"__data__": {"id_": "e56a9f4e-b03d-4277-b41f-30c4222edd0f", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "031c20b8-1b75-4e84-b63c-3b43b2c4d3d2", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "a371635359ce2a108f33fe4c6c08e8b1a0aeb9808cdb52f08818e6cce83183dc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2a31221c-699a-4a12-b839-82276cce38ff", "node_type": "1", "metadata": {}, "hash": "80560983efba37dd7ab1732b18fe4ff3169be6659a07a58bcb84cf79dc36770e", "class_name": "RelatedNodeInfo"}}, "text": "Cholera is clinically manifested \n\nby rapid loss of water that accounts for dehydration, \n\npolydipsia, hollow tear troughs, decreased blood \n\npressure, weak pulse, renal failure, seizures, coma, and \n\ndeath [4]. \n\nV. cholerae  are known to be associated with several \n\nepidemics and pandemics. In underdeveloped nations, \n\nthe fecal contamination of food-water and poor sanitary \n\nhabits are associated with cholera epidemic [5]. \n\nTherefore, the aim of this study was to study the \n\nincidence of Vibrio species and devise simple method \n\nfor rapid detection of Vibrio cholera from water samples \n\nof Sunsari, Nepal. \n\nMethods and materials \nStudy site \nThe study area was Sunsari District (Latitude 26\u00b0 37' \n\n39.19\" N and longitude: 87\u00b0 10' 55.82\" E) of Province \n\nNo.1, Nepal. The district is situated in Terai region and \n\ncovers an area of 1,257 km\u00b2. Sunsari district is situated at \n\nan altitude up to 6600 ft. from sea level. \n\nStudy design \nThis study was designed to setup from November 2018 \n\nto January 2019. In this study water samples were \n\ncollected from different regions of Sunsari district, \n\nNepal. During the study period a total of 100 water \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nmailto:interfacebj@gmail.com\nhttps://orcid.org/0000-0002-6542-829X\nmailto:interfacebj@gmail.com\nhttps://orcid.org/0000-0002-9397-4511\n\n\nNepal J Biotechnol. 2021Dec.9 (2): 33-38 Shrestha and Shakya \n\n\u00a9NJB, BSN 34 \n\nsamples were collected from four available sites (Pond, \n\nsewage, tap and tube well) of districts rich in water \n\nresources. In this study, same frequency of samples was \n\ncollected from four different places in which 25 water \n\nsamples were collected from sewage, 25 from pond, 25 \n\nfrom tap and 25 from tube well from random areas of \n\nSunsari district to have uniform distribution of different \n\nsamples. Sites of water samples were selected by simple \n\nrandom sampling and lottery method. About 100 mL \n\nwater was collected in a sterile BOD sample bottle and \n\nwas transported to microbiology laboratory of Central \n\nCampus of Technology, Dharan on the same day \n\nmaintained in ice cold chain and processed within 3 \n\nhours of collection.  \n\nSocietal information \nThe socio-demographic activities of people affecting \n\nwater sanitation were observed and information were \n\ncollected from local residents through a semi- structured \n\nQuestionnaire. \n\nInclusive and exclusive criteria \nWater samples from rivers, ponds, tap and sewage were \n\nincluded in this study.  \n\nScreening V. cholerae  from water \nIsolation of V. cholerae  from water was performed as \n\ndescribed by CDC (1994) [6]. Membrane filters \n\ntechnique was most appropriate for clear sample that \n\ndid not contain debris, mud or silt. Clean, non-cloudy \n\nand sediment free water from tap and tube well was \n\nfiltered through membrane filtration technique. In this \n\nmethod, 100 mL of water sample was passed through \n\n0.22 \u00b5m membrane filter (HiMedia, India) attached to \n\nsuction flask. With the help of a sterile forceps, the \n\nMembrane filter was inoculated in test tube containing \n\n10mL of sterile alkaline peptone water of pH-8.6 (APW) \n\n(HiMedia, India) and vortexed for 30 sec. The \n\nenrichment of bacteria from pond and sewage was \n\nperformed by inoculating 1 mL of sample water in 9 mL \n\nalkaline peptone water.", "start_char_idx": 3853, "end_char_idx": 7296, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2a31221c-699a-4a12-b839-82276cce38ff": {"__data__": {"id_": "2a31221c-699a-4a12-b839-82276cce38ff", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e56a9f4e-b03d-4277-b41f-30c4222edd0f", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "e3c2431439748a367efc08e4661c3b8d980039e08ac8a76b70fa1bc038814057", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "821858b8-ba8a-496d-ba9b-ec6e82523c9c", "node_type": "1", "metadata": {}, "hash": "7eac59c6aa5bcfd100fb8281ddb933277f50b396dd8cd590f44ab85b8980d3ce", "class_name": "RelatedNodeInfo"}}, "text": "The enriched APW culture tubes were incubated at 37\u00b0C \n\nfor 6 hours and then the topmost layer of enriched \n\naliquot was inoculated on thiosulfate citrate bile salts \n\nsucrose (TCBS) agar media (HiMedia, India) and \n\nstreaking over the TCBS agar surface was done with the \n\nhelp of sterile inoculating loop. The inoculated culture \n\nmedia were incubated at 37\u00b0C for 18-24 hours. After \n\novernight incubation, the Vibrio like yellow colony, 2 to \n\n4 mm in diameter, slightly flattened with opaque centers \n\nwere chosen and cultured on nutrient agar without \n\nNaCl and incubated at 37\u00b0C for 16-24 hours. In routine \n\nmicrobiology tests, the bacterial colonies obtained from \n\nTCBS are cultured in Nutrient Agar with NaCl \n\nalongside overnight incubation and further the isolated \n\ncolonies are biochemically tested which requires lengthy \n\ncost, reagents, time and labor. Most of the biochemical \n\ntests require overnight or 18-24 hours long incubation \n\nperiod. In this regard, suspected isolated Vibrio culture \n\nfrom TCBS directly grown in NA devoid of NaCl \n\nprovides presumptive identification of V. cholerae \n\nwithout further biochemical tests.  \n\nBiochemical test for identification of V. \ncholerae  \nAfter incubation, the nutrient agar (NA) plate with the \n\ngrowth of colorless, glistening, translucent colonies \n\nprovided presumptive identification of V. cholerae . For \n\nfurther confirmation, Gram staining and biochemical \n\ntests like oxidase test, string test, MR test, citrate test, \n\nindole test, esculine hydrolysis and arginine dihydrolase \n\nactivity were performed as described by CDC (1994) [6]. \n\nFor Gram staining and biochemical tests, the culture \n\nwas taken from nutrient agar without NaCl (non-\n\nselective media). Sucrose fermenting colonies (yellow \n\npigmented) from TCBS which were not recovered in NA \n\ndevoid of NaCl were further cultured in Nutrient agar \n\nwith 0.5% NaCl and subjected for biochemical tests. \n\nQuality Control \nV. cholerae  O1 El Tor strain N16961 was used in this \n\nstudy as positive control. Reagents and culture media \n\nwere regularly monitored for their performance. \n\nEquipment was standardized, optimized and its \n\nperformance was checked through pretesting of positive \n\nand negative controls. \n\nData analysis \nThe data was documented in MS-EXCEL 2010 and was \n\nanalyzed using SPSS version 16.0 software. Statistical \n\nsignificance was established at P <0.05 with 95% \n\nconfidence interval. \n\nResults \nOut of 100 water samples collected, sucrose fermenting \n\nVibrio species were isolated only from 16 water samples \n\n(9 isolates from raw sewage, 6 isolates from pond \n\ndevoid of any sewage connections and 1 isolate from \n\npublic tap water). None of the non-sucrose fermenting \n\nVibrio species were isolated from TCBS. Further, the \n\nselective isolation of V. cholerae  from nutrient agar \n\nwithout NaCl isolated 6 isolates from sewage samples \n\nand 3 isolates from pond samples. The incidence of", "start_char_idx": 7299, "end_char_idx": 10246, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "821858b8-ba8a-496d-ba9b-ec6e82523c9c": {"__data__": {"id_": "821858b8-ba8a-496d-ba9b-ec6e82523c9c", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2a31221c-699a-4a12-b839-82276cce38ff", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b16d8aa7602287a82e15ded81362d8caebc20af72957bb140033326d4c40ff08", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4a8884b4-9e3d-41f5-97b6-eaf1ca88b1ef", "node_type": "1", "metadata": {}, "hash": "651bffd157215c5d49362ad1c4e90bcb6e4d1d43a9cee1376fd28905af707dd1", "class_name": "RelatedNodeInfo"}}, "text": "The incidence of  \n\n\n\nNepal J Biotechnol. 2021Dec.9 (2): 33-38 Shrestha and Shakya \n\n\u00a9NJB, BSN 35 \n\nVibrio cholera in the study sample was reported to be 9%, \n\nV. alginolyticus to be 4% and V. fluvialis to be 3%. \n\nHowever, in this study V. cholerae  was not isolated from \n\ntap and tube well water (Table 1). The biochemical tests: \n\noxidase test, string test, citrate test, indole test, MR test, \n\nesculine hydrolysis, arginine dihydrolase test confirmed \n\nthe isolates recovered from NA without NaCl to be V. \n\ncholerae  (Table 2). The biochemical tests of sucrose \n\nfermenting 7 Vibrio isolates from TCBS which did not \n\ngrow in nutrient agar devoid of NaCl reported 4 isolates \n\nto be V. alginolyticus and remaining 3 isolates to be V. \n\nfluvialis. There was significant difference in distribution \n\nof Vibrio cholera from different water samples (P=0.001). \n\nTable 2. Biochemical tests of isolated V. cholerae . \n\nBiochemical Bacterial isolates \n\nTest S1 S2 S3 S4 S5 S6 P1 P2 P3 C \n\nOxidase Test + + + + + + + + + + \n\nString Test + + + + + + + + + + \n\nCitrate Test + + + + + + + + + + \n\nIndole Test + + + + + + + + + + \n\nMR - - - - - - - - - - \n\nEsculine \nhydrolysis \n\n- - - - - - - - - - \n\nArginine \ndihydrolase \n\n- - - - - - - - - - \n\nNote: Isolates S1, S2, S3, S4, S5, S6 were isolated from sewage whereas \nisolates P1, P2 and P3 were isolated from Pond. C is the quality control \nstrain; V. cholerae  O1 El Tor N16961 strain used in the study \n\nTable 3. Characteristics pertaining to water contamination at \nstudy sites. \n\nCharacteristics Pond Tap water Tube well water \n\n1.Open human\nDefecation \n\nYes No No \n\n2.Animal\nwashing and \ndefecation \n\nYes No No \n\n3. Wastes\ndumping sites \n\nYes No No \n\n4. Source of\ndrinking water \n\nYes Yes Yes \n\n5. Use of sample\nsite for religious \npractices \n\nYes No No \n\n6. Water\ntreatment before \ndrinking \n\nNo No No \n\nFigure 1. Vibrio sp. on TCBS media with yellow colony, 2-\n\n4mm in diameter, slightly flattened with opaque centers.  \n\nFigure 2. V. cholerae with colorless, glistening, translucent \n\ncolonies on Nutrient Agar without NaCl. \n\nDiscussion \nCholera has been a disease of great epidemic potential \n\nfor the last many centuries and now has become \n\nendemic in many countries [7]. Cholera outbreaks have \n\nbeen reported frequently from different parts of Nepal \n\n[8]. V. cholerae 01 strain associated with epidemic \n\noutbreak of Cholera has been known to establish in \n\naquatic environment posing threat to public health [9]. \n\nTable 1. Vibrio species from culture media. \n\nSources \n(Total sample) \n\nSucrose \nfermenters \n\nIsolates from NA without \nNaCl (V. cholerae) \n\nIsolates from NA with \nNaCl (V. alginolyticus) \n\nIsolates from NA with \nNaCl (V. fluvialis) \n\nP-\nvalue \n\n1. Sewage (S) (25) 9 6 2 1 \nP=0.0\n\n01 \n2. Pond (P) (25) 6 3 1 2 \n3. Tap water (25) 1 0 1 0 \n4. Tube well (25) 0 0 0 0 \nTotal 16 9 4 3 \n\n\n\nNepal J Biotechnol.", "start_char_idx": 10230, "end_char_idx": 13113, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4a8884b4-9e3d-41f5-97b6-eaf1ca88b1ef": {"__data__": {"id_": "4a8884b4-9e3d-41f5-97b6-eaf1ca88b1ef", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "821858b8-ba8a-496d-ba9b-ec6e82523c9c", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "c12d3a0d7d7cc26d6de3642bfddff1235ed8c9970802304b778d3021d2ae4f00", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "144102a8-aec5-407e-a7a9-4996c671d018", "node_type": "1", "metadata": {}, "hash": "d5db52e2cce38b541d120f609343d83510528dd9618adfe306efb7505a7f3f25", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021Dec.9 (2): 33-38 Shrestha and Shakya \n\n\u00a9NJB, BSN 36 \n\nMultidrug Resistance V. cholerae  EL Tor possessing \n\ncholera toxin (ctx) identified with diarrheal illness from \n\nthree districts from Nepal was similar with strains \n\nrelated with cholera outbreak in Bangladesh and Haiti \n\n[10].  \n\nV. cholerae and V. mimicus can only grow in media that \n\nlacks sodium chloride while other species of Vibrio are \n\nstrictly halophilic in nature [11]. V. cholerae  can ferment \n\nsucrose in TCBS medium producing yellow colony \n\nwhereas Vibrio mimicus fails to ferment sucrose and \n\nhence produce green pigmented colony on TCBS [12]. \n\nAll the colonies growing on TCBS media with yellow \n\ncolony are not necessarily V. cholerae. The ability of V. \n\ncholerae  to grow on culture media devoid of sodium \n\nchloride helps in selective isolation and identification of \n\nV. cholerae  from other Vibrio species [13]. \n\nIn this study the presumptive isolation and \n\nidentification of V. cholerae was made from media \n\ndevoid of NaCl within two days of sample collection \n\nand processing. This study was consistent with the \n\nstudy that has demonstrated that V. cholerae  can be \n\nselectively isolated from media devoid of sodium \n\nchloride [14]. The main principle of this study was the \n\nenrichment of Vibrio in alkaline peptone water and its \n\nselective isolation from TCBS and NA without NaCl. \n\nThe biochemical tests like esculine hydrolysis and \n\narginine dihydrolase are efficient techniques for \n\nidentifying Vibrio cholera from aquatic samples [15]. In \n\nthis study, even arginine dihydrolase test stood key tool \n\nfor differentiating V. alginolyticus (Arginine dihydrolase-\n\nnegative) from V. fluvialis (Arginine dihydrolase-\n\npositive). The Gram staining, string test and oxidase test \n\nalong with serogrouping, biotyping and serotyping can \n\nbe performed minimum from second day of sample \n\nprocessing. However, PCR based methods can provide \n\nsensitive result much rapidly within 6-12 hours but \n\nconventional methods are only the option in resource \n\nlimited areas. However, antimicrobial susceptibility test \n\ncan only be accessed through conventional culture \n\nmethod which is most important in clinical settings. \n\nSerogrouping, biotyping and serotyping of V. cholerae \n\nneed to be performed whenever there are reported and \n\noutbreak cases which provides evidence for clinical and \n\nepidemiological studies. \n\nV. cholerae differs from other Vibrio species in that it can \n\ngrow in nutritional broth without the addition of \n\nsodium chloride [16]. In this study the identification of \n\nV. cholerae from other Vibrio species was based on three \n\nmajor trait features like sucrose fermentation, non-\n\nrequirement of additional Na+ for growth, and presence \n\nof oxidase which was in consistent to the findings of \n\nother similar study [17]. Most sucrose-positive \n\nhalotolerant or halophilic Vibrio were eradicated when \n\ngrown on nutritional agar without additional NaCl. It is \n\nalso concluded that isolation of V. cholerae  from media \n\ndevoid of NaCl is simple and rapid method of isolation \n\nand identification which was in agreement to the \n\npresent study [14]. However, some limiting factors may \n\nexists, study reports that some V. cholerae strains, have \n\nbeen reported of not being able to ferment sucrose. \n\nThese strains would not be detected in conventional \n\nstudies as only sucrose fermenting Vibrio from TCBS are \n\nconsidered for further identification of V. cholerae [18]. \n\nSome V. cholerae strains which are strictly halophiles \n\nmight also remain in shadow during isolation through \n\nmedia devoid of NaCl. In addition, routine conventional \n\nmicrobiological techniques are not capable of isolating \n\nViable but non-culturable (VBNC) state of V. cholerae \n\n[19]. In this regard, only the resource rich settings with \n\nthe polymerase chain reaction provides high specificity \n\nand accuracy for identifying Vibrio species and can even \n\ndistinguish its biotypes.", "start_char_idx": 13094, "end_char_idx": 17088, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "144102a8-aec5-407e-a7a9-4996c671d018": {"__data__": {"id_": "144102a8-aec5-407e-a7a9-4996c671d018", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4a8884b4-9e3d-41f5-97b6-eaf1ca88b1ef", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "68780ecb5fd2b757b8e2523e92db005d07a34af923ed27b58e4f2aeabe6a72d1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9c10f0da-4b4e-4124-9574-3810aefb08ab", "node_type": "1", "metadata": {}, "hash": "b02c3a2d0e940fc67f59a82a9107f70d1ec81ad95326007769e52700b283977a", "class_name": "RelatedNodeInfo"}}, "text": "According to the study, the prevalence rate of V. cholerae \n\nfrom water samples of Bhaktapur was 5% [20] and from \n\nKathmandu, the prevalence was 11.11% [21] which were \n\nin agreement to the prevalence of the current study. \n\nHowever, the prevalence of V. cholerae  was 0.84% in one \n\nstudy from drinking water of Kathmandu [22]. The \n\nfilthy pond with algae growth might be an organism's \n\nreservoir [23]. In Nepal, the period between mid-june to \n\nmid-july had the highest incidence of Cholera [24]. In \n\n2012 A.D., diarrhea outbreaks in three districts of Nepal \n\nwere due to transmission of multidrug resistant V. \n\ncholerae El Tor possessing cholera toxin (ctx) B-7 allele, \n\nwhich is clonal and related closely with V. cholerae \n\nassociated with cholera in Bangladesh and Haiti [25]. \n\nThe increased prevalence and isolation rates of V. \n\ncholerae in Nepalgunj (Banke), Dang, and Dhangadhi \n\n(Kailali) might be attributed to contaminated drinking \n\nwater, poor management of water pipes, sewage, and \n\nexcreta, and a less sanitary environment [26]. \n\nIn one study V. cholerae  was found in two of the patients \n\nfrom Sunsari. In the same study, eight of the thirteen \n\nwater samples were determined to be unfit for human \n\nconsumption. Contamination of the source of drinking \n\nwater and the major reasons of such epidemics were \n\nunsanitary behaviors. It is, thus, necessary that gastro \n\nenteritis outbreaks may be avoided simply by \n\nencouraging cleanliness and sanitary practices behaviors \n\ninvolving drinking water and defecation [27]. The \n\npresence of heterotrophs and the coliform in the \n\ndrinking water marketed in eastern Nepal is a serious \n\n\n\nNepal J Biotechnol. 2021Dec.9 (2): 33-38 Shrestha and Shakya \n\n\u00a9NJB, BSN 37 \n\nconcern for public health [28]. V. cholerae  isolated from \n\npond and sewage water samples of sunsari district \n\ncould pose a severe health hazard to humans who either \n\ncome in contact with or consume water from the \n\ncontaminated site. In addition, the lack of safe drinking \n\nwater, awareness and poor sanitary practices among \n\npeople of rural areas of sunsari makes them more prone \n\nto the infections. Water borne diseases among \n\nunderprivilege family and community remains in \n\nshadow until it leads to community burden leading \n\nheavy mortality and morbidity. Microbiological water \n\nsurveillance in these rural settings have never gained \n\nimportance and attention from the concerned authority \n\nthat alarms the possibility of future outbreak of disease.  \n\nIn this study the possible cause behind the water \n\ncontamination was also studied. The necessary \n\ninformation was collected from the local residents \n\nthrough Questionnaire. It was found that the local \n\nvillagers of Terai observe chhath festival (local ethnic \n\nfestival that includes Fasting, Prayers and religious \n\nrituals by taking bath in ponds/ rivers water). In this \n\ncourse of ritual, the activity of open defecation results in \n\nfecal contamination of water. In this report, the pond of \n\nthis study sites was even used for many ritual \n\nperformance, open defecation, animal washing and even \n\nfor drinking purpose. Human activities like disposing \n\nhuman and animal wastes have been identified as the \n\nmain cause behind deteriorating water resource that \n\nleads pond water to be a potent source for cholera \n\ntransmission [8]. Outbreak of Cholera in Saptari VDC of \n\nNepal was associated with use of pond water which was \n\ncontaminated by V. cholerae  [23]. One study reported \n\nthat diarrheal outbreak in Rautahat District of Nepal \n\nwas associated with V. cholerae O1 Ogawa serotype, \n\nwhich was result of fecal contamination of drinking \n\nwater from nearby sewage [29]. Hence, these finding \n\naddresses the cause and route behind fecal \n\ncontamination of water which can lead to cholera \n\noutbreak. Therefore, Studies suggest promotion in safe \n\ndrinking water is fundamental approach to prevent \n\nmorbidity and mortality induced by cholera in near \n\nfuture [30].", "start_char_idx": 17091, "end_char_idx": 21081, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9c10f0da-4b4e-4124-9574-3810aefb08ab": {"__data__": {"id_": "9c10f0da-4b4e-4124-9574-3810aefb08ab", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "144102a8-aec5-407e-a7a9-4996c671d018", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "ce29055a28e6311d8fba261da7bfea0be4157e6bac990e03dc96f5da744f040c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9cf9b585-fddc-4174-9cbf-d57693145357", "node_type": "1", "metadata": {}, "hash": "c885f847c1e579584c75afbdbbce53d8365d31ff138bf0c3c1237abca2d0a310", "class_name": "RelatedNodeInfo"}}, "text": "Maintenance of safe water chain a key \n\napproach for preventing cholera outbreak in near future. \n\nConclusions \nThis study concludes that yellow colonies (sucrose-\n\nfermenting) of Vibrio from TCBS agar medium that can \n\ngrow on nutrient agar without added NaCl and which \n\nexhibit a positive oxidase reaction can be confidently \n\nidentified as presumptive V. cholerae. This method can \n\nreduce the labor, reagents, cost and time-consuming \n\nprocess of conducting several biochemical and \n\nmolecular tests in resource limited settings. \n\nAbbreviations \nAPW: Alkaline Peptone Water \n\nBOD: Biological Oxygen Demand \n\nCDC: Centre for Disease Control and Prevention \n\nNA: Nutrient Agar \n\nNaCl: Sodium Chloride \n\nPCR: Polymerase Chain Reaction \n\nTCBS: Thiosulfate Citrate Bile Salts Sucrose  \n\nVDC: Village Development Committee \n\nLimitations \nMolecular methods were not performed for \n\nidentification of bacteria. Serotyping, Biotyping and \n\nAntibiotics susceptibility test of isolated pathogens were \n\nnot done.  \n\nAcknowledgement \nAuthors want to thank all the helping hands and \n\nDepartment of Microbiology, Central Campus of \n\nTechnology, Tribhuvan University, Hattisar Dharan, \n\nNepal.\n\nAuthor\u2019s Contribution: \nBKS participated in study design, sample collection, \n\nsample processing, organism identification, data \n\nanalysis, intellectual content design and result \n\ninterpretation. JS participated in sample collection, \n\nsample processing, and proof-reading manuscript for \n\nintellectual content. Both the authors drafted the \n\nmanuscript and agreed for its publication. \n\nConflict of interest \nAuthor declares no conflict of study. \n\nFunding source \nThis study did not receive any fund. \n\nReferences \n1. Cabral JP. Water microbiology. Bacterial pathogens and water. \n\nInternational journal of environmental research and public \nhealth. 2010; 7(10):3657-703. DOI: https://doi.org/ \n10.3390/ijerph7103657 \n\n2. Reidl J, Klose KE. Vibrio cholerae and cholera: out of the water and \ninto the host. FEMS microbiology reviews. 2002; 26(2):125-39. \nDOI: 10.1111/j.1574-6976.2002.tb00605.x. \n\n3. Alexakis LC. Cholera-\u201cRice water stools. The Pan African \nmedical journal. 2017, 26(147). DOI: https: \nhttps://doi.org/10.11604/pamj.2017;26(147).11874. \n\n4. Harris JF, Ryan ET, Calderwood SB. Cholera. The Lancet. 2012; \n379: 2466-76. DOI: https: https://doi.org/10.1016/S0140-\n6736(12)60436-X. \n\n5. Rabbani GH, Greenough III WB. Food as a vehicle of \ntransmission of cholera. Journal of diarrhoeal diseases research. \n1999; 17(1):1-9. \n\n6. Centers for Disease Control and Prevention. Laboratory methods \nfor the diagnosis of Vibrio cholerae. Atlanta; 1994:27-37. \n\nhttps://doi.org/10.1111/j.1574-6976.2002.tb00605.x\nhttps://doi.org/10.11604/pamj.2017;26(147).11874\nhttps://doi.org/10.11604/pamj.2017;26(147).11874\nhttps://doi.org/10.1016/S0140-6736(12)60436-X\nhttps://doi.org/10.1016/S0140-6736(12)60436-X\n\n\nNepal J Biotechnol. 2021Dec.9 (2): 33-38 Shrestha and Shakya \n\n\u00a9NJB, BSN 38 \n\n7. Lim VK. Cholera: A re-emerging infection. Med J Malaysia. 2001; \n56(1):1\u20133. \n\n8. Gautam S, Jha P, Khanal B, Tamrakar D, Yadav DK.", "start_char_idx": 21082, "end_char_idx": 24192, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9cf9b585-fddc-4174-9cbf-d57693145357": {"__data__": {"id_": "9cf9b585-fddc-4174-9cbf-d57693145357", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9c10f0da-4b4e-4124-9574-3810aefb08ab", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "e1f63bc3c4e675f17a3a384decfacb6555de7e71e87d428762d249ed866c1bd9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3a545784-fef2-4797-9bb4-98a563dceed9", "node_type": "1", "metadata": {}, "hash": "36843af816c03dd9535aa651e7cdf3a3ba946505091e6f0dadca2f5901389bd2", "class_name": "RelatedNodeInfo"}}, "text": "Cholera: \nsmall outbreak in winter season of eastern Nepal. North \nAmerican journal of medical sciences. 2012; 4(12):657-658. DOI: \nhttps://doi.org/10.4103/1947-2714.104321\n\n9. Alam MT, Weppelmann TA, Weber CD, Johnson JA, Rashid MH, \nBirch CS, Brumback BA, de Rochars VE, Glenn J, Ali A. \nMonitoring water sources for environmental reservoirs of \ntoxigenic Vibrio cholerae O1, Haiti. Emerging Infectious Diseases. \n2014; 20(3):356-363. DOI: 10.3201/eid2003.131293.\n\n10. Dixit SM, Johura FT, Manandhar S, Sadique A, Rajbhandari RM, \nMannan SB, Rashid MU, Islam S, Karmacharya D, Watanabe H, \nSack RB. Cholera outbreaks (2012) in three districts of Nepal \nreveal clonal transmission of multi-drug resistant Vibrio \ncholeraeO1. BMC infectious diseases. 2014; 14(392). DOI: \nhttps://doi.org/10.1186/1471-2334-14-392. \n\n11. Elliot EL, Kaysner CA, Jackson L, Tamplin ML. V. cholerae, V. \nparahaemolyticus, V. vulnificus and other Vibrio sp. Chapter 9. \nUS Food and Drug Administration. Bacteriological Analytical \nManual Online. AOAC International, Gaithersburg, Md; 1998. \n\n12. Tille P. Bailey & Scott\u2019s diagnostic microbiology-E-Book. Elsevier \nHealth Sciences; 2015. \n\n13. Baron S, Chevalier S, Lesne J. Vibrio cholerae in the environment: a \nsimple method for reliable identification of the species. Journal of \nhealth, population, and nutrition. 2007; 25(3):312-318. \n\n14. Robert-Pillot A, Baron S, Lesne J, Fournier JM, Quilici ML. \nImproved specific detection of Vibrio cholerae in environmental \nwater samples by culture on selective medium and colony \nhybridization assay with an oligonucleotide probe. FEMS \nMicrobiology Ecology. 2002; 40(1): 39-46.DOI: https://doi.org/ \n10.1111/j.1574-6941.2002.tb00934.x \n\n15. Choopun N, Louis V, Huq A, Colwell RR. Simple procedure for \nrapid identification of Vibrio cholerae from the aquatic \nenvironment. Applied and environmental microbiology. \n2002;68(2):995-8.DOI: https://doi.org/10.1128/AEM.68.2.995-\n998.2002. \n\n16. Nair GB. National Institute of Cholera and Enteric Diseases, \nCalcutta, India, with contributions from J. Bartram, Water, \nSanitation and Health. World Health Organization, Geneva, \nSwitzerland. \n\n17. Baron S, Chevalier S, Lesne J. Vibrio cholerae in the environment: a \nsimple method for reliable identification of the species. Journal of \nhealth, population, and nutrition. 2007;25(3):312.\n\n18. Desmarchelier PM, Reichelt JL. A phenotypic and genetic study \nof sucrose nonfermenting strains of Vibrio mimicus and Vibrio \ncholerae. Current Microbiology. 1984;10(1):41-7. \n\n19. Ramamurthy T, Das B, Chakraborty S, Mukhopadhyay AK, Sack \nDA. Diagnostic techniques for rapid detection of Vibrio cholerae \nO1/O139. Vaccine. 2020;38:A73-82.\n\n20. Shrestha UT, Sujakhu H. Coliform and Vibrio cholerae analysis of \ndrinking water collected from cholera outbreak region of \nBhaktapur Municipality. International Journal of Environment.", "start_char_idx": 24193, "end_char_idx": 27087, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3a545784-fef2-4797-9bb4-98a563dceed9": {"__data__": {"id_": "3a545784-fef2-4797-9bb4-98a563dceed9", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9cf9b585-fddc-4174-9cbf-d57693145357", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "baa51313d22427682ab197a013bb4d6f1bc899a7c95e90b4ad330df08de3fd51", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a98bbcf5-d70d-4402-9ff9-b8c24cae4052", "node_type": "1", "metadata": {}, "hash": "c083801eda4c39f2e4e9e1e67aac573770deb1c0d0e283dfd381734455dbfe7c", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of Environment. \n2014;3(3):139-45.. \n\n21. Kansakar P, Baral P, Malla S, Ghimire GR. Antimicrobial \nsusceptibilities of enteric bacterial pathogens isolated in \nKathmandu, Nepal, during 2002-2004. The Journal of Infection in \nDeveloping Countries. 2011;5(03):163-8. \n\n22. Prasai T, Lekhak B, Joshi DR, Baral MP. Microbiological analysis \nof drinking water of Kathmandu Valley. Scientific world. \n2007;5(5):112-4. \n\n23. Yadav DK, Tamrakar D, Baral R, Jha, P, Gautam S, Pokharel PK. \nOutbreak of Cholera in Tilathi VDC Saptari Nepal. Kathmandu \nUniversity Medical Journal. 2012; 10 (4):36-39. \n\n24. Karki R, Bhatta DR, Malla S, Dumre SP. Cholera incidence among \npatients with diarrhea visiting National Public Health \nLaboratory, Nepal. Japanese journal of infectious diseases. \n2010;63(3):185-7. \n\n25. Dixit SM, Johura FT, Manandhar S, Sadique A, Rajbhandari RM, \nMannan SB, Rashid MU, Islam S, Karmacharya D, Watanabe H, \nSack RB. Cholera outbreaks (2012) in three districts of Nepal \nreveal clonal transmission of multi-drug resistant Vibrio cholerae \nO1. BMC infectious diseases. 2014;14(1):1-1. \n\n26. Shah BK, Sharma S, Shakya G, Upadhyay BP. Multiple drug \nresistant Vibrio cholerae, Salmonella and Shigella from Nepalgunj \nCholera outbreak and different hospitals of Nepal. Nepalese \njournal of Biosciences. 2012;2:31-9. \n\n27. Sharma AK, Jha N, Ramachandran VG, Shariff M, Deb M, \nKannan AT, Paudel IS, Yadav BK. Descriptive epidemiology of a \ngastroenteritis outbreak in Sunsari district, Nepal. J Nepal Med \nAssoc. 2002;41(143):383-7. \n\n28. Rai R, Kumal B, Rai D, Keshari A, Bhandari R. Bacteriological \nevaluation of bottled water commercially available in Eastern \nNepal. Sunsari Technical College Journal. 2015;2(1):54-7. \n\n29. Rai KR, Mukhiya RK, Thapa S, Rai G, Sabina KC, Thapa PM, \nShrestha P, Rai SK. Diarrheal disease outbreak in Gaidatar village \nof Rautahat District, Nepal. BMC research notes. 2019; 12(124). \nDOI: DOI: https://doi.org/10.1186/s13104-019-4156-9. \n\n30. Rahman Z, Rahman MD, Rashid MU, Monira S, Johura FT, \nMustafiz M, Bhuyian SI, Zohura F, Parvin T, Hasan K, Saif-Ur-\nRahman KM. Vibrio cholerae Transmits Through Water Among \nthe Household Contacts of Cholera Patients in Cholera Endemic \nCoastal Villages of Bangladesh, 2015\u20132016 (CHoBI7 Trial). \nFrontiers in Public Health. 2018;6(238):1-9.  DOI: \nhttps://doi.org/10.3389/fpubh.2018.00238.", "start_char_idx": 27050, "end_char_idx": 29449, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a98bbcf5-d70d-4402-9ff9-b8c24cae4052": {"__data__": {"id_": "a98bbcf5-d70d-4402-9ff9-b8c24cae4052", "embedding": null, "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-2", "node_type": "4", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "b08c7b7c9c658b5015045bff1a71116e3d2c1fd5216b46987f37cc17b0238cac", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3a545784-fef2-4797-9bb4-98a563dceed9", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "8ea010070b3b441055d8ebca917fda19a9c5f35a6b35b7d518bb344ce28c6966", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b97a6b74-d58b-4c61-8ddc-bc1269ffa4ff", "node_type": "1", "metadata": {}, "hash": "b944ce10d68000fb55acd410946a5bdfec94177cb42bc3f04ba119eaf836a392", "class_name": "RelatedNodeInfo"}}, "text": "https://doi.org/10.4103/1947-2714.104321\nhttps://doi.org/10.1186/1471-2334-14-392\nhttps://doi.org/10.1111/j.1574-6941.2002.tb00934.x\nhttps://doi.org/10.1128/AEM.68.2.995-998.2002\nhttps://doi.org/10.1128/AEM.68.2.995-998.2002\nhttps://doi.org/10.1186/s13104-019-4156-9\nhttps://doi.org/10.3389/fpubh.2018.00238", "start_char_idx": 29452, "end_char_idx": 29759, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b97a6b74-d58b-4c61-8ddc-bc1269ffa4ff": {"__data__": {"id_": "b97a6b74-d58b-4c61-8ddc-bc1269ffa4ff", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a98bbcf5-d70d-4402-9ff9-b8c24cae4052", "node_type": "1", "metadata": {"identifier": "njb-2", "author": "Shrestha, Bijay Kumar; Shakya, Jenish", "title": "Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal", "date": "2021-12-30", "file": "njb-2.pdf"}, "hash": "fab2fcb78f93622c18abba648e16c778511d621b4485bae16f7e0a311b6b7d4f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cf8b89bc-a4a5-4423-8bb1-6f837e378798", "node_type": "1", "metadata": {}, "hash": "463b8334d8c8a11d103b52489775ebd8aee795a6e1d272b42d797f04cf69e09d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91   ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\n REVIEW ARTICLE \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              74                                                Nepjol.info/index.php/njb \n\n \n\nTechnologies for the Extraction, Separation and Purification of \npolyphenols \u2013 A Review \n\nShyam Suwal1* and Alice Marciniak2 \n1 Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej Denmark \n2Institute of Nutrition and Functional Foods (INAF), Department of Food Sciences, Universit\u00e9 Laval, \n\nQu\u00e9bec, Canada \n\nAbstract \nPolyphenols are high molecular weight, organic molecules mainly found in plant kingdom. They \nare mostly known for their positive impact on health, specifically for their antioxidant activity. \nIndeed, they are widely studied for the prevention of multiple diseases such as cancer, \ninflammatory, cardiovascular and neurodegenerative diseases. Nevertheless, extractions of these \ngrowing interest molecules remain challenging using conventional methods such as solvent \nextraction. That is why recent researches have focused on improving the extraction of polyphenol \nby using different technologies such as ultrasound, microwave, pressurized liquid, pulsed electric \nfield, supercritical fluid and high hydrostatic pressure. In the current context, the assisted-\nextraction should demonstrate their potential to improve the extraction efficiency while being \ncost-effective and with a low environmental impact. To this end, technologies ought to, for \ninstance, increase the solubility of polyphenol and the permeability of the cell wall. Consequently, \nthis review is focused on the use and potential of these technologies to improve polyphenol \nextractions from plants as well as their purification using various methods. It discusses of the \nadvantages and disadvantages with some examples of all these technologies assisted-extraction in \ncomparison with conventional extraction method as well as purification technology.  \nKeywords: Polyphenols, Conventional extraction process, Emerging technologies \n*Corresponding Author \n\nEmail: shyam@food.ku.dk \n\nIntroduction \nThe prevention and treatment of chronic diseases \n\nmainly cardiovascular diseases (CVD) and cancer \n\nare the major challenge in modern day health care \n\nsystem. About 60% of deaths were caused by the \n\nchronic diseases in 2001 and according to the \n\nstudy of World Health Organization (WHO) the \n\nheart disease will overcome the infectious disease \n\nby 2020 [1]. Therefore, the burden (economical as \n\nwell as social) of chronic diseases is huge and \n\ntends to increase day by day alarming an urgent \n\nneed of preventive measures. Undoubtedly, the \n\nprimary cause of these diseases is associated with \n\nunhealthy diets. \n\nThere is an increasing interest in diets and \n\nnutrition which has resulted in an increased \n\nimportance and beliefs in functional foods and \n\nnutraceuticals [2]. Several bioactive molecules \n\n(peptides, vitamins, polyunsaturated fatty acids \n\nand polyphenols) have been isolated from food \n\nsources of plant as well as animal origin and are \n\nclaimed to possess health benefits against CVD \n\nand cancer [3-5]. Very diverse nature of \n\npolyphenol in their chemical composition and \n\ntheir wide spread prevalence in edible plant make \n\nthem very interesting as an ingredient of \n\nfunctional foods or nutraceuticals [3]. Different \n\nbeneficial roles on human health such as \n\nantihypertensive anticancer, neuroprotector etc. \n\nhave been confirmed [6]. Therefore, an immense \n\ninterest of researchers on polyphenols mainly in \n\ntheir physiological roles (both in vitro and in vivo), \n\ntheir extraction processes and product \n\ndevelopment in an industrial scale can be noticed. \n\nHowever, extraction of such molecules from the \n\ncomplex plant matrices is the rate limiting factor. \n\nSeveral methods of polyphenol extraction from \n\nvariety of food and food by-products exist such as \n\nsolvent (Soxhlet), ultrasound assisted, microwave \n\nassisted, pressurized liquid, pulse electric field, \n\nsupercritical fluid and high hydrostatic pressure \n\nextractions. The extraction methods not only affect \n\nthe input cost, final productivity and purity but \n\nalso their biological activity in laboratory assay [7, \n\n8]. A cost effective and environment friendly \n\nmethod are thus desired for the isolation of \n\npolyphenols. A separation and purification \n\nprocess is another vital step in product \n\ndevelopment with polyphenols. Therefore, the", "start_char_idx": 43, "end_char_idx": 4615, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cf8b89bc-a4a5-4423-8bb1-6f837e378798": {"__data__": {"id_": "cf8b89bc-a4a5-4423-8bb1-6f837e378798", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b97a6b74-d58b-4c61-8ddc-bc1269ffa4ff", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "5859a16dcfadaec10d69884b51aec1fb8c148c6e675038b232b7b4a57f046f2c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "144812ba-80b6-4b4d-b9e6-1a7e289f1884", "node_type": "1", "metadata": {}, "hash": "50963004e9c4262ce4f105b1bb08d2abff8d4def27bae00b3db1dd055a32892a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              75                                                Nepjol.info/index.php/njb \n\n \n\npresent study aims to review different techniques used for the isolation of  \n\nFigure 1: Chemical structure of principal polyphenols (adapted from [3]). .\n\npolyphenols published in the literature. In \n\naddition, methods of fractionation and \n\npurification of extracted polyphenol or free \n\npolyphenols present in liquid beverages are also \n\ndiscussed \n\nPolyphenols and health claims \nPolyphenols are readily available in the plant-\n\nderived food sources consumed by human in \n\ndaily basis such as fruits, vegetables, cereals, tea, \n\nwine etc. [3]. They are responsible for the major \n\norganoleptic properties such as color and taste of \n\nplant-derived food and beverages [9]. Plant \n\npolyphenols comprise of diverse groups of \n\ncompounds that make them difficult to be \n\ncharacterized except for some well-known ones \n\nwhose structure, chemical composition and \n\nfunctionality are well defined. Ample of \n\npolyphenol structures (having several hydroxyl \n\ngroups in aromatic ring) are found in plants are \n\ntherefore classified according to the number of \n\nphenol rings to one another. They can be mainly \n\nclassified into the phenolic acids, flavonoids \n\n(flavones, anthocyanidins), stilbenes, and lignans \n\n(cinamic acid) as shown in figure 1. Each of these \n\nphenolic compounds can be subclassified \n\naccording to the type of heterocycle involved. The \n\nchemical nature of phenolic compound can vary \n\nfrom a simple to polymerized structure and may \n\nbe found in a complex form with some \n\ncarbohydrate, protein or plant substances making \n\nthem highly insoluble [10]. Furthermore, a single \n\nmay contain several polyphenols whose \n\nconcentration (several mg to few g per kg or L), \n\nstability and activity vary from one to another. \n\nMore than 8000 phenolic compounds are known \n\nto exist and about 4000 flavonoids have been \n\nidentified [11]. Further details about different \n\ntype of phenolic compounds in food sources, their \n\ncharacteristics and relevance can be found in the \n\nliterature [3, 12]. \n\nPolyphenols are very well known as the most \n\nabundant antioxidant in the diet and they have \n\nbeen claimed to possess potential benefits against \n\ncardiovascular diseases, cancer and \n\nneurodegenerative diseases in vitro as well as in \n\nvivo [6, 13, 14]. Due to the vast structural diversity \n\nof polyphenols many of them have not yet been \n\nidentified and characterized consequently the \n\nfunctional mechanism are not well established. \n\nAmongst them, the tea polyphenol: catechin also \n\nknown as epigallocatechin-3-gallate (EGCG) is \n\nwell characterized and a large number of \n\nresearches are focused on its mode of action. \n\nLorenz [14] discussed the different mechanisms of \n\nhttp://en.wikipedia.org/wiki/Anthocyanidin", "start_char_idx": 4620, "end_char_idx": 7618, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "144812ba-80b6-4b4d-b9e6-1a7e289f1884": {"__data__": {"id_": "144812ba-80b6-4b4d-b9e6-1a7e289f1884", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cf8b89bc-a4a5-4423-8bb1-6f837e378798", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "90db0fab13f8fcb74751ebc5e6120947d69598fcf1a836dec4a51422e5fb6858", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "044cbf65-1b71-41f7-a2fa-62115740024b", "node_type": "1", "metadata": {}, "hash": "dff08d5491dc24cea46c4513e18345576b028570a8cadbe7167ba2dffada79ca", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              76                                                Nepjol.info/index.php/njb \n\n \n\nanticancer (apoptosis, inhibition of tumor growth \n\nand inhibition of telomerase), antioxidant (free \n\nradical scavenging, chelation of metal ions, \n\ninhibition of ROS producing enzyme and \n\nreduction of inflammatory cytokines), \n\nantihypertensive (vasodilation) and \n\nneuroprotective (antineuro inflammatory effect,  \n\nactivity of EGCG in the cellular and molecular \n\nlevels. In addition, a detail pharmacokinetic study \n\n(absorption, distribution metabolism and \n\nelimination) of tea polyphenols is recently \n\nreviewed by Clifford et al. [15].  \n\nExtraction process  \nDue to positive effects on human health, phenolic \n\ncompounds (isolated form or in extract) could be \n\nused as food supplements (functional food) or \n\nnutraceuticals. To do so, a cost-effective methods \n\nfor the extraction and separation of polyphenols is \n\nnecessary. A lot of research has focused on the \n\nanalysis of biological activities and \n\ncharacterization of phenolic compound and the \n\nextraction process are often ignored. The \n\nextraction and isolation of polyphenols remain \n\nchallenging especially due to structural \n\ncomplexity and instability (degradation and \n\nreaction during processing) [9, 11]. Therefore, the \n\nefficiency of the extraction of phenolic \n\ncompounds from plant material are influenced by \n\nseveral parameters: the chemical nature and \n\nlocation within the plant matrix of the phenolics, \n\nthe extraction method used, the size of sample \n\nparticle, storage conditions and presence of \n\ninterfering substance and consequential \n\nbiochemical and chemical reactions [10, 16]. \n\nPhenolic compound are essentially found in cell \n\nwalls, vacuoles, associated with nuclei, leaves, \n\ngymnosperm and rhizomes [17]. In addition, most \n\nof the phenolic compounds in plant sources are \n\nbound to plant material by covalent bond making \n\nit a real challenge to liberate them into the \n\nextractable form [18]. Therefore, different \n\nextraction methods have been developed in this \n\ndomain. The rate limiting step for phenolic \n\ncompound extraction from plant material is the \n\n(a) solubility, (b) diffusibility through the cell \n\nwalls and (c) washing out (rinsing) of the cell wall \n\n[16, 19].  \n\n1. Conventional Solvent extraction \nSolvent extraction (SE) is one of the conventional \n\nmethods of extraction of bioactive molecules from \n\nplant sources but is still widely used for the \n\nextraction of various natural bioactive \n\ncompounds specially the phenolic compounds \n\nfrom various sources [20]. Pretreated plant \n\nmaterial (washing, drying, etc.) are exposed to \n\nvarieties of solvents such as, water, hexane, ether, \n\nchloroform, acetonitrile, benzene, ethanol and \n\nmethanol which take up the molecules of interest \n\n(polyphenols). However, the efficiency of solvent \n\nextraction methods is affected by the solvent used \n\n(solubility of phenolic compounds depend on the \n\nsolvent type or its polarity) and the nature of \n\nphenolics (degree of polymerization) and their \n\ninteraction with other plant or food constituents \n\n[10]. The choice of extraction techniques have to \n\ntake into consideration of the location of phenolic \n\ncompounds in the plant, most of which are stored \n\nin the vacuoles only extractable by alcoholic or \n\norganic solvents except that are bound to \n\ninsoluble carbohydrate or protein [21]. Soxhlet \n\nextraction also known as solid-liquid extraction is \n\none of the first techniques known to be used for \n\nthe extraction of polyphenols compounds [22]. \n\nThis technique is more often considered as a \n\nleaching or lixiviation. The working principal of \n\nSoxhlet apparatus is simple as shown in figure 2. \n\nThe sample is placed in a thimble-holder and \n\nsolvent (extractant) and is filled with condensed \n\nsolvent from a distillation flask. A siphon \n\naspirator is used to unload the excess of solvents \n\nwhen it reaches to overflow level that carries the \n\nextracted molecules to the bulk liquid. This \n\nprocess is repeated until a complete extraction is \n\nachieved.", "start_char_idx": 7621, "end_char_idx": 11877, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "044cbf65-1b71-41f7-a2fa-62115740024b": {"__data__": {"id_": "044cbf65-1b71-41f7-a2fa-62115740024b", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "144812ba-80b6-4b4d-b9e6-1a7e289f1884", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "5c25b3eb727bf52c60157820b5352f49a16c89bc8b03bc9dd0a97e2904f8404e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ecdcece4-6803-4823-a1a2-be260979aaa9", "node_type": "1", "metadata": {}, "hash": "e19a5fd199c3fc4103d88fb0a2a02ca055e34e57683ad5149072ba688fca4116", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              77                                                Nepjol.info/index.php/njb \n\n \n\n \n\nFigure. 2: Conventional Soxhlet extractor (adapted \n\nfrom [23]) \n\nThe Soxhlet extraction process is easy to operate \n\nnot requiring any professional expertise. In \n\naddition, extraction productivity could be \n\nincreased by simultaneous extraction in parallel \n\nand further treatment such as filtration is not \n\nrequired as no plant residues are present.  \n\nHowever this method of extraction is often \n\ncriticized as a large volume of solvent used which \n\neventually cause environmental problems in \n\naddition to higher cost involved, long treatment \n\nperiod and thermal degradation (due to excessive \n\nexposure to heat during extraction and \n\nevaporation post extraction) of extracted \n\nmolecules as the extraction is carried out at \n\nboiling point of solvent [22]. In addition, excessive \n\nexposure to light and oxygen lead to the \n\ndegradation of other sensitive compounds [24]. \n\nTherefore, various other improved methods of \n\nsolvent extraction are researched and rapidly \n\nbeing developed which mainly focus on \n\ndecreasing the quantity of organic solvent, \n\ntreatment time and energy and increase the yield. \n\nThese methods are discussed in the following \n\nsections in details.  \n\n2 Novel and emerging methods of \n\nextraction \n\n2.1 Ultrasound assisted extraction \nThe use of ultrasound (wave frequencies of more \n\nthan 20 kHz to 10MHz) in the extraction of \n\nbioactive molecules known as ultrasound-assisted \n\nextraction (UAE) are believed to reduce the \n\norganic solvent, find other alternative solvents \n\nwhich are economically, environmentally sound \n\nand safe to human health [25].  \n\nThe irradiation of plant tissues with ultrasound \n\nincrease the number of extraction solvent exposed \n\nto the molecules by breaking down the cell wall,  \n\nincreasing swelling and hydration degree thus \n\nimproving the diffusion and mass transfer [19]. \n\nThe use of UAE have been found to improve the \n\nextraction efficiency up to 35 % during the \n\nextraction of phenolic compounds from various \n\nplant sources [25]. Chen et al. [26] found an \n\noptimized condition of UAE treatment (solvent to \n\nsolute ratio of 4:1 (ml/g), extraction time 200 s \n\nand ultrasonic power was 400 W) and found 3.45 \n\nmg of anthocyanins per gram of fresh raspberries \n\nat moderate temperature (less than 40oC. Another \n\nstudy was carried out for the retention (stability) \n\nof anthocyanins in grape juice as function of \n\nultrasound amplitude during ultrasound \n\ntreatment [27]. At higher amplitude and \n\ntreatment time degradation of certain \n\nanthocyanins were observed due to the \n\nmechanism called sonolysis of water molecules \n\ndue to cavitation inducing the formation of \n\nhydroxyl radicals which eventually lead to \n\nchemical degradation. A comparative study of \n\ndifferent method of extraction namely UAE, high \n\nhydrostatic pressurized extraction (HHPE) and \n\npulse electric field (PEF), which will be discussed \n\nlater, showed higher total phenolic content as \n\ncompared to that of control (water as solvent at \n\n70oC) [28]. But least yield and antioxidant activity \n\nwas observed in case of UAE as compared to \n\nHHPE and PEF. However, no reason was \n\ndiscussed for the least antioxidant activity \n\nobserved. This could be large quantity of plant \n\nresidues (impurities) present in the extracts.  \n\nUAE is a non-thermal process which could be \n\neasily integrated to other existing methods \n\nwithout major modification to improve the \n\nextraction efficiency and yield. Therefore, the \n\nmain advantage of UAE over other technique \n\ncould be mild temperature thus minimum \n\nthermal degradation [29]. However, the drawback \n\nof this technique is the requirement of post \n\ntreatment like filtration to get rid of resulting \n\nplant residues during the process and inability to \n\nreuse the extraction solvent [22].", "start_char_idx": 11883, "end_char_idx": 15943, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ecdcece4-6803-4823-a1a2-be260979aaa9": {"__data__": {"id_": "ecdcece4-6803-4823-a1a2-be260979aaa9", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "044cbf65-1b71-41f7-a2fa-62115740024b", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "89091a28b17c81250ca50483c4598082143d51d2d5771aeae6ce15fcd80420c6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "41bf7679-8626-4f76-b2f9-8a6dea41cd1f", "node_type": "1", "metadata": {}, "hash": "552afc75b605cac82f5932e722c61234899dc27a5bfc077b14bebfa8f6f06bc2", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              78                                                Nepjol.info/index.php/njb \n\n \n\n2.2 Microwave assisted extraction \nMicrowave assisted extraction (MAE), developed \n\nin 1980s, is a process which involves the heating \n\nof solvent by in contact with sample with \n\nmicrowave energy to partition the molecules of \n\ninterest from plant matrix to the solvent used [30]. \n\nIn comparison to other traditional extraction \n\nmethod, MAE possesses advantages like shorter \n\ntime, controlled temperature and other extraction \n\nenvironment, less solvent, higher extraction rate, \n\nenergy saving and better quality product (higher \n\nantioxidant activity) with lower cost [31]. In \n\naddition, MAE method could adapt easily the \n\nalready existing extraction process for example \n\nthat of Soxhlet. The extraction method is different \n\nfrom other conventional method because the \n\nextraction occurs due to the changes in cell \n\nstructure caused by electromagnetic waves [32]. \n\nUnlike the conventional method where the heat is \n\ntransferred from the solvent to the interior of \n\nsample, in MAE the heat is dissipated \n\nvolumetrically inside the irradiated medium \n\ntherefore the distribution of temperature is equal \n\nall over the sample  \n\nThe increase in free phenolic acid content with \n\nincrease in microwave power and treatment time \n\nwas observed in citrus mandarin pomace and \n\npeels consequently increasing their antioxidant \n\ncapacity [33, 34]. This study also demonstrated \n\nthat at 250W of microwave power, the total \n\nflavonoid compounds decreased with increase in \n\nthe duration of treatment suggesting a possible \n\ndegradation due to longer exposure to heat. \n\nTherefore, microwave treatment could be \n\neconomically viable, rapid and used in an \n\nindustrial scale production but careful choice of \n\nheating temperature and duration is needed.  \n\n2.3 Pressurized liquid extraction \nA pressurized liquid extraction (PLE) method \n\nwhich is comparatively a new method, is \n\noperated at elevated temperature (50 to 200\u00b0C) \n\nand pressure (3.5 to 20 MPa) and is supposed to \n\nenhance the extraction efficiency as compared to \n\nconventional methods carried out at room \n\ntemperature and atmospheric pressure [16]. This \n\ntechnique uses a solid samples packed in an \n\nextraction cell and uses organic solvent or water \n\nat high pressure and temperature above the \n\nboiling point [35]. A typical instrumental set up of \n\na pressurized liquid or fluid extraction (PLE) is \n\nshown in figure 3. The system consists of a \n\npumping system for the supply of solvent from \n\nthe solvent vessel which enters to a heating \n\nsystem to increase the temperature of the solvent, \n\nan extraction cell containing sample, a back \n\npressure controller and a collector at the end [35]. \n\nThe extraction efficiency using PLE are improved \n\nby increasing the solubility of phenolic \n\ncompounds and mass transfer to the extracting \n\nsolvent. [36]. In addition, increasing the pressure \n\nat elevated temperature, the diffusivity of a \n\nsolvent could be increased which will better \n\npenetrate through the plant matrix and disrupt \n\nthe solute-matrix interaction due to der Waals \n\nforces, hydrogen bonding and dipole attractions \n\nbetween solute molecules and active sites on the \n\nmatrix [37].  \n\nIn the study of Denery et al. [24] similar extraction \n\nefficiency of caretenoids were observed using  \n\nPLE and conventional (which used only \n\nsonication and centrifugation) extraction method \n\nreducing the extraction solvent by 50% and \n\nextraction time by  about 77 %. In addition, PLE \n\nwas advantageous for the oxygen and light liable \n\ncarotenoids.  \n\nFig. 3: Schematic diagram of pressurized liquid \n\nextraction system adapted from [35] \n\nThe thermal degradation which even favors the \n\noxidation of phenolic compound such as \n\nanthocyanins could be main issue related to PLE. \n\nHowever, Palma et al. [38] have demonstrated \n\nthat among 9 phenolic compounds tested most of \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al.", "start_char_idx": 15948, "end_char_idx": 20182, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "41bf7679-8626-4f76-b2f9-8a6dea41cd1f": {"__data__": {"id_": "41bf7679-8626-4f76-b2f9-8a6dea41cd1f", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ecdcece4-6803-4823-a1a2-be260979aaa9", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "ccf0e9a89705160c803d7aff69d74f0cc05bee3de6715ba0256f4ad342c13031", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ecc717c7-0e75-4299-982e-186a4ebb3741", "node_type": "1", "metadata": {}, "hash": "8e87600b5a54bf45f4325bc9f4ebaa3fbdaa4576ef2fc988649928c1fc3c94a5", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, Biotechnology Society of Nepal                              79                                                Nepjol.info/index.php/njb \n\n \n\nthem are stable except for catechin and \n\nepicatechin at 150 \u00b0C and maintain the recovery \n\nrate above 85 % with methanol as extracting \n\nsolvent. Similar result was observed by Pi\u00f1eirol et \n\nal. [39] during the extraction of catechin and \n\nepicatechin from tea leaves and grape seeds with \n\nrecovery rate below 95% at 130oC. However, this \n\nstudy demonstrated the best recovery of catechin \n\nand epicatechin from grape seeds using PLE \n\nusing methanol as solvent as compared to simple \n\nstirring assisted extraction and UAE.  \n\nWater is an extremely polar solvent, therefore not \n\nusually used as solvent for the extraction of \n\norganic compounds. However, water is non-\n\nflammable, non-toxic, widely distributed \n\necological solvent [36]. At high temperature and \n\npressure water can act as good solvent as the \n\npolarity approaches (decreases) to that of \n\nalcohols. To maintain the water in liquid state a \n\npressure ranging from 15 (at 200oC) to 85 (at \n\n300oC) bars has to be applied [37]. Therefore, \n\npressurized hot water extraction (PHWE) method \n\nis getting interest in the extraction of bioactive \n\nmolecules from plant and food material. It could \n\nalso be possible to combine the PLE with PHWE \n\nin order to increase the efficiency of extraction \n\nprocess. The temperature of solvent, pressure \n\napplied and extraction time vary widely \n\ndepending strongly upon the type of phenolics, \n\nthe solvent used and extraction instrument. \n\nTherefore, at this stage nothing could be \n\nconcluded about the optimum PLE parameters \n\n(time, solvent, pressure and temperature). \n\n2.4 Pulsed Electric field \nPulsed electric field (PEF) is one of the newest \n\ntechnologies developed as a non-thermal process \n\nwhich have been widely studied in the food \n\nprocessing sectors. Pretreatment of plant material \n\nwith PEF enhance the extraction of bioactive \n\nmolecules by electro-permeabilization (also \n\nknown as electroporation) of plant cell increasing \n\nthe mass transfer even at lower electric field [40]. \n\nBiological membrane plays an important role to \n\namply the electrical signal as it is more conductive \n\nas compared to its surrounding environment \n\n(cytoplasm and extracellular medium). The \n\nmechanism of electroporation is shown in the \n\nfigure 4. When an external electric field (E) is \n\napplied to a cell with certain thickness, the \n\nelectrical potential across the membrane (called \n\ntransmembrane electric field, which is far greater \n\nthan applied electric field) increases and when the \n\nE exceeds the critical value a reversible or \n\nirreversible pores are formed. Effectively, the \n\nnumber pore formation depends directly on the \n\nelectric field applied as well as the number of \n\npulse.  \n\nA significant improvement by 21.5 % and 28.6 % \n\nin anthocyanins content in wine with the grape \n\nskin pretreated with PEF (50 pulses and 1 Hz) \n\nusing 5 and 10 kV/cm electric fields as compared \n\nto that of untreated one was shown by L\u00f3pez et \n\nal. [41]. In addition, application of PEF (50 pulses \n\nat 122 Hz) at 5 kV/cm electric field have shown to \n\nmaintain higher anthocyanins and polyphenols \n\nconcentration during the aging process of red \n\nwine \n\n[42]. Furthermore, treatment with PEF of merlot \n\ngrapes was found to improve the sensorial and \n\nvisual quality of wine in addition to increase in \n\nthe polyphenols and anthocyanins [43, 44]. The \n\nfour fold increase in antioxidant activity was \n\nobserved for PEF (30 pulses, 2 Hz and 3 kV/cm) \n\ntreated grape as compared to untreated grape by-\n\nproducts [28]. The increase in antioxidant activity \n\nwas directly related to significant increase in \n\nphenolics as compared to the untreated samples.  \n\nTherefore, PEF can be regarded as a promising \n\ntool for valorization of low cost industrial waste \n\nto nutraceuticals or functional food. The PEF \n\ntreatment to enhance the extraction and increase \n\nthe phenolic compound concentration during \n\nmaceration and fermentation process of red wine \n\nmaking is extensively researched. \n\n\n\nNepal Journal of Biotechnology.", "start_char_idx": 20200, "end_char_idx": 24379, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ecc717c7-0e75-4299-982e-186a4ebb3741": {"__data__": {"id_": "ecc717c7-0e75-4299-982e-186a4ebb3741", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "41bf7679-8626-4f76-b2f9-8a6dea41cd1f", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "366a7175ddabb962042ec8783b9d5afd2f7313b141d7eda83034723243529b47", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bd516900-9249-4d99-a630-337fa3d13df2", "node_type": "1", "metadata": {}, "hash": "5a86b20719376897111dd91639188c82e3c91db51b71489e34cbb7440b104cdb", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              80                                                Nepjol.info/index.php/njb \n\n \n\n \nFig. 4: Mechanism of electroporation of biological \n\nmembrane in an external electric field (E). Ec represent \n\nthe critical value of electric field applied (adapted from \n\n[40]). \n\n2.5 Supercritical fluid extraction \nSupercritical fluid extraction (SFE) has been \n\nwidely used in large scale in order to extract \n\nnatural solid materials especially for food \n\ningredients, supplementation and \n\nphytopharmaceuticals. SFE is getting its \n\npopularity due to a very low or no organic \n\nsolvent, innovative, high valued and quality final \n\nproduct, reduced extraction time and higher \n\nselectivity but often criticized for high investment \n\ncost [45, 46]. At the critical values of pressure and \n\ntemperature a fluid behaves like a supercritical \n\nfluid having an intermediate properties \n\n(diffusivity, solubility and density) of gas and \n\nliquid [46]. Carbon dioxide and water are most \n\nwidely used as supercritical fluid. In comparison \n\nto water CO2 is comparatively easier to operate \n\nand commonly used as it has a moderate critical \n\ntemperature (31.2oC as compared to 101.1oC for \n\nwater) and pressure (72.9 atm vs. 217.6 atm for \n\nwater). In addition, CO2 is generally regarded as \n\nsafe (GRAS) and does not leave the residue after \n\nextraction simply by depressurization at room \n\ntemperature. However, CO2 is not very suitable \n\nfor extraction of highly polar compounds (such as \n\nflavonoids) as polarity of CO2 is lower thus, other \n\nsolvent (e.g. methanol and ethanol) at low \n\nconcentration could be used as co-solvent, known \n\nas modifiers, which subsequently increase the \n\nextraction efficiency. Le Floch et al. [47] used \n\nsupercritical CO2 extraction using methanol as \n\nmodifiers to extract polyphenols from olive leaves \n\nat different temperature, pressure, concentration \n\nof modifiers and extraction time. The phenolic \n\ncontent increased with temperature, pressure and \n\nconcentration of modifiers but a plateau was \n\nobserved at 140 minutes. At the same time, a \n\ncomparison was made with a UAE with different \n\nsolvents. The SFE with 10 % modifier (methanol) \n\ngave rise higher phenolic concentration as \n\ncompared to UAE with low polarity solvent (n-\n\nhexane, diethyl ether or ethyl acetate) but \n\nsignificantly lower as compared to UAE with high \n\npolarity solvent (methanol).  \n\nOnly 45 % extraction efficiency was observed \n\nusing SFE. Another comparative study for the \n\nextraction of isoflavones from soybean flour were \n\nconducted between SFE (CO2) with methanol as \n\nmodifiers (10 %), soxhlet (80 % ethanol) and UAE \n\n(70 % methanol) [56]. As before, a strong \n\ninteraction between pressure and temperature \n\nwas noticed. Similarly, least amount of \n\nisoflavones (86.28 \u00b5g/g of dry weight) were \n\nextracted using SFE than UAE (311.55 \u00b5g/g) and \n\nSoxhlet  \n\n(212.86 \u00b5g/g). Despite of lower recovery of \n\nisoflavones using SFE techniques, it was the most \n\nselective process as very less co-extracts were \n\npresent and require fewer steps for the extraction. \n\nMoreover, the selectivity of the SFE process was \n\nfurther proven by the results found by Kitzberger \n\net al. [57] who demonstrated that only the shiitake \n\nmushroom extracts (which contained flavonoids) \n\nobtained by SFE (at 40 \u00b0C, 20 MPa and 15% \n\nethanol) with ethanol as a co-solvent had an \n\nantibacterial activity against Micrococcus luteus \n\nand Bacillus cereus as compared to the \n\nconventional extraction with n-hexane, ethyl \n\nacetate and dichloromethane.", "start_char_idx": 24348, "end_char_idx": 28074, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bd516900-9249-4d99-a630-337fa3d13df2": {"__data__": {"id_": "bd516900-9249-4d99-a630-337fa3d13df2", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ecc717c7-0e75-4299-982e-186a4ebb3741", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "c2beb5bd2ab3bcdb72d0cac894cc5c4cb57748fb28443c9ef8483293e0298629", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9495a286-f2c5-48a4-b179-bb851f25b0b0", "node_type": "1", "metadata": {}, "hash": "deeae553938630fecc666ce080e03cf4564652e68acfac0e210866be0f89d527", "class_name": "RelatedNodeInfo"}}, "text": "However, the \n\nantibacterial activity was supposed to be due to \n\nselective extraction of flavonoids with SFE-\n\nethanol however, the authors did not compare the \n\nresults with conventional extraction with only \n\nethanol as solvent. Thus, it was not clear if the \n\nselective extraction of flavonoids and thus \n\nbioactivity was dependent on extraction \n\ntechnique or also with the type of solvent (more \n\npolar solvent like ethanol. Therefore, SFE \n\ntechnique undoubtedly carries a huge application \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              81                                                Nepjol.info/index.php/njb \n\n \n\non polyphenols extraction but considerable \n\ninnovations are needed for their industrial and \n\neconomic feasibility. \n\n2.6 High hydrostatic pressure \nHigh hydrostatic pressure is a non-thermal and \n\ngreen process governed by Le Chatelier\u2019s \n\nprinciple (Mozhaev 1994). While first application \n\nof HHP dates from few decades, the technology is \n\nconsidered as an emerging process since the first \n\nindustrial-scale systems were available in 1990 in \n\nJapan. High hydrostatic pressure processing \n\nconsists to apply a uniform pressure (100 - 1000 \n\nMPa) which is instantaneously and uniformly \n\ntransmitted, independent of the size and \n\ngeometry of food, on flexible packaging materials \n\nfilled with liquid or solid food products. The \n\npressure transmitter fluid is generally water and \n\nthe process can be used with or without \n\nutilization of heat. Its main application concerns \n\nthe pasteurization of food product in order to \n\nimprove their shelf-life and preserve components \n\nof a wide range of food products. Nowadays, a \n\nlarge number of studied focused on innovative \n\napplications of HHP such as the improvement of \n\npolyphenol\u2019s extraction. \n\nJun et al., (2009) compared the assisted extraction \n\nof polyphenol from green tea leaves by HHP \n\n(HHPE) with conventional methods, using \n\ndifferent solvents, pressure parameters and ratio \n\nliquid:solid. They stated an increase of \n\npolyphenol extraction yields is governed by an \n\nincrease in pressure level (from 15 to 30% from \n\n100 to 600", "start_char_idx": 28075, "end_char_idx": 30345, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9495a286-f2c5-48a4-b179-bb851f25b0b0": {"__data__": {"id_": "9495a286-f2c5-48a4-b179-bb851f25b0b0", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bd516900-9249-4d99-a630-337fa3d13df2", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "2814ff78513ff83aacd90d13c09f559c86144b1eaedd2c0164f346115a497d42", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2dd7c7a9-e73d-46e2-adeb-d62fd4be0150", "node_type": "1", "metadata": {}, "hash": "cd96e48d122c265d71a3ed53a8e80bbc57eedd0383a3be1abf0591734bf1412e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              82                                                Nepjol.info/index.php/njb \n\n \n\nTable 1: Methods of polyphenol extraction from different food sources and byproducts.   \n\nSource Extraction method \nTotal phenolic content \n\n(mg/g) or mg/ml \nReference \n\nCitrus mandarin peels MAE-methanol 6.37 \u00b1 0.200 [34] \n\nCitrus mandarin pomace MAE-methonol 6.18\u00b1 0.12 [33] \n\nMedicinal plant-Lien Tze Hsin IL-MAE-methanol 15.76\u00b10.329 [48] \n\nPeanut skin MAE-ethanol 144 [49] \n\nGrape marcs SE-ethanol 28.06 [50] \n\nBlueberry SE-ethanol (50%) 1.48 [51] \n\nLemon peel Enzyme \u2013assisted 1.13 \u00b1 0.0076 [52] \n\nBlack tea \n\nSolvent-acetone \n\n-N,N-dimethylformamide \n\n-ethonol \n\n-methanol \n\n98.19\u00b11.10 \n\n109.36\u00b10.71 \n\n86.31\u00b13.50 \n\n68.69\u00b11.34 \n\n[8] \n\nAlgae and Cynobacteria PLE-SPE - [53] \n\nGreen algae PLE (1500-2000 psi) 11.4 [24] \n\nHerbal medicine (Epimedium sp.) PLE- 70% ethanol (1500Psi) 48.51 [54] \n\nGrape seeds \n\n \n\nTea leaves \n\nPLE-methanol \n\n1.82 \n\n0.65 \n\n0.62 \n\n3.31 \n\n[39] \n\nRaspberries UAE (200W), 22kHz 3.45 [26] \n\n \n\nGrape seeds \nUAE (200W), 24kHz \n\n0.23 \n\n0.07 \n[39] \n\nGrape skin \n\nPEF (50 pulses, 122 Hz) -\n\n5kV/cm \n\n-10 kV/cm \n\n \n\n9.65 \n\n10.6 \n\n[41] \n\nRed grapes \n\nPEF (50 pulses, 122 Hz) -5 \n\nkV/cm \n\n-7 kV/cm \n\n \n\n6.02 \n\n5.20 \n\n[55] \n\nGrape byproduct \nPEF (30 pulses, 2 Hz, \n\n3kV/cm) \n61.2 [28] \n\nOlive leaves \nSFE-10 % methanol, 330 atm \n\nand 100 oC \n7.6 \u00b1 0.5 [47] \n\nSoybean flour \nSFE-10 % methanol, 355 atm \n\nand 50 oC \n0.086 [56] \n\nShiitake mushroom \nSFE-15 % ethanol, 296 atm \n\nand 50 oC \n10.2 [57] \n\nGrape skin HHP-50% ethanol (200 MPa) 8.91 \u00b1 0.13 [58] \n\nChilean papaya seeds \nHHP-500 MPa 15 min 50% \n\nethanol \n- [59] \n\nGreen tea leaves \nHHP-500 MPa 1 min 50% \n\nethanol \n- [60] \n\nWatercress \nHHP-600 MPa 3.1 min 100% \n\nethanol \n64.68 \u00b1 2.97 [61] \n\nMPa). Globally, they achieve in 1 min - 500 MPa \n\nthe same extraction yield obtained by 90 min \n\nultrasonic extraction, 45 min heat reflux extraction \n\nor 20h room temperature extraction. More \n\nrecently, a research on Chilean papaya seeds have \n\nreach the same conclusion concerning the \n\nefficiency of HHP to improve the extraction of \n\npolyphenol. Indeed, Briones-Labarca et al., (2014) \n\ncould extract more efficiently polyphenol by \n\nHHPE (5, 10 and 15 min) in comparison with \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al.", "start_char_idx": 30356, "end_char_idx": 32878, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2dd7c7a9-e73d-46e2-adeb-d62fd4be0150": {"__data__": {"id_": "2dd7c7a9-e73d-46e2-adeb-d62fd4be0150", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9495a286-f2c5-48a4-b179-bb851f25b0b0", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "0afb3ab2d27d84caee340470a62e06e28f603bd30ebbfd07436deb5f303849ce", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fbc0b645-fcc9-4a53-a4ea-2162a3b76290", "node_type": "1", "metadata": {}, "hash": "4a4ffd8adf62810929f5a9389fafb40af8bbb1a77d344e71b24a1b9d8beef7d0", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, Biotechnology Society of Nepal                              83                                                Nepjol.info/index.php/njb \n\n \n\nconventional extraction (30 min) or even \n\nultrasound assisted-extraction (5, 10 and 15 min). \n\nLastly, Pinela et al., (2018) determined by \n\nresponse surface methodology, the optimal HHPE \n\nparameters to achieve the higher extraction yields \n\nof polyphenol from watercress. Once again, there \n\nis a significant positive correlation between the \n\nlevel of pressure and the extraction yield. Broadly \n\nspeaking, HHP enable the solvent to enter cells \n\nand lead to an increase of the cell permeability. \n\nDuring the instantaneous release of pressure, the \n\ncell wall is disrupted leading to a spill of \n\ncytoplasm and target molecules such as \n\npolyphenols (Jun et al., 2009). The different \n\nmethods of extraction process used for \n\npolyphenol extraction from various food sources \n\nand their by-products are summarized in Table 1.  \n\nTo this point, it is not very clear which is the best \n\nextraction method due to the complexity in plant \n\nsource, wide nature of polyphenols (size and \n\nphysicochemical) and varieties of solvent used. \n\nVarious studies made the comparison among \n\ndifferent methods. However, the same method \n\nshowed different yield which largely depended \n\non type of source and solvent used. Table 2 shows \n\nthe comparison between different extractions \n\nmethods discussed above. Therefore, more \n\ncomprehensive studies are needed that can clearly \n\ndistinguish the mechanism of extraction and its \n\nrelation to different type source, molecules of \n\ninterest, time and cost.  \n\n4. Separation and purification processes \n\n4.1 Ion exchange chromatography \nIon exchange chromatography (IEC) is widely \n\nused technique in food processing which \n\nseparates the molecules according to their charge \n\n[62]. The later can be modified by changing the \n\npH or ionic strength of the solution. Ottens et al. \n\n[62] emphasized that for a preparative \n\npurification of bio-molecules an IEC process in \n\nindispensable that need to take into consideration \n\nof the choice of resins, the adsorbent (cost, resin \n\ncapacity, regeneration cycle, life time  and safety). \n\nAdsorption and desorption of molecules to be \n\nseparated (target molecules) on resins packed in a \n\ncolumn are the main steps in the IEC process. \n\nEquilibration of resins (pH, ionic strength and \n\nsolvent concentration), loading of samples with \n\nimpurities (that will adsorb onto the resin), \n\nwashing (to desorb the impurities) and elution (of \n\ntarget molecule to elution buffer) that can be \n\ncarried out in isocratic or gradient mode, are the \n\nmain steps of a typical IEC process.  \n\nA high recovery efficiency of EGCG was found by \n\nusing cyclic IEC techniques that uses less volume \n\nof solvents (30% ethanol) as compared to \n\nconventional maceration extraction using \n\nultrasound [63]. Anthocyanins from strawberry \n\nwere purified by IEC using Amberlite XAD-7 as \n\nion exchange resin before analytic separation [64]. \n\nSimilarly, cation exchange chromatography \n\ntechnique was used to purify polyphenols from \n\nred wine after solvent extraction [65]. As this \n\ntechnique uses solvents as an eluent to desorb the \n\nadsorbed polyphenols from the ion exchange \n\nresin further purification is needed to ensure the \n\npurity of the final product. In addition, the use of \n\nnon-solvent IEC process could be a better option. \n\nPolyphenols such as epigallocatechin-gallate \n\n(EGCG), epicatechin-gallate (ECG) and \n\nepigallocatechin (EGC) were separated by using \n\nweakly acidic cation exchange gels (dextran \n\nbased) from crude tea extracts without using any \n\nsolvent [66] \n\n4.2 Membrane based process \nA membrane based separation process has been \n\nwidely used in an industrial scale. In addition, all \n\nthe above mentioned extraction processes \n\ncontained more or less organic solvent which are \n\nproven to be toxic to human health, thus has an \n\nimportant role in consumer acceptance of these \n\nproduct.", "start_char_idx": 32896, "end_char_idx": 36923, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fbc0b645-fcc9-4a53-a4ea-2162a3b76290": {"__data__": {"id_": "fbc0b645-fcc9-4a53-a4ea-2162a3b76290", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2dd7c7a9-e73d-46e2-adeb-d62fd4be0150", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "567961d850822937c233f5ed09c9dd5aeb3dde52011d9124d7beb5906bdd3de4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7a424b62-ee92-42c6-802d-79920feacc79", "node_type": "1", "metadata": {}, "hash": "5fba10b9fb66261a3ad2c4128d311296f9f415a9cf7f0861b7f61c4ca1112dca", "class_name": "RelatedNodeInfo"}}, "text": "In addition, the separation and \n\npurification efficiency of membrane process \n\ndepends upon the molecular weight polyphenols \n\nto be concentrated which ranges from 290 to 1200 \n\ng/mol, membrane molecular weight cut off \n\n(MWCO) sizes and the transmembrane pressure \n\n(the driving force) applied in the system. \n\nGenerally, ultrafiltration (UF), nanofiltration (NF) \n\nand reverse osmosis (RO) process are used. The \n\nmembrane process are particularly interesting to \n\nseparate and concentrate polyphenols from or in   \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              84                                                      Nepjol.info/index.php/njb \n\n \n\nTable 2: Comparison between different extraction process adapted and modified from [29] \n\n \nSolvent extraction \n\n \n\nMicrowave \nassisted \n\nextraction \n \n\nSupercritical fluid \nextraction \n\nUltrasound-\nassisted extraction \n\nPulsed electric \nfield \n\nPressurized \nsolvent extraction \n\nHigh Hydrostatic \nPressure \n\nextraction \n\nBrief description \n\nSolvent is heated \nby a conventional \n\noven \nand passed by the \n\nsample \n\nImmersion of the \nsample in \n\nsolvent and  \nmicrowave energy \n\nis submitted \n\nA high pressure \nvessel is filled with \nsample and crossed \n\ncontinuously by \nthe supercritical \n\nfluid \n\nImmersion of the \nsample in solvent \nand submission to \nultrasound using a \n\nUS probe or US \nbath \n\nPulses of high \nelectric \n\nvoltages are \napplied to the \nsample placed \n\nin between \ntwo electrodes \n\nHeat of the sample \nby a conventional \noven and crossed \nby the extraction \n\nsolvent under \npressure \n\nSample is \npressurized (100 \u2013 \n\n1000 MPa) \nthrough a \npressure \n\ntransmitter liquid \n\nExtraction time 6-8 hours 3\u201330 min 10\u201360 min 10\u201360 min - 10\u201320 min 1 \u2013 30 min \n\nSample size - 1\u201310 g 1\u20135 g 1\u201330 g  1\u201330 g - \n\nSolvent volume - \n10\u201340 ml 2\u20135 ml \n\n(solid trap) \n30\u201360 ml (liquid \n\ntrap) \n50\u2013200 ml - 15\u201360 ml - \n\nCost  Moderate High Low High High High \n\nAdvantages \nRapid and easy to \n\nhandle \n\nRapid \nEasy to handle \n\nModerate solvent \nconsumption \n\nRapid \nLow solvent \nconsumption \n\nConcentration of the \nextract \n\nNo filtration \nnecessary \n\nPossible high \nselectivity \n\nEasy to use \nRapid and \n\nnon-thermal \nprocess \n\nRapid \nNo filtration \n\nnecessary \nLow solvent \nconsumption \n\nRapid \nGreen technology \nHigh selectivity \nHigh extraction \n\nyield \nNo degradation of \ntarget molecules \n\nDisadvantages \n\nHigh solvent \nconsumption, long \ntreatment time and \n\nthermal \ndegradation \n\nExtraction solvent \nmust \n\nabsorb microwave \nenergy \n\nFiltration step \nrequired \n\nMany parameters to \noptimize \n\nLarge amount of \nsolvent \n\nconsumption \nFiltration step \n\nrequired \n\nMechanism \nnot well \n\nknown and \nprocess \n\nintensification \nis difficult \n\nPossible \ndegradation of \nthermolabile \n\nanalytes \n\nHigh cost \nequipment \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              85                                                      Nepjol.info/index.php/njb \n\n \n\n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              86                                                          Nepjol.info/index.php/njb \n\n \n\naqueous solutions (fruit juices) or from the \n\ncomplex solute-solvent after the extraction \n\nprocess using conventional methods can \n\nsignificantly minimize the amount of solvent \n\nbeing used [67, 68].", "start_char_idx": 36924, "end_char_idx": 40598, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7a424b62-ee92-42c6-802d-79920feacc79": {"__data__": {"id_": "7a424b62-ee92-42c6-802d-79920feacc79", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fbc0b645-fcc9-4a53-a4ea-2162a3b76290", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "e023b1603fa93c190228eb22a84abaa7d9cfaa28cd44063dfbf05f856de90912", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "47b8c217-f154-4ef8-9e23-9e1a24a4bffa", "node_type": "1", "metadata": {}, "hash": "c47eb224077fe96af11b6866c8db7cb97c65cc4555f09ee97f773b0e74e23404", "class_name": "RelatedNodeInfo"}}, "text": "The efficiency of \n\nconcentration of polyphenols from grape seeds by \n\nultrafiltration (dead end filtration) with 50 kDa \n\nmembrane was found to depend on the method of \n\nextraction used [68]. In fact, the different \n\nextraction processes result to in different quality \n\nfinal product in terms of solute (polyphenols) \n\ncontent. Significantly higher concentration (40-\n\n50%) of total polyphenols was recovered in \n\nretentate after the ultrafiltration. The studies with \n\ndifferent MWCO membranes have shown that \n\nlow molecular weight phenolic compounds are \n\nmostly present in permeate solution however in \n\nthe concentrate of reverse osmosis process [69].  \n\nAnother possibility of concentration of \n\npolyphenols with higher selectivity is to use the \n\nsequential filtration process. For the low \n\nmolecular free polyphenols found in olive mill \n\nwastewater (OMW) were extracted by sequential \n\nfiltration process [70]. The first step, \n\nmicrofiltration was followed by nanofiltration \n\nand osmotic distillation (also known as isothermal \n\nmembrane distillation) and vacuum membrane \n\ndistillation. The preliminary step of \n\nmicrofiltration reduced significantly total organic \n\ncarbon and 0.5 g/L of low molecular weight \n\npolyphenols. The osmotic distillation process is \n\nrelatively a new method which can potentially be \n\nused in the concentration of beverages and other \n\nliquid food stuffs. In this process, a micro-porous, \n\nnon-liquid-wettable membrane (hydrophobic) \n\nseparates a mixture of liquid containing a volatile \n\nin one side and a second liquid phase capable of \n\nabsorbing the volatile component. Membrane \n\ndistillation process could be advantageous to \n\nselectively remove the solvent (ethanol) used \n\nduring the extraction process.  The details about \n\nthe membrane distillation process are discussed \n\nin the literature [71]. This process was used to \n\nconcentrate polyphenols from olive mill \n\nwastewater using micro-porous membranes with \n\nup to 99 % separation coefficient [72].  \n\nHowever, the membrane processes are frequently \n\ncriticized due to membrane fouling and reduced \n\nselectivity for polyphenols separation. Fouling by \n\npolyphenols and therefore significant reduction in \n\nwater flux was observed by Susanto et al. [73]. \n\nThey proposed different mechanisms of \n\ninteraction between polyphenols molecule (or \n\naggregate of polyphenols) with membrane \n\nmaterials. The different interaction could be \n\nhydrophobic, hydrogen bonding and benzene \n\nring interaction via \u03c0\u2013\u03c0 stacking when the \n\nmembrane contains benzene rings \n\n(polyethersulfone, PES). The polyphenols can \n\nreduce the size of pore or block entirely. \n\n4.3 Electromembrane process- \n\nElectrodialysis with ultrafiltration \n\nmembrane \nElectromembrane process is widely used to \n\nseparate charged molecules from a complex \n\nmixture of solution. One of the recent \n\nelectromembrane processes is the combination of \n\nthe conventional electrodialysis process with \n\nmembrane filtration known as electrodialysis \n\nwith filtration membrane (EDFM), with a diverse \n\napplication in food processing, biotechnology and \n\nbiopharmaceutical [74]. In this method, the \n\nelectric field applied acts as the driving force for \n\nthe migration of charged molecules (ionic species) \n\nacross a porous membrane (microfiltration) from \n\none solution to another. As there is no pressure \n\napplied in the system in contrast to the pressure \n\ndriven filtration process, there are no or very less \n\naccumulation of solutes on the membrane surface. \n\nBazinet et al. [75] reported the first application of \n\nelectrodialysis (ED) process in the separation of \n\npolyphenols from tobacco extract. The authors \n\nsuccessfully separated chlorogenic acid with \n\nmigration rate up to 28 % using in 3 hours of \n\ntreatment. A significant improvement in \n\nmigration rate of 90.8 % for chlorogenic acid and \n\n86.5 % and 81.3 % for scopoletin and rutin \n\nrespectively by increasing the membrane stacks \n\nby 3 folds in the same system treated for 4 hours \n\nas shown in figure 5 [76]. \n\nLater on the EDFM process was used to separate \n\ncatechins from green tea. Labb\u00e9 et al.", "start_char_idx": 40599, "end_char_idx": 44732, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "47b8c217-f154-4ef8-9e23-9e1a24a4bffa": {"__data__": {"id_": "47b8c217-f154-4ef8-9e23-9e1a24a4bffa", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7a424b62-ee92-42c6-802d-79920feacc79", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "135b15974ae712aff9acf771fb52d2e66ff831a612f8b4483bfa35c45ec09255", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4594c5a-8de8-4c19-827d-8b11b37907de", "node_type": "1", "metadata": {}, "hash": "dc8439082740415024112dc4434c8ee89ab354d0f42fb36353a3286c5a594964", "class_name": "RelatedNodeInfo"}}, "text": "Labb\u00e9 et al. [77] \n\nsuccessfully and selectively separated  EGCG and \n\nEGC from green tea infusion by in an ED system \n\nwith the migration rate as high as 50 % within 1 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              87                                                          Nepjol.info/index.php/njb \n\n \n\n  \n\nFig. 5: Schematic diagram of electrodialysis system used for polyphenol separation from tobacco extract (adapted from \n\n[74]) \n\nhour of treatment. This study, in contrast to the \n\nstudy of Bazinet et al. [75], a better migration rate \n\nwas observed using ultrafiltration membrane \n\n(1000 Da)  in comparison to anion selective \n\nmembranes. Therefore, it was concluded that \n\nseparation and purification process using ED \n\ndepend on the type (physicochemical and \n\nmolecular cut off values) of the membrane used.  \n\nCatechins are negatively charged at their initial \n\npH (5.6 to 5.8) and thus migrate towards anode. In \n\naddition to isolating the polyphenols, another \n\ninteresting application of EDFM is to concentrate \n\npolyphenols with antioxidant activity in fruit \n\njuices. The anthocyanins and proanthocyanins \n\nwere concentrated to 52.9 and 34.8 % respectively  \n\nfrom natural cranberry juice by [78]. The authors \n\nalso proposed a feasibility of direct integration of \n\nEDFM system in the cranberry juice processing \n\nplant in order to produce anthocyanins enriched \n\ncranberry juice.  However, attenuation polyphenol \n\nconcentration in the feed cranberry juice leads to \n\nthe degradation of juice quality and membrane \n\nfouling during the treatment are major issues that \n\nshould be solved for its successful application. \n\nAnother study was carried out with larger volume \n\nof raw cranberry juice in the diluate compartment \n\nand smaller volume in the concentrate \n\ncompartment [79]. This study demonstrated no \n\nchange in physicochemical properties (sugar, \n\norganic acid, vitamin C, color index pH and \n\nconductivity) of raw cranberry juice in the diluate \n\nsolution. Another study were carried out by \n\nincreasing the number of membrane stacking and \n\nusing different volume ratio (diluate to \n\nconcentrate side) of 30 which increased the \n\nanthocyanins content by 24 % [80]. A continuous \n\nbut single passage of cranberry juice in diluate \n\nside of EDFM system was found to increase the \n\npolyphenol content of another cranberry juice \n\ncontinuously circulated in the system. Moreover, \n\ntitrable acidity was found to significantly decrease \n\nin the enriched juice thus increase the organoleptic \n\ncharacteristic.  \n\nHowever, above mentioned studies clearly \n\ndemonstrated a problem related to the membrane \n\nfouling. Therefore, optimisation of process for a \n\nproper membrane type (both MWCO as well as \n\nmembrane material), pH, other electrodialytic \n\nparameters are needed. In addition, this process \n\nseems very promising for the separation and \n\npurification of polyphenols present in industrial \n\nbyproducts such as grape seeds and marcs, olive \n\noil wastewater, etc after an appropriate extraction \n\nprocess. \n\nConclusion \nSignificant improvements in polyphenol \nextraction have been made as compared to \ntradition method of solvent extraction. However, \nalthough the quantity solvent used is significantly \nreduced, the use of solvent at less concentration is \nvital for better productivity and yield. The studies \nclearly show that the efficiency of an extraction \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              88                                                          Nepjol.info/index.php/njb \n\n \n\nprocess is largely \n\nFig. 6: Configuration of the EDFM cell and of the \nglobal system used to enrich cranberry juice \npolyphenols (anthocyanins). AEM: anion-\nexchange membrane, FM: filtration membrane, \nCEM: cation-exchange membrane (adapted from \n[77]).", "start_char_idx": 44720, "end_char_idx": 48796, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4594c5a-8de8-4c19-827d-8b11b37907de": {"__data__": {"id_": "d4594c5a-8de8-4c19-827d-8b11b37907de", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "47b8c217-f154-4ef8-9e23-9e1a24a4bffa", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "1d56a8092020bd9505119bf89d78079c93bc93a73fc9f47c5e418d8e6eadc9df", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5160a3a8-c592-4bf3-b7c7-185cf2cbb785", "node_type": "1", "metadata": {}, "hash": "5f24eb8828a177d66658098c881e7d1fdfefe91f893e216a965560023d15de66", "class_name": "RelatedNodeInfo"}}, "text": "dependent on the type of polyphenol to be \n\nextracted, its location in plant as well as type of \n\nplant material, selectivity and quality desired. The \n\nextraction process design therefore has to take into \n\naccount of the source, the total yield, the \n\nproductivity and selectivity. Moreover, more and \n\nmore researches are inclined in the valorization of \n\nindustrial by products such as from wine making \n\nprocess, olive oil mill and other fruit juices. This \n\nunquestionably shows both economical as well as \n\nenvironmental benefits. Once the polyphenol has \n\nbeen extracted and for the polyphenols that are \n\nalready present in an aqueous solution such as \n\njuices and wines, different separation and \n\npurification could be employed. Ion exchange \n\nresins and pressure driven membrane process are \n\nwidely used. However, electromembrane process \n\nknown as electrodialysis with ultrafiltration could \n\nbe a best alternative for the conventional \n\nfractionation methods because this is a green and \n\nvery selective (no solvent used) technique and has \n\nno or less membrane fouling  \n\nReferences \n1. Lopez AD, Murray CCJL: The global burden \n\nof disease, 1990-2020. Nat Med 1998, \n4(11):1241-1243. \n\n2. Wansink B, Westgren RE, Cheney MM: \nHierarchy of nutritional knowledge that \n\nrelates to the consumption of a functional \nfood. Nutrition 2005, 21(2):264-268. \n\n3. Manach C, Scalbert A, Morand C, R\u00e9m\u00e9sy C, \nJim\u00e9nez L: Polyphenols: food sources and \nbioavailability. Am J Clin Nutr 2004, 79(5):727-\n747. \n\n4. Korhonen H, Pihlanto A: Bioactive peptides: \nproduction and functionality. Int Dairy J 2006, \n16(9):945-960. \n\n5. Siriwardhana N, Kalupahana NS, Moustaid-\nMoussa N, Se-Kwon K: Chapter 13 - Health \nBenefits of n-3 Polyunsaturated Fatty Acids: \nEicosapentaenoic Acid and Docosahexaenoic \nAcid. In: Advances in Food and Nutrition \nResearch. vol. Volume 65: Academic Press; \n2012: 211-222. \n\n6. Scalbert A, Johnson IT, Saltmarsh M: \nPolyphenols: antioxidants and beyond. Am J \nClin Nutr 2005, 81(1):215S-217S. \n\n7. Yu J, Ahmedna M, Goktepe I: Effects of \nprocessing methods and extraction solvents \non concentration and antioxidant activity of \npeanut skin phenolics. Food Chem 2005, 90(1-\n2):199-206. \n\n8. Turkmen N, Sari F, Velioglu YS: Effects of \nextraction solvents on concentration and \nantioxidant activity of black and black mate \ntea polyphenols determined by ferrous \ntartrate and Folin-Ciocalteu methods. Food \nChem 2006, 99(4):835-841. \n\n9. Cheynier Vr: Polyphenols in foods are more \ncomplex than often thought. Am J Clin Nutr \n2005, 81(1):223S-229S. \n\n10. Naczk M, Shahidi F: Extraction and analysis \nof phenolics in food. J Chromatogr 2004, \n1054(1-2):95-111. \n\n11. Tsao R: Chemistry and Biochemistry of \nDietary Polyphenols. Nutrients 2010, \n2(12):1231-1246. \n\n12. El Gharras H: Polyphenols: food sources, \nproperties and applications \u2013 a review. Int J \nFood Sci Tech 2009, 44(12):2512-2518. \n\n13.", "start_char_idx": 48798, "end_char_idx": 51721, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5160a3a8-c592-4bf3-b7c7-185cf2cbb785": {"__data__": {"id_": "5160a3a8-c592-4bf3-b7c7-185cf2cbb785", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4594c5a-8de8-4c19-827d-8b11b37907de", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "3a912143dc7180ff64906db60f680f79b0a96e72297ab480b7b4e135aaf23545", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "01cbe329-41d3-4cd1-8eae-064e3a383fbc", "node_type": "1", "metadata": {}, "hash": "06a73e9fb9a320cd5039ff02893181faa95092cf54f4193161db845ac773b223", "class_name": "RelatedNodeInfo"}}, "text": "13. Rodriguez-Mateos A, Rendeiro C, Bergillos-\nMeca T, Tabatabaee S, George TW, Heiss C, \nSpencer JP: Intake and time dependence of \nblueberry flavonoid-induced improvements \nin vascular function: a randomized, \ncontrolled, double-blind, crossover \nintervention study with mechanistic insights \ninto biological activity. Am J Clin Nutr, \n98(5):1179-1191. \n\n14. Lorenz M: Cellular targets for the beneficial \nactions of tea polyphenols. Am J Clin Nutr \n2013. \n\n15. Clifford MN, van der Hooft JJ, Crozier A: \nHuman studies on the absorption, \ndistribution, metabolism, and excretion of tea \npolyphenols. Am J Clin Nutr 2013. \n\n16. Mustafa A, Turner C: Pressurized liquid \nextraction as a green approach in food and \nherbal plants extraction: A review. Anal Chim \nActa 2011, 703(1):8-18. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              89                                                          Nepjol.info/index.php/njb \n\n \n\n17. Hutzler P, Fischbach R, Heller W, Jungblut TP, \nReuber S, Schmitz R, Veit M, Weissenb\u00f6ck G, \nSchnitzler J-P: Tissue localization of phenolic \ncompounds in plants by confocal laser \nscanning microscopy. J Exp Bot 1998, \n49(323):953-965. \n\n18. Xu G, Ye X, Chen J, Liu D: Effect of Heat \nTreatment on the Phenolic Compounds and \nAntioxidant Capacity of Citrus Peel Extract. J \nAgric Food Chem 2006, 55(2):330-335. \n\n19. Vinatoru M: An overview of the ultrasonically \nassisted extraction of bioactive principles \nfrom herbs. Ultrason Sonochem 2001, 8(3):303-\n313. \n\n20. Joana Gil-Ch\u00e1vez G, Villa JA, Fernando Ayala-\nZavala J, Basilio Heredia J, Sepulveda D, Yahia \nEM, Gonz\u00e1lez-Aguilar GA: Technologies for \nExtraction and Production of Bioactive \nCompounds to be Used as Nutraceuticals and \n\nFood Ingredients: An Overview. Compr Rev \nFood Sci Food Saf 2013, 12:5-23. \n\n21. Robbins RJ: Phenolic Acids in Foods: An \nOverview of Analytical Methodology. J Agric \nFood Chem 2003, 51(10):2866-2887. \n\n22. Luque de Castro MD, Garc\u0131 \u0301a-Ayuso LE: \nSoxhlet extraction of solid materials: an \noutdated technique with a promising \ninnovative future. Anal Chim Acta 1998, 369(1-\n2):1-10. \n\n23. Luque de Castro MD, Priego-Capote F: Soxhlet \nextraction: Past and present panacea. J \nChromatogr 2010, 1217(16):2383-2389. \n\n24. Denery JR, Dragull K, Tang CS, Li QX: \nPressurized fluid extraction of carotenoids \nfrom Haematococcus pluvialis and Dunaliella \nsalina and kavalactones from Piper \nmethysticum. Anal Chim Acta 2004, 501(2):175-\n181. \n\n25. Vilkhu K, Mawson R, Simons L, Bates D: \nApplications and opportunities for \nultrasound assisted extraction in the food \n\nindustry - A review. Innov Food Sci Emerg \nTechnol 2008, 9(2):161-169. \n\n26.", "start_char_idx": 51718, "end_char_idx": 54516, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "01cbe329-41d3-4cd1-8eae-064e3a383fbc": {"__data__": {"id_": "01cbe329-41d3-4cd1-8eae-064e3a383fbc", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5160a3a8-c592-4bf3-b7c7-185cf2cbb785", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "44014d264eeaaa563b28687234d781acadae1405ab9f28dccfa9fc65d80821b3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d3abb849-1685-4970-9f7d-01b806d2ec32", "node_type": "1", "metadata": {}, "hash": "23c91e37307465a32482ed67ec2325c885b9f25c7056ea6197baa8c4e6f4ea98", "class_name": "RelatedNodeInfo"}}, "text": "26. Chen F, Sun Y, Zhao G, Liao X, Hu X, Wu J, \nWang Z: Optimization of ultrasound-assisted \nextraction of anthocyanins in red raspberries \nand identification of anthocyanins in extract \nusing high-performance liquid \n\nchromatography-mass spectrometry. Ultrason \nSonochem 2007, 14(6):767-778. \n\n27. Tiwari BK, Patras A, Brunton N, Cullen PJ, \nO\u2019Donnell CP: Effect of ultrasound processing \non anthocyanins and color of red grape juice. \nUltrason Sonochem 2010, 17(3):598-604. \n\n28. Corrales M, Toepfl S, Butz P, Knorr D, \nTauscher B: Extraction of anthocyanins from \ngrape by-products assisted by ultrasonics, \nhigh hydrostatic pressure or pulsed electric \nfields: A comparison. Innov Food Sci Emerg \nTechnol 2008, 9(1):85-91. \n\n29. Chemat F, Zill e H, Khan MK: Applications of \nultrasound in food technology: Processing, \n\npreservation and extraction. Ultrason Sonochem \n2011, 18(4):813-835. \n\n30. Renoe BW: Microwave assisted extraction. \nAmerican Laboratory 1994, 26:34-34. \n\n31. Hayat K, Hussain S, Abbas S, Farooq U, Ding \nB, Xia S, Jia C, Zhang X, Xia W: Optimized \nmicrowave-assisted extraction of phenolic \nacids from citrus mandarin peels and \nevaluation of antioxidant activity in vitro. Sep \nPurif Technol 2009, 70(1):63-70. \n\n32. Veggi PC, Martinez J, Meireles MAA: \nFundamentals of Microwave Extraction. In: \nMicrowave-assisted Extraction for Bioactive \nCompounds. Springer; 2013: 15-52. \n\n33. Hayat K, Zhang X, Farooq U, Abbas S, Xia S, \nJia C, Zhong F, Zhang J: Effect of microwave \ntreatment on phenolic content and \nantioxidant activity of citrus mandarin \n\npomace. Food Chem 2010, 123(2):423-429. \n34. Hayat K, Zhang X, Chen H, Xia S, Jia C, Zhong \n\nF: Liberation and separation of phenolic \ncompounds from citrus mandarin peels by \nmicrowave heating and its effect on \nantioxidant activity. Sep Purif Technol 2010, \n73(3):371-376. \n\n35. Ong E-S, Woo S-O, Yong Y-L: Pressurized \nliquid extraction of berberine and \naristolochic acids in medicinal plants. J \nChromatogr 2000, 904(1):57-64. \n\n36. Ong ES, Cheong JSH, Goh D: Pressurized hot \nwater extraction of bioactive or marker \ncompounds in botanicals and medicinal plant \n\nmaterials. J Chromatogr 2006, 1112(1-2):92-102. \n37. Teo CC, Tan SN, Yong JWH, Hew CS, Ong ES: \n\nPressurized hot water extraction (PHWE). J \nChromatogr 2010, 1217(16):2484-2494. \n\n38. Palma M, Pi\u00f1eiro Z, Barroso CG: Stability of \nphenolic compounds during extraction with \nsuperheated solvents. J Chromatogr 2001, \n921(2):169-174. \n\n39. Pi\u00f1eiro Z, Palma M, Barroso CG: \nDetermination of catechins by means of \nextraction with pressurized liquids. J \nChromatogr 2004, 1026(1-2):19-23. \n\n40. Dons\u00ec F, Ferrari G, Pataro G: Applications of \nPulsed Electric Field Treatments for the \nEnhancement of Mass Transfer from \nVegetable Tissue. Food Eng Rev 2010, 2(2):109-\n130. \n\n41.", "start_char_idx": 54513, "end_char_idx": 57328, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d3abb849-1685-4970-9f7d-01b806d2ec32": {"__data__": {"id_": "d3abb849-1685-4970-9f7d-01b806d2ec32", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "01cbe329-41d3-4cd1-8eae-064e3a383fbc", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "902a673ea1b7915dcd157f9ba7a719585a370d67d43f4a6a1f65d9c8d4ee9991", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "23340354-3c0e-4aa9-969c-f8a7e701e7a6", "node_type": "1", "metadata": {}, "hash": "658f48966181c85a2ea5754d6353b41223881fe090a3f8ff6f93f718d60ef816", "class_name": "RelatedNodeInfo"}}, "text": "Food Eng Rev 2010, 2(2):109-\n130. \n\n41. L\u00f3pez N, Pu\u00e9rtolas E, Cond\u00f3n S, \u00c1lvarez I, \nRaso J: Effects of pulsed electric fields on the \nextraction of phenolic compounds during the \n\nfermentation of must of Tempranillo grapes. \nInnov Food Sci Emerg Technol 2008, 9(4):477-482. \n\n42. Pu\u00e9rtolas E, Salda\u00f1a G, Cond\u00f3n S, \u00c1lvarez I, \nRaso J: Evolution of polyphenolic compounds \nin red wine from Cabernet Sauvignon grapes \nprocessed by pulsed electric fields during \naging in bottle. Food Chem 2010, 119(3):1063-\n1070. \n\n43. Delsart C, Ghidossi R, Poupot C, Cholet C, \nGrimi N, Vorobiev E, Milisic V, Peuchot MM: \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              90                                                          Nepjol.info/index.php/njb \n\n \n\nEnhanced Extraction of Valuable Compounds \nfrom Merlot Grapes by Pulsed Electric Field. \nAm J Enol Viticult 2012. \n\n44. L\u00f3pez N, Pu\u00e9rtolas E, Hern\u00e1ndez-Orte P, \nIgnacio \u00c1lvarez I, Raso J: Effect of a pulsed \nelectric field treatment on the anthocyanins \ncomposition and other quality parameters of \nCabernet Sauvignon freshly fermented model \nwines obtained after different maceration \n\ntimes. LWT - Food Sci Technol 2009, 42(7):1225-\n1231. \n\n45. Perrut M: Supercritical fluid applications: \nIndustrial developments and economic \n\nissues. Ind Eng Chem Res 2000, 39(12):4531-\n4535. \n\n46. Herrero M, Cifuentes A, Ibanez E: Sub-and \nsupercritical fluid extraction of functional \ningredients from different natural sources: \nPlants, food-by-products, algae and \n\nmicroalgae: A review. Food Chem 2006, \n98(1):136-148. \n\n47. Le Floch F, Tena MT, R\u0131 \u0301os A, Valc\u00e1rcel M: \nSupercritical fluid extraction of phenol \n\ncompounds from olive leaves. Talanta 1998, \n46(5):1123-1130. \n\n48. Lu Y, Ma W, Hu R, Dai X, Pan Y: Ionic liquid-\nbased microwave-assisted extraction of \nphenolic alkaloids from the medicinal plant \nNelumbo nucifera Gaertn. J Chromatogr 2008, \n1208(1-2):42-46. \n\n49. Ballard TS, Mallikarjunan P, Zhou K, O\u2019Keefe \nS: Microwave-assisted extraction of phenolic \nantioxidant compounds from peanut skins. \nFood Chem 2010, 120(4):1185-1192. \n\n50. Celia M. Libr\u00e1n LM, Esperanza M. Garcia-\nCastello, Daniel Vidal-Brotons: Polyphenol \nextraction from grape wastes: Solvent and pH \neffect. Agric Sci 2013, 9B:56-62. \n\n51. Oancea S, Stoia M, Coman D: Effects of \nExtraction Conditions on Bioactive \nAnthocyanin Content of Vaccinium \nCorymbosum in the Perspective of Food \n\nApplications. Procedia Eng 2012, 42(0):489-495. \n52. Li BB, Smith B, Hossain MM: Extraction of \n\nphenolics from citrus peels: II. Enzyme-\n\nassisted extraction method. Sep Purif Technol \n2006, 48(2):189-196. \n\n53.", "start_char_idx": 57289, "end_char_idx": 60056, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "23340354-3c0e-4aa9-969c-f8a7e701e7a6": {"__data__": {"id_": "23340354-3c0e-4aa9-969c-f8a7e701e7a6", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d3abb849-1685-4970-9f7d-01b806d2ec32", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "e9e94a0a0d56cfbd7ac4d7c2092c67959d36488676402fef4bc05b5383d85e17", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "707aea6a-eefa-4aeb-a565-e815d6737ee3", "node_type": "1", "metadata": {}, "hash": "003aee73418faa553b2f03a38fefb407dd059b3597bd61bebb1830a927dcfa84", "class_name": "RelatedNodeInfo"}}, "text": "53. Onofrejov\u00e1 L, Va\u0161\u00ed\u010dkov\u00e1 J, Klejdus B, Stratil P, \nMi\u0161urcov\u00e1 L, Kr\u00e1\u010dmar S, Kopeck\u00fd J, Vacek J: \nBioactive phenols in algae: The application of \npressurized-liquid and solid-phase extraction \ntechniques. J Pharm Biomed Anal 2010, \n51(2):464-470. \n\n54. Chen XJ, Guo BL, Li SP, Zhang QW, Tu PF, \nWang YT: Simultaneous determination of 15 \nflavonoids in Epimedium using pressurized \nliquid extraction and high-performance \nliquid chromatography. J Chromatogr 2007, \n1163(1-2):96-104. \n\n55. Pu\u00e9rtolas E, L\u00f3pez N, Salda\u00f1a G, \u00c1lvarez I, \nRaso J: Evaluation of phenolic extraction \nduring fermentation of red grapes treated by \n\na continuous pulsed electric fields process at \npilot-plant scale. J Food Eng 2010, 98(1):120-\n125. \n\n56. A Rostagno Mc, Ara\u00fajo JMA, Sandi D: \nSupercritical fluid extraction of isoflavones \nfrom soybean flour. Food Chem 2002, 78(1):111-\n117. \n\n57. Kitzberger CSG, Sm\u00e2nia Jr A, Pedrosa RC, \nFerreira SRS: Antioxidant and antimicrobial \nactivities of shiitake (Lentinula edodes) \nextracts obtained by organic solvents and \nsupercritical fluids. J Food Eng 2007, 80(2):631-\n638. \n\n58. Corrales M, Garc\u00c3-a AFn, Butz P, Tauscher B: \nExtraction of anthocyanins from grape skins \nassisted by high hydrostatic pressure. J Food \nEng 2009, 90(4):415-421. \n\n59. Briones-Labarca V, Plaza-Morales M, \nGiovagnoli-Vicu\u00f1a C, Jamett F: High \nhydrostatic pressure and ultrasound \nextractions of antioxidant compounds, \nsulforaphane and fatty acids from Chilean \npapaya (Vasconcellea pubescens) seeds: \n\nEffects of extraction conditions and methods. \nLWT - Food Sci Technol 2015, 60(1):525-534. \n\n60. Jun X: Caffeine extraction from green tea \nleaves assisted by high pressure processing. J \nFood Eng 2009, 94(1):105-109. \n\n61. Pinela J, Prieto MA, Barros L, Carvalho AM, \nOliveira MBPP, Saraiva JA, Ferreira ICFR: \nCold extraction of phenolic compounds from \nwatercress by high hydrostatic pressure: \n\nProcess modelling and optimization. Sep Purif \nTechnol 2018, 192:501-512. \n\n62. Ottens M, Chilamkurthi S, Rizvi S (eds.): \nAdvances in process chromatography and \napplications in the food, beverage and \nnutraceutical industries; 2010. \n\n63. Wang L, Gong L-H, Chen C-J, Han H-B, Li H-\nH: Column-chromatographic extraction and \nseparation of polyphenols, caffeine and \ntheanine from green tea. Food Chem 2012, \n131(4):1539-1545. \n\n64. Andersen \u00d8M, Fossen T, Torskangerpoll K, \nFossen A, Hauge U: Anthocyanin from \nstrawberry (Fragaria ananassa) with the novel \naglycone, 5-carboxypyranopelargonidin. \nPhytochemistry 2004, 65(4):405-410. \n\n65. Vitrac X, Castagnino C, Waffo-T\u00e9guo P, \nDelaunay J-C, Vercauteren J, Monti J-P, \nDeffieux G, M\u00e9rillon J-M: Polyphenols Newly \nExtracted in Red Wine from Southwestern \nFrance by Centrifugal Partition \nChromatography.", "start_char_idx": 60053, "end_char_idx": 62817, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "707aea6a-eefa-4aeb-a565-e815d6737ee3": {"__data__": {"id_": "707aea6a-eefa-4aeb-a565-e815d6737ee3", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "23340354-3c0e-4aa9-969c-f8a7e701e7a6", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "e86be9200d74dd0868e707e938cd0dd3924191ff254ac44991965566061035e2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ee5d5305-dc8e-4a42-aa08-4b88c97046ef", "node_type": "1", "metadata": {}, "hash": "8eaf19c30efd83177c251e4557618c1765f82776506df6ea8b35930e6e66ea4a", "class_name": "RelatedNodeInfo"}}, "text": "J Agric Food Chem 2001, \n49(12):5934-5938. \n\n66. Feng L, Zhao F: Separation of Polyphenols in \nTea on Weakly Acidic Cation-Exchange Gels. \nChromatographia 2010, 71(9-10):775-782. \n\n67. Nawaz H, Shi J, Mittal GS, Kakuda Y: \nExtraction of polyphenols from grape seeds \nand concentration by ultrafiltration. Sep Purif \nTechnol 2006, 48(2):176-181.", "start_char_idx": 62818, "end_char_idx": 63162, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ee5d5305-dc8e-4a42-aa08-4b88c97046ef": {"__data__": {"id_": "ee5d5305-dc8e-4a42-aa08-4b88c97046ef", "embedding": null, "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-203", "node_type": "4", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "4fc9f2c93f2e3f055e29c44474e0f8f28fbf8f6df18836a6e541700f1d3f2d3d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "707aea6a-eefa-4aeb-a565-e815d6737ee3", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "3f5802ee28b1881e34ec744242553fc48e142b126564cdb02e74603e46421469", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ced44410-4106-40a7-98c1-e929aba93afd", "node_type": "1", "metadata": {}, "hash": "e77861774447d1e3c482aa25e51e89886417c4fed7d54ec32b172ea63fc8b0b2", "class_name": "RelatedNodeInfo"}}, "text": "Sep Purif \nTechnol 2006, 48(2):176-181. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 74-91        Surname of autheor et al. \n\n \n \n\n \n        \u00a9NJB, Biotechnology Society of Nepal                              91                                                          Nepjol.info/index.php/njb \n\n \n\n68. D. Liu, E. Vorobiev, R. Savoire, Lanoisell\u00e9 J-L: \nExtraction of polyphenols from grape seeds \nby unconventional methods and extract \nconcentration through polymeric membrane. \nIn: 11th International Congress on Engineering \nand Food (ICEF 11); Athens, Greece.  2011. \n\n69. Russo C: A new membrane process for the \nselective fractionation and total recovery of \npolyphenols, water and organic substances \n\nfrom vegetation waters (VW). J Membr Sci \n2007, 288(1-2):239-246. \n\n70. Garcia-Castello E, Cassano A, Criscuoli A, \nConidi C, Drioli E: Recovery and \nconcentration of polyphenols from olive mill \n\nwastewaters by integrated membrane system. \nWater Res 2010, 44(13):3883-3892. \n\n71. Lawson KW, Lloyd DR: Membrane \ndistillation. J Membr Sci 1997, 124(1):1-25. \n\n72. El-Abbassi A, Hafidi A, Garc\u00eda-Payo M, \nKhayet M: Concentration of olive mill \nwastewater by membrane distillation for \n\npolyphenols recovery. Desalination 2009, \n245(1):670-674. \n\n73. Susanto H, Feng Y, Ulbricht M: Fouling \nbehavior of aqueous solutions of \npolyphenolic compounds during \nultrafiltration. J Food Eng 2009, 91(2):333-340. \n\n74. Aider M, de Halleux D, Bazinet L: Potential of \ncontinuous electrophoresis without and with \nporous membranes (CEPM) in the bio-food \nindustry: review. Trends Food Sci Technol 2008, \n19(7):351-362. \n\n75. Bazinet L, DeGrandpr\u00e9 Y, Porter A: \nElectromigration of tobacco polyphenols. Sep \nPurif Technol 2005, 41(1):101-107. \n\n76. Bazinet L, DeGrandpr\u00e9 Y, Porter A: Enhanced \ntobacco polyphenol electromigration and \n\nimpact on membrane integrity. J Membr Sci \n2005, 254(1\u20132):111-118. \n\n77. Labb\u00e9 D, Araya-Farias M, Tremblay A, Bazinet \nL: Electromigration feasibility of green tea \ncatechins. J Membr Sci 2005, 254(1\u20132):101-109. \n\n78. Bazinet L, Cossec Cl, Gaudreau Hln, \nDesjardins Y: Production of a Phenolic \nAntioxidant Enriched Cranberry Juice by \n\nElectrodialysis with Filtration Membrane. J \nAgric Food Chem 2009, 57(21):10245-10251. \n\n79. Bazinet L, Brianceau S, Dub\u00e9 P, Desjardins Y: \nEvolution of cranberry juice physico-\nchemical parameters during phenolic \nantioxidant enrichment by electrodialysis \nwith filtration membrane. Sep Purif Technol \n2012, 87(0):31-39. \n\n80. Husson E, Araya-Farias M, Desjardins Y, \nBazinet L: Selective anthocyanins enrichment \nof cranberry juice by electrodialysis with \n\nultrafiltration membranes stacked. Innov Food \nSci Emerg Technol 2013, 17(0):153-162", "start_char_idx": 63123, "end_char_idx": 65857, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ced44410-4106-40a7-98c1-e929aba93afd": {"__data__": {"id_": "ced44410-4106-40a7-98c1-e929aba93afd", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ee5d5305-dc8e-4a42-aa08-4b88c97046ef", "node_type": "1", "metadata": {"identifier": "njb-203", "author": "Suwal, Shyam; Marciniak, Alice", "title": "Technologies for the Extraction, Separation and Purification of polyphenols \u2013 A Review", "date": "2019-01-15", "file": "njb-203.pdf"}, "hash": "5f19d2fd9acc8006a5629d1cd0cfd0381dc7e3ac584588c27a5c23a42a007eb5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c1ad6bf9-f67b-461c-ba09-c845ad7cd6cd", "node_type": "1", "metadata": {}, "hash": "26ea720cb4f1e17fd0cf146050e60473df5e38acb38e9f062b6893103f5066f0", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 69-73    ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\nREVIEW ARTICLE \n\n \u00a9NJB, Biotechnology Society of Nepal                              69                                                Nepjol.info/index.php/njb \n\n \n\nNipah Virus (NiV) Infection: Is Nepal Prepared for the \nPossible Outbreak? \n\nDhiraj Shrestha1, 2*, Balkrishna Bhattachan3 \n1Department of Microbiology, Tri-Chandra Multiple College, Kathmandu, Nepal \n\n2Department of Microbiology, Shi-Gan International College of Science and Technology, Kathmandu, \n\nNepal \n3Siddhi Memorial Hospital, Bhaktapur, Nepal \n\nAbstract \nAfter 20 years of the first Nipah Virus (NiV) outbreak in the world, it re-emerged as the \noutbreak in India. WHO has recognized NiV as a potent epidemic threat to human health. \nBoth animal-to-human and human-to-human transmission of zoonotic NiV has been \ndocumented. Fruit bat of Pteropodidae family is the natural reservoir of the virus. Thus, the \nterritorial habitat of these bats is the high risk zone of NiV outbreak. The symptoms are very \nnonspecific and the pathogenicity of NiV is yet to be fully understood. Diagnosis of NiV \ninfection still relies on molecular techniques. Till date, no drugs or vaccines against NiV has \nbeen approved. Some research have presented arrays of the possible treatment and \nprevention option, but without sure shot implications. So, appropriate precautions are the \nonly currently available prevention option. Nepal is yet to experience a NiV outbreak but that \ndoes not undermine the risk posed to the general population. High risk countries including \nNepal should be well prepared to tackle the possible outbreak in future. \nKeywords: Nipah virus, NiV, outbreak, Nepal \n\n*Corresponding Author \n\nEmail: hiraj.diamond@gmail.com  \n\nIntroduction \nOutbreaks in glance \nIn the past few years, the outbreak of Ebola virus \n\nwas on high rise throughout the world. In \n\ncounter response against the virus, vaccine was \n\ndeveloped and was being tested in high risk \n\nzones of Democratic Republic of the Congo on \n\n19 May, 2018 [1]. Coincidentally on the same \n\nday, this was overshadowed by the news of the \n\noutbreak of the Nipah virus (NiV) in India. \n\nInitially three deaths were reported due to NiV \n\ninfection. Since then 15 people have been tested \n\npositive for NiV, of which 13 are already dead. \n\nOther 16 suspected cases identified through \n\ncontact tracing are under observation and at \n\nleast 753 additional people are quarantined [2]. \n\nNiV is featured in the WHO list of blueprint \n\npriority diseases 2018 with potent epidemic \n\nthreat demanding urgent research and \n\ndevelopment (R&D) action [3]. The first \n\nrecorded outbreak of NiV occurred in 1998 in \n\nMalaysia following in Singapore in 1999. The \n\noutbreak involved severe respiratory illness in \n\npigs and encephalitis in humans. Later outbreak \n\ninvolving human infections was reported from \n\nBangladesh and India in 2001 [4,5]. Till date, \n\nmore than 600 cases of NiV human infections \n\nhas been documented. The outbreaks in Indian \n\nsubcontinent have been recurrent [6]. Higher \n\nmortality of around 70% has been observed in \n\nBangladesh and India, compared to mortality in \n\nMalaysia and Singapore outbreak [5]. This is \n\nprobably due to higher engagement of the \n\nrespiratory tract in the Bangladesh and India \n\noutbreaks, differences in pathogenicity of the \n\nviral strains and lack of advanced healthcare \n\nfacilities [5]. \n\nNipah Virus (NiV) \nNiV is a member of the family Paramyxoviridae, \n\ngenus Henipavirus [4,7]. The name Nipah virus \n\noriginated from Sungai Nipah, a Malaysian \n\nvillage reporting the onset of an outbreak in \n\n1998 [7]. NiV is a zoonotic virus.", "start_char_idx": 48, "end_char_idx": 3781, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c1ad6bf9-f67b-461c-ba09-c845ad7cd6cd": {"__data__": {"id_": "c1ad6bf9-f67b-461c-ba09-c845ad7cd6cd", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ced44410-4106-40a7-98c1-e929aba93afd", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "2cfacb052d75b278566012e699522d7aabcaae33a7847f0d5518b8846c2fc7ca", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7c46a6a0-9aed-4a3b-961a-cf2de2f2b923", "node_type": "1", "metadata": {}, "hash": "8a57ba0b909d4f421b1579d7f0a4e8c86cb165b06622aeb9946a9c672436a601", "class_name": "RelatedNodeInfo"}}, "text": "NiV is a zoonotic virus. Both animal-to-\n\nhuman transmission (from infected bats/pigs) \n\nand human-to-human transmission have been \n\ndocumented. During Malaysian and \n\nSingaporean outbreaks transmission occurred \n\nfrom infected pigs, an intermediate host. \n\nHowever during Bangladeshi and Indian \n\noutbreak, transmission occurred through \n\nconsumption of fruits/sap contaminated with \n\ninfectious bat secretions and also through \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 69-73 Shrestha and Bhattachan et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                              70                                                Nepjol.info/index.php/njb \n\n \n\nhuman-to-human transmission. During this \n\noutbreak, no any intermediate host was reported \n\nowing to the lack of pig farms in the region [4,7].  \n\n \nFigure 1. Adapted from \u201cNipah Virus Distribution \nMap,\u201d by Centers for Disease Control and Prevention \n(CDC) 2018, available at: https://www.cdc.gov \n/vhf/nipah/outbreaks/distribution-map.html. \nCopyright 2018 by the CDC. Adapted with \npermission. \n\nNatural host \nThe natural hosts of NiV are fruit bats belonging \n\nto the Pteropodidae family, Pteropus spp. in \n\nparticular [4,8]. Geographical distribution of \n\nNiV and Pteropus overlaps, ranging from \n\nMadagascar to Australia; covering South Asia, \n\nSouth-East Asia and Oceania region (Figure 1). \n\nBesides, African fruit bats of Pteropodidae \n\nfamily were also tested positive for NiV \n\nantibodies indicating further geographical \n\ndistribution of NiV to African territory. NiV has \n\nalso been tested positive in other animals \n\nincluding pigs, horses, goats, sheep, cats and \n\ndogs but only during the outbreaks [4]. \n\nDistribution map of these bats published by \n\nWHO includes Nepal however exact \n\ndistribution and population of these bats are still \n\nto be studied and reported from Nepal. \n\nPathogenesis \nNiV contains a negative-strand RNA. The non-\n\nsegmented RNA contain six genes which \n\nencodes structural proteins of virus namely, \n\nfusion protein (F), glycoprotein (G), polymerase \n\n(L), matrix protein (M), nucleocapsid (N) and \n\nphosphoprotein (P). The P gene encodes \n\nadditional three accessory proteins namely, C, V \n\nand W proteins [9,10]. \n\nV protein was the key determinants for \n\npathogenesis in hamster and ferret infection \n\nmodel [11-13]. V proteins target multiple host \n\nproteins and thus suppress the host antiviral \n\nresponse. In addition, V proteins interact and \n\nsuppress different signaling pathways. V protein \n\nsuppress the dephosphorylation of melanoma \n\ndifferentiation-associated protein 5 (MDA5) thus \n\ninhibiting the activation of the interferon (IFN) \u03b2 \n\npromoter [14-16]. V proteins also suppress the \n\nretinoic acid-inducible gene-I (RIG-I) dependent \n\ninduction of IFN [17]. V proteins also block \n\nsignaling through Toll-like receptors 7/9 (TLRs \n\n7/9) and suppress IFN induction [18,19]. V \n\nproteins interact with IFN-responsive signaling \n\npathway preventing activation and nuclear \n\naccumulation [20,21]. Similarly, C proteins \n\nregulate early host proinflammatory response \n\nthereby contributing virulence [22]. C protein \n\nwas responsible for respiratory diseases in a \n\nferret infection model [11,12]. The exact \n\nmolecular mechanism of the pathogenicity of \n\nNiV is still to be revealed. \n\nSigns and symptoms \nThe incubation time of NiV ranges between 4-14 \n\ndays, and upto 45 days in some cases. Infections \n\nin human ranges from asymptomatic infection to \n\nacute respiratory infection, and fatal encephalitis \n\nin severe cases. Initial symptoms include \n\nheadaches, influenza-like fever, myalgia (muscle \n\npain), sore throat and vomiting. These \n\nsymptoms are followed by acute encephalitis. \n\nFinally in severe cases, encephalitis and seizures \n\noccurs, leading to coma within 24-48 hrs.", "start_char_idx": 3757, "end_char_idx": 7592, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7c46a6a0-9aed-4a3b-961a-cf2de2f2b923": {"__data__": {"id_": "7c46a6a0-9aed-4a3b-961a-cf2de2f2b923", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c1ad6bf9-f67b-461c-ba09-c845ad7cd6cd", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "09a1e96e4649fb97ee5d538a9df8ee4dbde2cd5e0a7c0e33253aa11d896bce6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1681e353-352b-4896-a4b4-079453860869", "node_type": "1", "metadata": {}, "hash": "63057f1a986057d156fcfab8553b21a3288cbd8adfd31ae056890819cdd6486f", "class_name": "RelatedNodeInfo"}}, "text": "Fatality \n\nvaries from 40% to 75%. Surviving cases are \n\nreported to demonstrate long-term sequel \n\nincluding persistent convulsions, personality \n\nchange, seizure disorder, relapse and delayed \n\nonset encephalitis [4,7]. \n\nDiagnosis \nInitial signs and symptoms of NiV infection are \n\nnonspecific, thus accurate diagnosis is \n\nchallenging especially during an outbreaks. IgM \n\nElisa for NiV, Real-time polymerase chain \n\nreaction (RT-PCR) and viral isolation are the \n\ntests employed for diagnosis [4]. These tests \n\ndemands higher technical expertise and \n\nresources challenging the effectiveness of \n\nimmediate counter measures during an \n\noutbreak. \n\nTreatment \nCurrently, no drugs or vaccines are approved \n\nfor NiV infection. Supportive care with intensive \n\ncare is the only recommended treatment [4,7]. \n\nhttps://www.cdc.gov/\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 69-73 Shrestha and Bhattachan et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                              71                                                Nepjol.info/index.php/njb \n\n \n\nFavipiravir (T-705) has been demonstrated to \n\ninhibit NiV replication and transcription in \n\nSyrian hamster model, with twice daily oral \n\nadministration for 14 days [23]. Ribavirin, which \n\nhas broad spectrum anti-DNA and anti-RNA \n\nvirus activity and can transverse the blood-brain \n\nbarrier, was also reported to reduce mortality in \n\nan open-label trial [24]. \n\nPrevention \nVaccines have not been developed against NiV. \n\nHowever, routine and thorough disinfection of \n\npig farms can prevent the infection. Also, \n\navoiding the consumption of the bat eaten fruits \n\nor saliva/urine contaminated fruits/sap can \n\nprevent the infection. Thus, public awareness of \n\nthe associated risk factors is the only measure to \n\nreduce risk of NiV infection. During outbreaks, \n\nsuspected animal premises should be \n\nquarantined and infected animals should be \n\nculled. Also, healthcare workers caring \n\nsuspected or confirmed NiV infected patients \n\nshould adhere to standard infection control \n\nprecautions [4]. Personal protection, such as \n\nmasks, goggles, gloves, gowns, and boots, is \n\nadvocated for field and farm workers of NiV \n\nrisk zones. Personal protection should be \n\naccompanied by hand-washing and disinfection \n\nof equipments for better prevention [5]. \n\nFuture prospects \nRespiratory route administration of lipopeptides \n\nprevented NiV infection in both hamsters and \n\nnon-human primates. Also, retention of peptides \n\nin respiratory tract avoided systemic delivery of \n\nNiV thus increasing safety and enhancing \n\ninterventions [25]. Vaccines using a vesicular \n\nstomatitis virus vector have demonstrated \n\nprotection against NiV in hamsters, ferrets and \n\nAfrican green monkeys [26]. Recently in \n\nAustralia, subunit vaccines using Hendra G \n\nprotein have demonstrated protection against \n\nNiV in horses by producing cross-protective \n\nantibodies. This vaccine has potential for NiV \n\nprotection in humans as well [7]. We could be on \n\nverge of testing vaccine against NiV soon. \n\nNepalese prospects \nTill date no outbreak of NiV or NiV confirmed \n\nmortality has been documented within the \n\nterritory of Nepal. No research has been done on \n\nsurveilling antibodies against NiV in Nepalese \n\nhuman and/or bat population. However, Nepal \n\nharbors well distribution of Pteropus bat. Pig \n\nfarms are widespread throughout the country. \n\nNepal also has significant pork eating ethnic \n\ncommunities. This clearly outlines the possibility \n\nof surveilling NiV in bats and/or pigs of Nepal. \n\nFurthermore, Nepal and India share open \n\nborder without medical surveillance and \n\npopulation mobility is higher in the border \n\nregion. This further adds possible risk of viral \n\ntransmission in Nepal. Thus, Nepal is at high \n\nrisk of potential NiV outbreak. \n\nConclusion \nTwenty years ago, NiV unveiled itself to the \n\nworld causing significant morbidity and \n\nmortality. Initially NiV shattered the pig-\n\nfarming industry in Malaysia, and is continually \n\ncausing outbreaks in Indian subcontinent.", "start_char_idx": 7593, "end_char_idx": 11685, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1681e353-352b-4896-a4b4-079453860869": {"__data__": {"id_": "1681e353-352b-4896-a4b4-079453860869", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7c46a6a0-9aed-4a3b-961a-cf2de2f2b923", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "f58fd51c77838c9caf4ddd488579117c4b3adab6e1e060819598d8080ede126b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "639c12fe-0a8b-4e5c-ad6b-be68e26a4acd", "node_type": "1", "metadata": {}, "hash": "ca092f50edc3eee86ce2062c22671e89f6691503baeae50ba3af3e8dad7c5e6d", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nnatural reservoir Pteropus bat is widespread, \n\nthus outbreaks can occur in any risk zones. The \n\nrecent outbreak in India has again increased the \n\nconcern of NiV as potential threat to human \n\nhealth. Governments and universities should \n\nfund research to develop vaccine or drug against \n\nNiV to neutralize the potential threat. All high \n\nrisk countries including Nepal, should be well \n\nprepared to tackle the possible future \n\ncatastrophe. \n\nConflict of Interest \nThe authors declare no any conflict of interest. \n\nFunding \nNone \n\nAuthors Contribution \nBoth authors DS and BB contributed to the work. \n\nReferences \n1 World Health Organization (WHO): Ebola \n\nvirus disease-Democratic Republic of the \n\nCongo: Update on Ring vaccination. 2018, \navailable at: \nhttp://www.who.int/csr/don/21-may-2018-\nebola-drc/en/ [accessed 23/05/2018]. \n\n2  World Health Organization (WHO): Nipah \nvirus - India. 2018, available at: \nhttp://www.who.int/csr/don/31-may-2018-\nnipah-virus-india/en/ [accessed15/06/2018]. \n\n3 World Health Organization (WHO): R&D \nBlueprint. List of Blueprint priority diseases. \n2018, available at: \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 69-73 Shrestha and Bhattachan et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                              72                                                Nepjol.info/index.php/njb \n\n \n\nhttp://www.who.int/blueprint/priority-\ndiseases/en/ [accessed 23/05/2018]. \n\n4 World Health Organization (WHO): Nipah \nvirus. 2018, available at: \nhttp://www.who.int/news-room/fact-\nsheets/detail/nipah-virus [accessed \n23/05/2018]. \n\n5 Ang BSP, Lim TCC, Wang L: Nipah Virus \nInfection. J Clin Microbiol 2018, 56(6):e01875-\n17. DOI: 10.1128/JCM.01875-17. \n\n6 World Health Organization (WHO): R&D \nBlueprint. Nipah R&D. 2018, available at: \nhttp://www.who.int/blueprint/priority-\ndiseases/key-action/nipah/en/ [accessed \n23/05/2018]. \n\n7 Centers for Disease Control and Prevention \n(CDC): Nipah Virus (NiV). 2018, available at: \nhttps://www.cdc.gov/vhf/nipah/index.html \n[accessed 23/05/2018]. \n\n8 Yob JM, Field H, Rashdi AM, Morrissy C, van \nder Heide B, Rota P, bin Adzhar A, White J, \nDaniels P, Jamaluddin A, Ksiazek T: Nipah \nvirus infection in bats (order Chiroptera) in \npeninsular Malaysia. Emerg Infect Dis 2001, \n7(3):439-441.   DOI: 10.3201/eid0703.017312. \nPMID: 11384522. \n\n9 Eaton BT, Broder CC, Middleton D, Wang LF: \nHendra and Nipah viruses: different and \ndangerous. Nat Rev Microbiol 2006, 4:23\u201335. \nDOI: 10.1038/nrmicro1323. PMID: 16357858. \n\n10    Harcourt BH, Tamin A, Ksiazek TG, Rollin \nPE, Anderson LJ, Bellini WJ, Rota PA: \nMolecular characterization of Nipah virus, a \nnewly emergent paramyxovirus. Virology \n2000, 271(2):334\u2013349. DOI: \n10.1006/viro.2000.0340. PMID: 10860887. \n\n11 Satterfield BA, Cross RW, Fenton KA, Agans \nKN, Basler CF, Geisbert TW, Mire CE: The \nimmunomodulating V and W proteins of \nNipah virus determine disease course.", "start_char_idx": 11686, "end_char_idx": 14639, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "639c12fe-0a8b-4e5c-ad6b-be68e26a4acd": {"__data__": {"id_": "639c12fe-0a8b-4e5c-ad6b-be68e26a4acd", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1681e353-352b-4896-a4b4-079453860869", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "15aca62a38542e599c212c837adb748bbfd9a6b39245e47587e78961992d0a5e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e4e19e9a-2d2a-48bc-acc5-5e73a051e460", "node_type": "1", "metadata": {}, "hash": "c0f0b624868959753ac367bb4a16c45f42cdc2c60d21a02f1cd295ac04e54c67", "class_name": "RelatedNodeInfo"}}, "text": "Nat \nCommun 2015, 6:7483. DOI: \n10.1038/ncomms8483. PMID: 26105519. \n\n12 Satterfield BA, Cross RW, Fenton KA, \nBorisevich V, Agans KN, Deer DJ, Graber J, \nBasler CF, Geisbert TW, Mire CE: Nipah \nVirus C and W Proteins Contribute to \nRespiratory Disease in Ferrets. J Virol 2016, \n90(14):6326\u20136343. DOI: 10.1128/JVI.00215-16. \nPMID: 27147733. \n\n13 Uchida S, Horie R, Sato H, Kai C, Yoneda M: \nPossible role of the Nipah virus V protein in \nthe regulation of the interferon beta \ninduction by interacting with UBX domain-\ncontaining protein. Sci Rep 2018, 8(1):7682. \nDOI: 10.1038/s41598-018-25815-9. PMID: \n29769705. \n\n14  Davis ME, Wang MK, Rennick LJ, Full F, \nGableske S, Mesman AW, Gringhuis SI, \nGeijtenbeek TB, Duprex WP, Gack MU: \nAntagonism of the phosphatase PP1 by the \nmeasles virus V protein is required for \ninnate immune escape of MDA5. Cell Host \n\nMicrobe 2014, 16(1):19\u201330. DOI: \n10.1016/j.chom.2014.06.007. PMID: 25011105. \n\n15 Andrejeva J, Childs KS, Young DF, Carlos TS, \nStock N, Goodbourn S, Randall RE: The V \nproteins of paramyxoviruses bind the IFN-\ninducible RNA helicase, mda-5, and inhibit \nits activation of the IFN-beta promoter. Proc \nNatl Acad Sci USA 2004, 101(49):17264\u201317269. \nDOI: 10.1073/pnas.0407639101. PMID: \n15563593. \n\n16 Childs K, Stock N, Ross C, Andrejeva J, Hilton \nL, Skinner M, Randall R, Goodbourn S: mda-\n5, but not RIG-I, is a common target for \n\nparamyxovirus V proteins. Virology 2007, \n359(1):190\u2013200. DOI: \n10.1016/j.virol.2006.09.023. PMID: 17049367. \n\n17   Childs K, Randall R, Goodbourn S: \nParamyxovirus V proteins interact with the \nRNA Helicase LGP2 to inhibit RIG-I-\n\ndependent interferon induction. J Virol 2012, \n86(7):3411\u20133421. DOI: 10.1128/JVI.06405-11. \nPMID: 22301134. \n\n18 Kitagawa Y, Yamaguchi M, Zhou M, Komatsu \nT, Nishio M, Sugiyama T, Takeuchi K, Itoh M, \nGotoh B: A tryptophan-rich motif in the \nhuman parainfluenza virus type 2 V protein \nis critical for the blockade of toll-like \nreceptor 7 (TLR7)- and TLR9-dependent \nsignaling. J Virol 2011, 85(9):4606\u20134611. DOI: \n10.1128/JVI.02012-10. PMID: 21345944. \n\n19 Shaw ML, Cardenas WB, Zamarin D, Palese P, \nBasler CF: Nuclear localization of the Nipah \nvirus W protein allows for inhibition of both \nvirus- and toll-like receptor 3-triggered \n\nsignaling pathways. J Virol 2005, 79(10):6078\u2013\n6088. DOI: 10.1128/JVI.79.10.6078-6088.2005. \nPMID: 15857993.", "start_char_idx": 14640, "end_char_idx": 17016, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e4e19e9a-2d2a-48bc-acc5-5e73a051e460": {"__data__": {"id_": "e4e19e9a-2d2a-48bc-acc5-5e73a051e460", "embedding": null, "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-204", "node_type": "4", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "cd5a02f07edac0607ace7455896300912521789bfd510a07491db0d8ac5f753b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "639c12fe-0a8b-4e5c-ad6b-be68e26a4acd", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "61e639db447ab3f4a6181b229ebd25fa23ddf44695e4acac9be6afe1444343b7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cdf4d2ae-2ffb-4359-a681-1f6291d415cd", "node_type": "1", "metadata": {}, "hash": "6c78227ad015cf8d2880c8e6223953ea884a2900b1651975d13cac0d22ffcd02", "class_name": "RelatedNodeInfo"}}, "text": "PMID: 15857993. \n\n20 Rodriguez JJ, Parisien JP, Horvath CM: Nipah \nvirus V protein evades alpha and gamma \ninterferons by preventing STAT1 and \nSTAT2 activation and nuclear accumulation. \nJ Virol 2002, 76(22):11476\u201311483. DOI: \n10.1128/JVI.76.22.11476-11483.2002. PMID: \n12388709. \n\n21  Rodriguez JJ, Wang LF, Horvath CM: Hendra \nvirus V protein inhibits interferon signaling \nby preventing STAT1 and STAT2 nuclear \n\naccumulation. J Virol 2003, 77(21):11842\u2013\n11845. DOI: 10.1128/JVI.77.21.11842-\n11845.2003. PMID: 14557668. \n\n22 Mathieu C, Guillaume V, Volchkova VA, Pohl \nC, Jacquot F, Looi RY, Wong KT, Legras-\nLachuer C, Volchkov VE, Lachuer J, Horvat B: \nNonstructural Nipah virus C protein \nregulates both the early host \nproinflammatory response and viral \nvirulence. J Virol 2012, 86(19):10766\u201310775. \nDOI: 10.1128/JVI.01203-12. PMID: 22837207. \n\n23 Dawes BE, Kalveram B, Ikegami T, Juelich T, \nSmith JK, Zhang L, Park A, Lee B, Komeno T, \nFuruta Y, Freiberg AN: Favipiravir (T-705) \nprotects against Nipah virus infection in the \nhamster model. Sci Rep 2018, 8(1):7604. DOI: \n10.1038/s41598-018-25780-3. PMID: 29765101. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 69-73 Shrestha and Bhattachan et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                              73                                                Nepjol.info/index.php/njb \n\n \n\n24 Chong HT, Kamarulzaman A, Tan CT, Goh KJ, \nThayaparan T, Kunjapan SR, Chew NK, Chua \nKB, Lam SK: Treatment of acute Nipah \nencephalitis with ribavirin. Ann Neurol 2001, \n49:810-813. DOI: 10.1002/ana.1062PMID: \n11409437. \n\n25 Mathieu C, Porotto M, Figueira T, Horvat B, \nMoscona A: Fusion Inhibitory Lipopeptides \nEngineered for Prophylaxis of Nipah Virus \nin Primates. J Infect Dis 2018,   218(2):218-227. \nDOI: 10.1093/infdis/jiy152. PMID: 29566184. \n\n26 Satterfield BA, Dawes BE, Milligan GN: \nStatus of vaccine research and development \nof vaccines for Nipah virus. Vaccine 2016, \n34(26):2971-2975. DOI: \n10.1016/j.vaccine.2015.12.075 PMID: 26973068.", "start_char_idx": 17001, "end_char_idx": 19047, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cdf4d2ae-2ffb-4359-a681-1f6291d415cd": {"__data__": {"id_": "cdf4d2ae-2ffb-4359-a681-1f6291d415cd", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e4e19e9a-2d2a-48bc-acc5-5e73a051e460", "node_type": "1", "metadata": {"identifier": "njb-204", "author": "Shrestha, Dhiraj; Bhattachan, Balkrishna", "title": "Nipah Virus (NiV) Infection: Is Nepal Prepared for the Possible Outbreak?", "date": "2019-01-15", "file": "njb-204.pdf"}, "hash": "36dab0e3c363e622095814210bdfe00e203c0663491905c5b49ad646236e1d95", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f69f123c-e9b9-41a8-bcbd-1b5c4dd5423e", "node_type": "1", "metadata": {}, "hash": "70599e3fec014c03ae16de3038d1bfad7769c62cd45467f172e05e32d0766a9d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: -62-68 ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\n ORIGINAL RESEARCH ARTICLE \n \n\n\u00a9NJB, Biotechnology Society of Nepal   62                                           Nepjol.info/index.php/njb \n\n \n\nIsolation of Polyhydroxybutyrate (PHB) Producing Bacteria, \nOptimization of Culture Conditions for PHB production, \n\nExtraction and Characterization of PHB \nChristina Thapa\u00b9, Pallavi Shakya\u00b9, Rabina Shrestha\u00b9, Sushovita Pal\u00b9, Prakash Manandhar\u00b2 \n\nDepartment of Biotechnology, SANN International College, Purbanchal University, Nepal1 \n\nDepartment of Microbiology, St. Xavier\u2019s College, Nepal2 \n\nAbstract \nPolyhydroxybutyrates (PHBs) are energy reserves synthesized by different micro-organisms such \nas Alcaligenes, Pseudomonas, Staphylococcus, Algae, in excess of carbon and limitation of nutrients \nlike nitrogen. These biopolymers are suitable alternate to synthetic carbon-based polymers. \nHowever, the high production cost limits their commercialization. The aim of this study was thus, \nfocused on optimization of culture condition for maximum PHB production in an attempt to reduce \nthe production cost. The micro-organisms for this purpose were isolated from 4 different soil \nsamples and screened for PHB production. Culture conditions for these organisms were optimized \nby changing the parameters, viz., incubation time, pH, carbon source and NaCl concentration. \nThus, optimized culture condition was used to culture the isolates for extraction of PHB and its \nanalysis. The extracted compounds on FTIR-analysis gave characteristic C=O peak of PHB, thus, \nconfirming the seven isolates to be PHB producers. Results for optimized parameters for the \nisolated PHB positive species showed that synthesis of PHB was maximum at 48 hours i.e. during \nthe early stages of stationary phase. However, different isolates favored different culture \nconditions. Highest PHB accumulation and growth of isolates were seen at pH 7 and 9. Similarly, it \nwas observed that glucose was favored by 4 isolates and sucrose was favored by 3 isolates. \nInterestingly, NaCl concentration did not cause significant effect on neither the bacterial growth \nnor the PHB production. During the extraction of PHB from the optimized culture conditions, \nextraction of PHB from broth gave significant yield than that from agar. A good PHB yield from \nbroth amounting to 36.41% and 34.59% was observed for Bacillus pasteurii and Micrococcus luteus \nrespectively, showing a potential for their exploitation in industrial PHB production. At optimized \nconditions, 7 isolates exhibited significant PHB yields, thus showing a potential for further \nexploitation. \nKEYWORDS: Bioplastics, Biopolymer, Polyhydroxybutyrates, PHB, Fourier Transform Infra-Red \nSpectroscopy (FTIR) \n*Corresponding Author \nEmail: rabeenastha@gmail.com \n\nIntroduction \nTraditional plastics are synthetic carbon-based \n\npolymers that are made from non-renewable \n\nsource, mostly from petroleum. Due to their \n\nrelatively low cost, ease of manufacture and \n\nflexibility, the demand of plastics is ever-\n\ngrowing. However, plastics, being man-made, \n\nare not recognized by micro-organisms. [13] Thus, \n\nthey take very long to degrade i.e. 450 years on \n\naverage for degradation of a plastic bottle. [7] \n\nPlastic debris also poses considerable threat by \n\nchoking and starving wildlife, distributing non-\n\nnative and potentially harmful organisms, \n\nabsorbing toxic chemicals and degrading to \n\nmicro-plastics that may subsequently be \n\ningested. [2] Also, due to high cost of recycling, \n\nplastics are rarely recycled leading to crammed \n\nup landfills.", "start_char_idx": 48, "end_char_idx": 3715, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f69f123c-e9b9-41a8-bcbd-1b5c4dd5423e": {"__data__": {"id_": "f69f123c-e9b9-41a8-bcbd-1b5c4dd5423e", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cdf4d2ae-2ffb-4359-a681-1f6291d415cd", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "a0561c7270ffc639142b9d0ba9018447583de1021ef128a325ab54eabf21f671", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63963742-c1b6-4aae-a34b-6402cb1a0a17", "node_type": "1", "metadata": {}, "hash": "710b45f34852c35abdaa11272d3eb2556c5f55eb88aba8f6309878e523d20788", "class_name": "RelatedNodeInfo"}}, "text": "For eradication of these and various \n\nother problems such as carbon emission during \n\nincineration, biodegradation of plastic is a must. \n\nConsiderable amount of interest in the \n\ndevelopment and production of an alternative, \n\nbiodegradable plastics or bioplastics is being \n\ndone. Among them polyhydroxy alkanoic acids \n\n(PHAs) are drawing much attention as they \n\nhave nontoxic residual products and low \n\nenvironmental permanence. [9] Depending on the \n\ntypes of carbon sources available and the \n\nbiochemical pathways operating in the cell, \n\nmicroorganisms are capable of synthesizing \n\nvarious types of PHAs. Poly [R- 3- \n\nhydroxybutyrate] (P[3HB]) is the first type of \n\nPHA identified and is the most common PHA \n\nfound in nature. [1] \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 62-28  Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   63                                           Nepjol.info/index.php/njb \n\n \n\nPHBs are carbon and energy reserve polymers \n\nproduced in bacteria, archaea, and in few \n\neukaryotes, such as yeasts and fungi when \n\ncarbon source is in plentiful and other nutrients \n\nsuch as nitrogen, phosphorous, oxygen or \n\nsulphur are limited. The storage molecule is \n\nthen metabolized under unfavorable conditions \n\nwhen other common energy sources are not \n\navailable. Some bacterial species which \n\nnaturally produce PHB are Ralstonia eutrophes, \n\nAlcaligenes, Pseudomonas, Bacillus, Rhodococcus, \n\nStaphylococcus and Micrococcus. [1] \n\nPHB is ecofriendly, biodegradable, \n\nbiocompatible and is accumulated up to 90% of \n\ncell dry weight. [10] PHB based plastics made by \n\ncombining PHB with other biocompatible \n\npolymers (like 3-hydroxyvalerate) [8] find many \n\napplications in agriculture, packaging, and \n\nmedical field including drug delivery and tissue \n\nengineering. [10] In spite of these interesting \n\nproperties, industrial production of PHB is still \n\nnot well established due to its high production \n\ncost. This has made it unable to compete with \n\nconventional plastics in the commercial market.  \n\nThe PHB content and its composition are \n\ninfluenced mainly by the strain of the \n\nmicroorganism, the type of substrate employed \n\nand its concentration, and other growth \n\nconditions such as pH, time and temperature. [11] \n\nTherefore, much research is needed to discover \n\nand identify novel species with vastly superior \n\nproduction capacity and optimization of \n\nconditions for maximal synthesis of PHB.  \n\nThis research focuses on isolation and \n\ncharacterization of PHB producing bacteria from \n\neasy and convenient sources i.e. sewage soil \n\nsample. The objective was to analyze the \n\nextracted PHB by different isolated organisms in \n\noptimized physical and chemical conditions. \n\nBiochemical and morphological tests are \n\nperformed for the identification purpose. In an \n\nattempt to overcome the limitations associated \n\nwith costly substrates, this research is designed \n\nto use and test the efficiency of readily available \n\nand relatively cheap carbon sources such as \n\nsucrose, glucose and fructose in PHB \n\nproduction. Moreover, it also focuses on \n\noptimization of various growth conditions such \n\nas incubation time, pH and NaCl concentration \n\nfor increase in the polymer production from \n\nrespective PHB positive organism. Considering \n\nthe optimum growth conditions and carbon \n\nsource of each organism, PHB extraction and its \n\ncharacterization using FTIR analysis was done. \n\nThe extracted PHB was calculated as percentage \n\nyield of the cell dry weight obtained. \n\nMaterials and Methods \nSample collection and isolation of pure \ncultures \nSoil samples were collected aseptically from \n\ntopsoil of four sites viz., Teku Dumping Site, \n\nBalaju Industrial Site, Banks of Dhobi Khola and \n\nBudhanilkantha Animal Waste Manure.  \n\nOne gram of each sample was dispersed in 10ml \n\nof sterile distilled water and heated at 80\u00b0C for \n\n10 minutes to isolate only endospore forming \n\nbacteria.", "start_char_idx": 3716, "end_char_idx": 7696, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63963742-c1b6-4aae-a34b-6402cb1a0a17": {"__data__": {"id_": "63963742-c1b6-4aae-a34b-6402cb1a0a17", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f69f123c-e9b9-41a8-bcbd-1b5c4dd5423e", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "5780892e763686cd48a2eeb24bb1f512e691343b067b1477412fc65bef3ab4a6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9f54d1b0-9fc0-4cb6-91bf-7b1165b53f3c", "node_type": "1", "metadata": {}, "hash": "9b329b2b5faa6a0c1599568a8a0c68c58d2fc09015fe16716ea5d3b102a4b175", "class_name": "RelatedNodeInfo"}}, "text": "Serial dilution of these samples was \n\ndone up to 10-3, followed by spread plating of \n\n100\u00b5l diluted samples on nutrient agar plates. \n\nThereafter, the plates were incubated at 30\u00b0C for \n\n48 hours. Pure culture of morphologically \n\ndistinct colonies was grown in modified agar \n\nplates. The constituents of Modified agar plates \n\nare: Beef extract (0.3%), Peptone (0.5%), Sodium \n\nChloride (0.8%), Glucose (1%), and Agar (1.5%). \n[12] \n\nPrimary screening of PHB producing \nbacteria \nDetection for PHB production was employed by \n\nusing lipophilic stain Sudan Black B. [3] Stain was \n\nprepared by dissolution of 0.3 gm powdered \n\nstain in 100 ml of 70% ethanol. For microscopic \n\nstudies, smears of colonies were heat-fixed on \n\nclean, grease-free glass slides, followed by \n\nstaining with 0.3% solution of the Sudan Black \n\nB. After leaving the slides undisturbed for 15 \n\nminutes, immersion in xylene and \n\ncounterstaining with safranin (5% w/v in sterile \n\ndistilled water) was performed. Cells appearing \n\nblue-black under microscope were accredited as \n\nPHB positive strains. \n\nPHB positive strains were preserved on two \n\nvials, viz., working and stock vials, containing \n\nagar slants with 2% glycerol for preservation.  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 62-28  Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   64                                           Nepjol.info/index.php/njb \n\n \n\nMorphological and Biochemical \nCharacterization of PHB positive \nIsolates \nDistinct morphological features of the isolates \n\nwere recorded on the basis of shape, color and \n\nsize. Similarly, cellular morphology was studied \n\nunder the microscope using Gram Staining and \n\nEndospore Staining. Standard microbiological \n\nmethods were employed for identification of \n\nisolated bacteria by biochemical tests. The tests \n\nperformed were IMViC test, nitrate test, sugar \n\nutilization test, catalase test, oxidase test, starch \n\nutilization test and oxidative-fermentative test.  \n\nGrowth Curve Study of Isolates \nPHB producing medium was used to study the \n\ngrowth and production of PHB. The \n\ncomponents of the media are: Glucose - 1g, \n\nPeptone - \n\n0.25g, Yeast extract \u2013 0.25g, NaCl \u2013 0.01g, \n\nKH2PO4 \u2013 0.05g, MgSO4 \u2013 0.02g and pH at 7. [6] \n\nOne percent inoculums from activated PHB \n\npositive isolates were inoculated in conical \n\nflasks containing PHB producing media, \n\nfollowed by incubation of the culture for 48 \n\nhours at 37\u00b0C with occasional shaking. At an \n\ninterval of every 4 hours, the samples were \n\ncollected to perform Sudan staining and the \n\nbiomass reading was done using \n\nspectrophotometer at 640nm. \n\nOptimization of PHB production \nEffect of pH \n\nEvery microorganism has a minimum, an \n\noptimum and a maximum pH for growth. To \n\nstandardize the optimum pH for the production \n\nof PHB, the PHB positive bacterial cultures were \n\ninoculated in PHB producing media at different \n\npH (2, 4, 7, 9 and 11) and incubated at 37\u00b0C for \n\n48 hours with occasional shaking. The pH \n\nvalues were taken in order to cover different \n\nacidic, neutral and basic pH ranges. After \n\nincubation, the samples were screened using \n\nSudan stain to confirm PHB production and \n\nturbidity of the media due to bacterial growth \n\nwas measured by spectrophotometer at 640nm.  \n\nEffect of NaCl Concentration \nMicroorganisms vary widely in their NaCl \n\ntolerance.", "start_char_idx": 7697, "end_char_idx": 11088, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9f54d1b0-9fc0-4cb6-91bf-7b1165b53f3c": {"__data__": {"id_": "9f54d1b0-9fc0-4cb6-91bf-7b1165b53f3c", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63963742-c1b6-4aae-a34b-6402cb1a0a17", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "68ac9d164c412df3ffae365577f27faeae20a3601b36ade43873d307d2deeecd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5f42ae23-b16b-4912-a436-c472104912c6", "node_type": "1", "metadata": {}, "hash": "8e858e10e2f0fd0156bc5aaca59c513f5719581e3a0a9f0208189e945753e860", "class_name": "RelatedNodeInfo"}}, "text": "Thus, PHB producing media with \n\ndifferent NaCl concentrations (0.1%, 0.5%, 2%, \n\n5%, and 10%) was prepared. After autoclaving, \n\n1% of activated culture was added to each tube \n\nand incubated at 37\u00b0C for 48 hours. The samples \n\nwere collected after 48 hours for Sudan Staining \n\nand for measurement of O.D. at 640nm. \n\nEffect of Carbon Sources \n 2% glucose, sucrose, and fructose were added \n\ninto PHB producing media as carbon sources \n\nand the selected isolates were grown in it. After \n\nincubation and screening by Sudan stain, the \n\nPHB produced by the isolates was quantified \n\nspectrophotometrically for the selection of \n\ncarbon source that showed highest PHB \n\nproduction. \n\nExtraction of PHB \nThe optimized pH and carbon source for each \n\nbacterium were used for the extraction of PHB \n\nby solvent extraction method [4] with slight \n\nmodifications. Firstly, 1% of PHB positive strain \n\nwas inoculated in PHB producing media of \n\noptimized pH and carbon source and it was \n\nincubated at 37\u00b0C. After each 4-hour, 1 ml of \n\nmedia was centrifuged at 11,800 rpm for 20 \n\nminutes and Sudan staining was done to \n\nconfirm PHB production. When the PHB \n\nproduction was confirmed, which mostly \n\nensued after 48 hours, 50 ml of bacterial cell \n\nculture growth was taken and pelleted at 5000 \n\nrpm for 25 minutes. The dry weight of the pellet \n\nwas taken and then it was washed with acetone \n\nand ethanol successively. For the recovery of \n\nPHB, equal volume of 6% sodium hypochlorite \n\nwas used to re-suspend the pellet and it was \n\nincubated at 37\u00b0C for 10 minutes. This was \n\nfollowed by centrifugation at 5000 rpm for 30 \n\nminutes to sediment the lipid granules. The \n\npellet obtained was washed with acetone and \n\nethanol followed by hot chloroform treatment. \n\nAfter the pellet dissolved in chloroform, \n\nWhatman filter paper was used to filter out the \n\ncell residues so that only PHB is present in the \n\nchloroform solution. Finally, the filtrate was \n\nevaporated in hot air oven at 40\u00b0C and dry \n\nweight of extracted PHB was measured. The \n\npercentage of PHB accumulation was calculated \n\nusing the formulae: \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 62-28  Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   65                                           Nepjol.info/index.php/njb \n\n \n\n\ud835\udc37\ud835\udc5f\ud835\udc66 \ud835\udc64\ud835\udc52\ud835\udc56\ud835\udc54\u210e\ud835\udc61 \ud835\udc5c\ud835\udc53 \ud835\udc52\ud835\udc65\ud835\udc61\ud835\udc5f\ud835\udc4e\ud835\udc50\ud835\udc61\ud835\udc52\ud835\udc51 \ud835\udc43\ud835\udc3b\ud835\udc35 (\n\ud835\udc54\n\n\ud835\udc5a\ud835\udc59\n) \u00d7 100\n\n\ud835\udc37\ud835\udc5f\ud835\udc66 \ud835\udc64\ud835\udc52\ud835\udc56\ud835\udc54\u210e\ud835\udc61 \ud835\udc5c\ud835\udc53 \ud835\udc4f\ud835\udc56\ud835\udc5c\ud835\udc5a\ud835\udc4e\ud835\udc60\ud835\udc60\n \n\nThis procedure was repeated for all the PHB \n\npositive strains and PHB was extracted from the  \n\nstrains inoculated in broth media as well as in \n\nagar media. \n\nCharacterization of PHB \nThe presence of prominent functional groups \n\nsuch as CH3, CH2, C=O, C-O, CH, and OH is a \n\ncritical decider of presence of PHB. ATR-FTIR \n\nspectroscopy (Spectrum65 FTIR spectrometer) \n\nwas used for qualitative identification and for \n\nchecking the presence of such groups in the \n\nextracted compounds. \n\n \n\nResult and Disscussion \nIsolation and Screening \nAltogether, 23 colonies, which were distinct, \n\nwere chosen based on their shapes and colors.", "start_char_idx": 11089, "end_char_idx": 14124, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5f42ae23-b16b-4912-a436-c472104912c6": {"__data__": {"id_": "5f42ae23-b16b-4912-a436-c472104912c6", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9f54d1b0-9fc0-4cb6-91bf-7b1165b53f3c", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "2fb4c011ca1663779af31595492cfcbd85b3c6e27fb038447b42d66b70759e11", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7fe57e8b-99ec-4d81-9152-e0296c78645b", "node_type": "1", "metadata": {}, "hash": "26d95885ec661be38a7067ad70288e1c5c8583f2bd1c818ee37f9b63fd878953", "class_name": "RelatedNodeInfo"}}, "text": "After 24-48 hours culture period, Sudan Black B \n\nstaining was done to confirm the presence of \n\nPHB granules. Among 23 bacteria, 7 were found \n\nto be Sudan positive, i.e. they were capable of \n\nproducing lipid granules which could have the \n\npresence of PHB.  \n\nMorphological Characterization \nThe result of morphological and biochemical \n\ncharacterization of the 7 Sudan positive bacteria \n\nis shown in Table 1 and Table 2 respectively. It \n\nwas found that five of the strains belonged to \n\nBacillus species and 2 strains, Y202 and K302, \n\nbelonged to Arthrobacter species and Micrococcus \n\nluteus respectively. \n\n \nFigure 1: Graph showing growth curve of isolates \nIt was found that high amounts of black stained \ngranules were obtained in the 48-hour period for all \nthe bacteria. Therefore, 48 hours was chosen as the \noptimized incubation time for PHB production. \n\nGrowth Curve Analysis \n\nThe inoculated cultures were incubated at 37\u00b0C \n\nfor 48 hours and readings were taken at 640 nm \n\nusing spectrophotometer. 640 nm was taken as \n\nthe required wavelength because the \n\nWavelength of Measurement (WM) of OD \n\ndepends on growth of the culture and here, we \n\nexpect higher growth. \n\nThe Absorbance vs. Incubation hour plot of \n\nbacterial strains plotted using R-programming \n\nhas been shown in the Figure 1. \n\nOptimization of Culture Conditions \nThe 7 strains of Sudan positive bacteria were \n\nsubjected to growth in the PHB producing \n\nmedia prepared with different pH, NaCl and \n\nTable 1. Morphological Characteristics of Strains \n\nMorphology T101 B139 Y202 K302 X102 D301 L402 \n\nGram\u2019s Test + + + + + + + \n\nCell Size L: 1\u03bcm \nB:2 \u03bcm \n\nL: 3\u03bcm \nB: 1\u03bcm \n\nDiameter: \n2 \u03bcm \n\nDiameter: \n2 \u03bcm \n\nL: 5 \u03bcm \nB:1 \u03bcm \n\nL:5 \u03bcm \nB:1 \u03bcm \n\nL: 5 \u03bcm \nB: 1 \u03bcm \n\nShape Rod Rod Cocci Cocci Rod Rod Rod \n\nSpore + + - - + + + \n\nMargin Irregular Smooth Smooth Smooth Wooly Smooth Smooth \n\nColor White Light \nYellow \n\nYellow Yellow Pinkish White White White \n\nElevation Flat Drop-like Convex Drop-like Flat Flat Convex \n\nOpacity Translucent Opaque Opaque Opaque Translucent Opaque Opaque \n\nTable 2. Identification of Strains \n\nS.N. Strain \nIdentification from Bergey\u2019s \nManual of Determinative \nBacteriology \n\n1. T101 Bacillus pumilus \n\n2. B139 Bacillus megaterium \n\n3. Y202 Arthrobacter sp.  \n\n4. K302 Micrococcus luteus \n\n5. X102 Bacillus pasteurii \n\n6.  D301 Bacillus cereus \n\n7. L402 Bacillus sphaericus", "start_char_idx": 14127, "end_char_idx": 16517, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7fe57e8b-99ec-4d81-9152-e0296c78645b": {"__data__": {"id_": "7fe57e8b-99ec-4d81-9152-e0296c78645b", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5f42ae23-b16b-4912-a436-c472104912c6", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "aea748b86d2bedb77e5ce87e2d0ab94bb21c73fe78af9347ce336cefd81ba5ce", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c1238244-8eba-44c5-ac3f-45f544ad4619", "node_type": "1", "metadata": {}, "hash": "24e9934cd6a33e3750fb0df71d196bdfeae24845d358d2b76d5ad343cc0084c2", "class_name": "RelatedNodeInfo"}}, "text": "D301 Bacillus cereus \n\n7. L402 Bacillus sphaericus \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:   Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   66                                           Nepjol.info/index.php/njb \n\n \n\ncarbon sources. The graphs of Absorbance vs the \n\noptimized conditions of bacterial strains were \n\nplotted using R-programming (Figure 4, 5 and 6 \n\nrespectively). \n\n \nFigure 2: Sudan Stained black PHB granules of strain \nL402 \n\n \nFigure 3: Sudan Stained black PHB granules of strain \nD301 \n\n \nFigure 4: Graph showing absorbance vs. pH plot of \nbacterial strains. It was established that, for Bacillus \nmegaterium (B), Arthrobacter sp.(Y), Bacillus cereus (D) \nand Bacillus sphaericus (L) the optimized pH was 9, for \nMicrococcus luteus (K) and Bacillus pasteurii (X), the \noptimized pH was 7 and for Bacillus pumilus (T), pH 7 \nand pH 9 were compatible. Thus, we can deduce that \nacidic pH is not suitable for PHB production. \n\nExtraction of PHB  \nExtraction was performed from both PHB \nproducing broth and agar. It was observed that \nextraction from broth gave much better results \nthan that from agar from all of the species except \nArthrobacter spp. The highest percentage of PHB \naccumulation from broth culture was shown by \nBacillus pasteurii and the lowest was shown by \nArthrobacter spp. Similarly, from agar, \n\n  \n\nFigure 5: Graph showing absorbance vs. NaCl \n\nConcentration plot of bacterial strains. It was \nestablished that Bacillus pumilus(T), Bacillus \nmegaterium (B) and Bacillus cereus (D) grew the most in \nthe concentration of 0.5%, Arthrobacter spp. (Y) and \nBacillus sphaericus (L) grew the most in the \nconcentration of 2% while Micrococcus luteus (K) and \nBacillus pasteurii (X) grew uniformly in all the \nconcentrations except in 10%. \n\n \nFigure 6: Graph showing effect of different carbon \nsources on bacterial growth. It was observed that \nBacillus species could grow and produce PHB in both \nGlucose and Sucrose. Bacillus pumilus (T), Bacillus \npasteurii(X) and Bacillus sphaericus preferred Sucrose \nwhile Bacillus megaterium (B) preferred glucose and \nBacillus cereus preferred both Glucose and Sucrose. \nArthrobacter spp (Y) and Micrococcus luteus (X) \npreferred glucose. \n\nArthrobacter spp. showed highest accumulation \n\nof PHB. Bacillus pasteurii, Bacillus pumilus, \n\nBacillus sphaericus did not show any growth in", "start_char_idx": 16467, "end_char_idx": 18870, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c1238244-8eba-44c5-ac3f-45f544ad4619": {"__data__": {"id_": "c1238244-8eba-44c5-ac3f-45f544ad4619", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7fe57e8b-99ec-4d81-9152-e0296c78645b", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "53d33d9ebe49e9ae579943cdde09e2a32128808ff0b1a6ce99de8ae3fb16b4b4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c5babce2-59bc-442c-b175-cd2200668729", "node_type": "1", "metadata": {}, "hash": "1b73f79c46098157281d558d1f06107921535461a45a6bdab422facd4055fc4a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:   Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   67                                           Nepjol.info/index.php/njb \n\n \n\nagar possibly due to limited availability of \n\nnutrients in their respective growth conditions.  \n\nTable 3. Comparison between percentage PHB \n\naccumulation from isolates grown in broth and \n\nagar \n\nFTIR Analysis \nThe functional groups of extracted PHB were \n\nidentified using FTIR Analysis. The functional \n\ngroups of PHB extracted from Bacillus pasteurii, \n\nArthrobacter spp., Micrococcus luteus and Bacillus \n\ncereus was confirmed as C=O groups. \n\n \n Figure 7: FTIR Analysis of extracted product from \nX102 (Bacillus pasteurii) \n\n \nFigure 8: FTIR Analysis of Standard PHB  \n\nConclusion \nThe present study was designed for the isolation \n\nof effective poly-hydroxybutyrate producing \n\nstrains from soil to yield maximum PHB under \n\noptimized conditions. From our research, we \n\nfound out that cosmopolitan \u201cEverything is \n\nEverywhere\u201d population such as Bacillus, \n\nArthrobacter and Micrococcus species were able to \n\nproduce PHB in considerably good quantity \n\ncompared to other isolated species. \n\nConsequently, the effect of various parameters \n\nlike carbon source, incubation time, pH and \n\nNaCl concentration on PHB production were \n\nseen to be species specific. Similarly, the \n\nproduction from broth and fermentation \n\nmethods gave much better results than that from \n\nagar in all of the isolated species except \n\nArthrobacter. PHB yield from broth amounting to \n\n36.41% and 34.59% was observed in Bacillus \n\npasteurii and Micrococcus luteus respectively, \n\nshowing a potential for their exploitation in \n\nTable 3. Comparison between percentage PHB accumulation from isolates grown in broth and agar \n\nS.N. Bacterial Code Bacteria %PHB accumulation \nfrom broth \n\n%PHB accumulation \nfrom agar \n\n1. X102 Bacillus pasteurii  36.41 - \n\n2. K302 Micrococcus luteus 34.59 17.65 \n\n3. B139 Bacillus megaterium  28.63 4.12 \n\n4. T101 Bacillus pumilus 21.46 - \n\n5. L402 Bacillus sphaericus 18.45 - \n\n6. D301 Bacillus cereus  14.91 9.35 \n7. Y202 Arthrobacter spp. 8.56 20.65 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:   Thapa et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   68                                           Nepjol.info/index.php/njb \n\n \n\nindustrial PHB production. Further \n\ncharacterization of extracted products with the \n\nhelp of ATR-FTIR analysis showed prominent \n\nfunctional groups CH3 , CH2 , C=O, C-O, CH \n\nand OH, which when compared with the \n\nstandard PHB curve, confirms the extracts as \n\nPHB. Hence, this project focused on the isolation \n\nof microorganisms from soil samples of polluted \n\nsites and the optimization of conditions for the \n\nproduction of PHB effectively and frugally. \n\nAcknowledgement \nWe sincerely thank the biotechnology \n\ndepartment and management of SANN \n\nInternational College for funding our research \n\nwork. We are also grateful to Mrs. Manjushree \n\nHada for her constant support and supervision.  \n\n \n\nReferences \n1. Ansari S, Fatma, T: Polyhydroxybutyrate - a \n\nBiodegradable Plastic and its Various \n\nFormulations. International Journal of \n\nInnovative Research in Science, Engineering and \n\nTechnology 2014, 3(2):9494\u20139499. \n\n2. Barnes DKA, Galgani F, Thompson RC, \n\nBarlaz M: Accumulation and \n\nFragmentation of Plastic Debris in Global \n\nEnvironments.", "start_char_idx": 18881, "end_char_idx": 22318, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c5babce2-59bc-442c-b175-cd2200668729": {"__data__": {"id_": "c5babce2-59bc-442c-b175-cd2200668729", "embedding": null, "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-205", "node_type": "4", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "85342b95e03e0b02f74ed1c9594be91addac8317d345c745aa4ddc4d974d34d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c1238244-8eba-44c5-ac3f-45f544ad4619", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "b22d95ee4b75316c46e72d910d6c1f30b23807e4cb37d102ed780035df5dad21", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c3489603-4a8f-4c20-9c5e-e0527a24abbf", "node_type": "1", "metadata": {}, "hash": "a5290ba4cd9a72d474b03d44973a0ce09064188690b0978580133ebc44a84f63", "class_name": "RelatedNodeInfo"}}, "text": "Philosophical Transactions \n\nof the Royal Society B: Biological Sciences \n\n2009; 364(1526): 1985\u20131998. \n\n3. Burdon KL, Stokes JC: Studies of the \n\ncommon aerobic spore-forming Bacilli \n\nstaining for fat with Sudan Black B stain. \n\nJournal of Bacteriology 1942, 43: 717-724 \n\n4. Chang Y, Hahn S, Kim B, Chang H: \nOptimization of Microbial Poly (3 \nhydroxybutyrate) Recovery Using \nDispersions of Sodium Hypochlorite \n\nSolution and Chloroform. Biotechnol. \nBioeng. 1994, 44(2): 256-261.  \n\n5. Godale C, Ambarshetti S: Media \noptimization for Phb production and its \napplication as precursor for bioplastics. \nEuropean Journal of Biotechnology and \nBioscience 2015, 3(12):49\u201351. \n\n6. Mikkili I, Karlapudi AP, Venkateswarulu, \n\nNath SB, Kodali VP: Isolation, Screening \n\nand Extraction of Polyhydroxybutyrate \n\n(PHB) producing bacteria from Sewage \n\nsample. International Journal of PharmTech \n\nResearch 2014, 6(2): 850-857 \n\n7. LeBlanc R: How Long Does It Take \n\nGarbage to Decompose?  the balance 2017. \n\n8. Nehra K, Chhabra N, Sidhu P, Lathwal P, \n\nRana J S: Molecular identification and \n\ncharacterization of Poly-\u03b2-hydroxybutyrate \n\n(PHB) producing bacteria isolated from \n\ncontaminated soils. Asian Journal of \n\nMicrobiology, Biotechnology and \n\nEnvironmental Sciences 2017, 17:281-290. \n\n9. Pachekoski WM, Agnelli JAM, Belem LP: \n\nThermal, mechanical and morphological \n\nproperties of poly (hydroxybutyrate) and \n\npolypropylene blends after processing. \n\nMaterials Research 2009, 12(2):159-164 \n\n10. Rydz J, Wanda S, Mariya K, and Darinka C: \nPolyester-Based (Bio) Degradable \nPolymers as Environmentally Friendly \n\nMaterials for Sustainable Development. \nInternational Journal of Molecular Sciences \n2015, 16(1): 564\u2013596. \n\n11. Saharan BS, Grewal A, Kumar P: \nBiotechnological Production of \nPolyhydroxyalkanoates\u202f: A Review on \nTrends and Latest Developments. Chinese \nJournal of Biology 2014. \n\n12. Singh G, Mittal A, Kumari A, Goel A, \nAggarwal NK, Yadav A: Optimization of \nPoly-B-Hydroxybutyrate Production from \n\nBacillus species. European Journal of \nBiological Sciences 2011, 3 (4): 112-116. \n\n13. Wolchover N: Why Doesn\u2019t Plastic \nBiodegrade? LiveScience 2011.", "start_char_idx": 22319, "end_char_idx": 24492, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c3489603-4a8f-4c20-9c5e-e0527a24abbf": {"__data__": {"id_": "c3489603-4a8f-4c20-9c5e-e0527a24abbf", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c5babce2-59bc-442c-b175-cd2200668729", "node_type": "1", "metadata": {"identifier": "njb-205", "author": "Thapa, Christina; Shakya, Pallavi; Shrestha, Rabina; Pal, Sushovita; Manandhar, Prakash", "title": "Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB", "date": "2019-01-15", "file": "njb-205.pdf"}, "hash": "6b94332c3390c6743cf884a6dedb6de96aee758979618ee3b676cee43e745858", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "867cec48-5d1b-4cae-9235-2e2186f56662", "node_type": "1", "metadata": {}, "hash": "e9ca2b1dc893aa9ea2fe3c17e4e6b536ff904d447b5bb23b4902b9031824ca01", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60    ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nCASE STUDY \n\n\u00a9NJB, Biotechnology Society of Nepal    54        Nepjol.info/index.php/njb \n\nPattern of Cancer in Nepal from 2003 to 2011 \nSunil Kumar Sah1, Naval Kishor Yadav2, Roshan Kurmi3, Ramanuj Rauniyar3, Krishna Das Manandhar4, \n\n Birendra Prasad Gupta4* \n1Department of Pathology, BP Koirala Memorial Cancer Hospital , Bharatpur, Chitwan Nepal \n\n2Department of Biochemistry, Manipal Medical College, Nepal \n3Bhawani Hospital, Birgunj, Parsa, Nepal \n\n4Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n\nAbstract  \nCancer is global burden of disease in developed and developing countries. It is one of the main causes of death. \n\nThe environmental factor and life styles are major causes of cancer.  \n\nThis hospital based retrospective study was carried out using data retrieved from the register maintained at \n\nseven cancer centers. The most common basis of diagnosis were microscopic (histopathological and \n\ncytopathological examination). The diagnosis was also based on clinical examination, radiological examination, \n\nendoscopy, biochemical and immunological tests.  \n\nMost of the cancer cases were diagnosed at BPKMCH (23908) followed by BPKIHS (9668) and BH (5959) and few \n\ncases from KCH (518) in 2003 to 2011. The total number of cancer cases were increasing from 2003 to 2011 and it \n\nbecome double in 2011.. Out of 75 district of Nepal, more number of cancer cases was found in Kathmandu, \n\nSunsari, Morang, Chitwan, Lalitpur, Jhapa, Kaski, Nawalparasi, Rupendehi and Kavrepalchowk in 2010. \n\nSimilarly, in 2011 more number of cancer cases was found in Kathmandu, Morang, Jhapa, Sunsari, Chitwan, \n\nLalitpur, Rupendehi, Kaski, Saptari, Bhaktapur. Lung cancer was the common cancer and similarly, other \n\nprevalent cancers were cervical, breast, stomach, ovarian and colo-rectum cancer in 2003 to 2011. The common \n\ncancers were lung, cervical, breast, stomach, ovarian and colo-rectum. The number of patients is increasing, \n\nwhich may be due to change in life style and lack of education. \n\nKey words: Cancer, Nepal, 2003-2011, Kathmandu,  \n\n*Corresponding Author \n\nEmail:- birendraphd@gmail.com \n\nIntroduction \nCancer is characterized by uncontrolled growth \n\nand spread of abnormal cells with multi-factorial \n\netiology [1].  It is one of the most dreaded non-\n\ncommunicable diseases that have become the \n\nimportant contributor to the global burden of \n\ndisease [2]. The causes of cancer is not only genetic \n\nfactor, which contribute 5-10%, while \n\nenvironmental factor and life styles cover 90-95% \n\n[3,4]. The lifestyle factors include cigarette \n\nsmoking, diet (fried foods, red meat), alcohol, sun \n\nexposure, environmental pollutants, infections, \n\nstress, obesity, and physical inactivity [5,6]  \n\nIn 2012, new cancer cases were 14.1 million and \n\nwith 8.2 million deaths. The lung cancer was the \n\nmost common cancer 16.7% of all new cases in men \n\nand breast cancer 25.2% in women [7]. According \n\nto estimates from the International Agency for \n\nResearch on Cancer (IARC), the global burden is \n\nexpected to grow to 21.4 million new cancer cases \n\nand 13.2 million cancer deaths by 2030.", "start_char_idx": 48, "end_char_idx": 3328, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "867cec48-5d1b-4cae-9235-2e2186f56662": {"__data__": {"id_": "867cec48-5d1b-4cae-9235-2e2186f56662", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c3489603-4a8f-4c20-9c5e-e0527a24abbf", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "12eff29a6cb50a9cff32dd7afc91612b20da9d2f2d482da5fd7cef5c6383f3ac", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f18577ae-d6a5-449f-b53c-a21fe3a4bde8", "node_type": "1", "metadata": {}, "hash": "6d9404b50c86c2a057a860734092aab0067177be9437f13a1929b302db592987", "class_name": "RelatedNodeInfo"}}, "text": "[8] \n\nIn developed countries, most common diagnosed \n\ncancers were prostate, lung and bronchus, and \n\ncolorectal among men while breast, colorectal, and \n\nlung among women. Similarly, lung, stomach, and \n\nliver cancer in men while breast, cervix uteri, and \n\nlung in women in developing countries [9]. Nepal \n\nis a developing country where cancer cases are \n\nincreasing day by day. The aim of this study is to \n\nsee the pattern of cancer cases from 2003 to 2011 in \n\nNepalese people.  \n\nMaterials and Methods \nThis hospital based retrospective study was carried \n\nout using data retrieved from the register \n\nmaintained at seven cancer centers of Nepal from \n\n2003 to 2011. The cancer centers were B.P. Koirala \n\nMemorial Cancer Hospital (BPKMCH) Bharatpur, \n\nChitwan, Bhaktapur Cancer Hospital (BCH) \n\nBhaktpur, Bir Hospital (BH) Kathmandu, TU \n\nTeaching Hospital (TUTH) Kathmandu, Kanti \n\nChildren\u2019s Hospital (KCH) Kathmandu, \n\nB.P.Koirala Institute of Health Sciences (BPKIHS) \n\nDharan and Manipal Teaching Hospital (MTH), \n\nPokhara, Nepal. The most common basis of \n\ndiagnosis were microscopic (histopathological and \n\ncytopathological examination). The diagnosis was \n\nalso based on clinical examination, radiological \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    55        Nepjol.info/index.php/njb \n\nexamination, endoscopy, biochemical and \n\nimmunological tests. The collected variables are \n\nage, sex, occupation, religion, ethnicity, region of \n\nresidence and type of cancer. The data were \n\nanalysed using Excel 2007.  \n\nResults \nMost of the cancer cases were diagnosed at \n\nBPKMCH (23908) followed by BPKIHS (9668) and \n\nBH (5959) and few cases from KCH (518) in 2003 to \n\n2011 (Figure 1).  \n\nFigure 1: The number of total cancer between 2003 to \n\n2011 from seven cancer reporting institute of Nepal.  \n\nThe total number of cancer cases were increasing \n\nfrom 2003 to 2011 and it became double in 2011 \n\n(Figure 2).  \n\nFigure 2: This data represents total number of cancer \n\ncases and sex wise distribution of cancer from 2003 to \n\n2011. \n\nCancer cases were more in female than male in all \n\nthe years (Table 2). Most of the cancer patients \n\nwere married (Table 3) and Hindu religion \n\nfollowed by Buddhist religion (table 4). More \n\ncancer patients were illiterate followed by literate \n\n(Table 5). Agriculture was main occupation of \n\nmost cancer patients followed by housework (table \n\n6). Most of cancer cases were prevalent in age \n\ngroup 40-75 years. Less number of cancer cases was \n\nfound in age group below 15 years (Table 7). \n\nBiopsy and histology were the major method for \n\nthe diagnosis of cancer cases followed by cytology \n\nand haematology. The clinical examination and \n\nbiochemical/immunological test were help to \n\nidentified only few cancer cases. (Table 8). Out of \n\n75 district of Nepal, more number of cancer cases \n\nwas found in Kathmandu, Sunsari, Morang, \n\nChitwan, Lalitpur, Jhapa, Kaski, Nawalparasi, \n\nRupendehi and Kavrepalchowk in 2010 (figure 4). \n\nSimilarly, in 2011 more number of cancer cases was \n\nfound in Kathmandu, Morang, Jhapa, Sunsari, \n\nChitwan, Lalitpur, Rupendehi, Kaski, Saptari, \n\nBhaktapur (figure 5).", "start_char_idx": 3329, "end_char_idx": 6585, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f18577ae-d6a5-449f-b53c-a21fe3a4bde8": {"__data__": {"id_": "f18577ae-d6a5-449f-b53c-a21fe3a4bde8", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "867cec48-5d1b-4cae-9235-2e2186f56662", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "d83f97e75c936f2421bf1292791fd3d2eedf7a4ad615be0a5048284ab7d09aff", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "32b4c753-b465-438b-bf70-fe1768995f35", "node_type": "1", "metadata": {}, "hash": "0874c639c73501ec817c091be24e05d4cd13ed368286526d3f703cbbc92966fa", "class_name": "RelatedNodeInfo"}}, "text": "Lung cancer was the common \n\ncancer and similarly, other prevalent cancers were \n\ncervical, breast, stomach, ovarian and colo-rectum \n\ncancer in 2003 to 2011(figure 5).  \n\nDiscussion \nNepal is a developing country and divided into \n\nbeautiful three region terai, mountain and \n\nHimalayan. People of different region have their \n\nown language, religion, festival and source of \n\nincome to run the family. This study reveals, \n\ndiagnosed cancer cases were mostly from terai \n\nregion and most of them were working in field. The \n\nsecond occupation was housework as cancer cases \n\ndiagnosed high in female compare to male. More \n\nthan half of the cancer cases were illiterate. Most of \n\ncancer cases were prevalent in age group 40-75 \n\nyears. Less number of cancer cases was found in \n\nage group below 15 years. The study conducted in \n\nindia in 1994 and 1955 showed prevalence of cancer \n\nin same age group [11]. It may be due to decreases \n\nin immune system, iliteracy and exposure to cance \n\ncausing agents in environmet. The most diagnosed \n\ncancer cases were hindus followed by buddhist, \n\nIslam and Christians. This is due to high number of \n\npopulation were Hindus in Nepal as we know \n\nNepal were Hindu country in world.  \n\nIn Nepal, among all diagnosed cancer cases, the \n\ncommon cancer were lung, cervical, breast, \n\nstomach, ovarian and colo-rectum [12]. The report \n\nby WHO showed common cancers was lung, breast \n\nand colo-rectum in the world [13]. Some studies \n\nshowed similar finding for these common cancers \n\n0\n\n2000\n\n4000\n\n6000\n\n8000\n\nN\nu\n\nm\nb\n\ne\nr \n\nYears\n\nTotal cancer cases Male Female\n\n0\n2000\n4000\n6000\n8000\n\n10000\n12000\n14000\n16000\n18000\n20000\n22000\n24000\n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    56        Nepjol.info/index.php/njb \n\n[13, 14, 15]. The lung cancer was estimated a total \n\nof 239,320 new cases in US in 2010 [16]. The lung \n\ncancer is due to maximum use of tobacco which \n\ncan be seen mainly in 25-60 yrs. age group in \n\nNepal. Tobacco smoking [16] and tobacco uses \n\nwere higher in mid-western, rural, far western and \n\nmountainous areas of Nepal [17].  \n\nConclusion \nThe cancer is becoming a major problem in Nepal \n\nwith the number rising continuously. All this may \n\nbe due to environmental factors and life style \n\nchanges i.e. tobacco use, food habits, alcohol use \n\nand physical inactivity.  \n\nReferences \n1. Peppas LB, Blanchette JO: Nanoparticle and \n\nTargeted Systems for Cancer Therapy. Adv. Drug. \n\nDelivery Rev. 2003 56: 1649-59. \n\n2. Binu V, Chandrashekhar T S, Subba S H, Jacob S, \n\nKakria A, Gangadharan P, Menezes R G: Cancer \n\nPattern in Western Nepal: A Hospital Based \n\nRetrospective Study. Asian Pac. J. Cancer. Prev. \n\n2007 8:183-186. \n\n3. Loeb KR, and Loeb LA: Significance of Multiple \n\nMutations in Cancer. Carcinogen. 2000 21:379\u201385. \n\n4. Hahn WC, and Weinberg RA: Modelling the \n\nMolecular Circuitry of Cancer. Nat. Rev. Cancer \n\n2002 2:331\u201341.  \n\n5. Anand P, Kunnumakara AK, Sundaram C et al: \n\nCancer is a Preventable Disease that Requires \n\nMajor Lifestyle Changes. Pharma. Res.", "start_char_idx": 6586, "end_char_idx": 9729, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "32b4c753-b465-438b-bf70-fe1768995f35": {"__data__": {"id_": "32b4c753-b465-438b-bf70-fe1768995f35", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f18577ae-d6a5-449f-b53c-a21fe3a4bde8", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "585bea6c8e209e1eb810806da552496e56f8e29f44c962bb3fa2c0d0479301a3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "368ce3de-7c28-4938-990b-85ddcca1f4c2", "node_type": "1", "metadata": {}, "hash": "d213d49763b91d4bb087f564de8089ff90b8e86a68f4dd79fcd504f9b56b6681", "class_name": "RelatedNodeInfo"}}, "text": "Pharma. Res. 2008 25 (9): \n\n2097-2116. \n\n6. Cogliano V, Baan R, Straif K, et al: Preventable \n\nexposures associated with human cancers. J. Natl. \n\nCancer Inst. 2011 103 :1827-39. \n\n7. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser \n\nS, Mathers C, Rebelo M, Parkin DM, Forman D, \n\nBray F: GLOBOCAN 2012: Cancer Incidence and \n\nMortality Worldwide: IARC Cancer Base No. 11 \n\n[Internet]. Lyon, France: International Agency for \n\nResearch on Cancer; 2013 \n\n8. Ferlay J, Shin HR, Bray F, Forman D, Mathers CD, \n\nParkin D: GLOBOCAN 2008: Cancer Incidence \n\nand Mortality Worldwide: IARC CancerBase \n\nNo.10 [Internet]. Lyon, France: International \n\nAgency for Research on Cancer. 2010 \n\n9. Irigaray JA, Newby R, Clapp L, Hardell V, \n\nHoward, L. Montagnier, S. Epstein, and D. \n\nBelpomme: Lifestyle-related factors and \n\nenvironmental agents causing cancer: an \n\noverview. Biomed. Pharmacother 2007 61:640\u201358. \n\n10. IARC, GLOBOCAN 2008 (2010): International \n\nAgency for Research on Cancer (IARC). \n\nhttp://globocan.iarc.fr/ factsheet.asp accessed on \n\n25.7.2012. \n\n11. Mitra S and Gupta AD: An Estimate of the \n\nPrevalence of Cancer in India. Bull. Org. inond. \n\nSante. 1960 22: 485-492. \n\n12. IARC: GLOBOCAN 2012, International Agency \n\nfor Research on Cancer (IARC). \n\nhttp://globocan.iarc.fr/ factsheet.asp accessed on \n\n12.12.2013 \n\n13. Jemal A, Bray F, Center MM, et al: Global Cancer \n\nStatistics. CA: Cancer J. Clin. 2011 61(2): 69\u201390. \n\n14. Behera D, Balamugesh T: Lung Cancer in India. \n\nIndian J. Chest Dis. Allied Sci. 2004 46(4): 269-81. \n\n15. Noronha V, Dikshit R, Raut N, Joshi A, Pramesh \n\nCS, George K, Agarwal JP, Munshi A, Prabhash K:  \n\nEpidemiology of Lung Cancer in India: Focus on \n\nthe Differences Between Non-smokers and \n\nSmokers: a Single-Centre Experience. Indian J. \n\nCancer 2012 49(1): 74-81 \n\n16. Jemal A, Ward E, Hao Y, et al: Trends in the \n\nLeading Causes of Death in the United States \n\n1970\u2013 2002. JAMA. 2005 294(10): 1255\u20131259. \n\n17. Chandrashekhar T, Sreeramareddy N , \n\nRamakrishnareddy HN, Kumar H, Sathian B, \n\nArokiasamy JT: Prevalence, Distribution and \n\nCorrelates of Tobacco Smoking and Chewing in \n\nNepal: a Secondary Data Analysis of Nepal \n\nDemographic and Health Survey-2006. Subst. \n\nAbuse Treat Prev.", "start_char_idx": 9717, "end_char_idx": 11965, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "368ce3de-7c28-4938-990b-85ddcca1f4c2": {"__data__": {"id_": "368ce3de-7c28-4938-990b-85ddcca1f4c2", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "32b4c753-b465-438b-bf70-fe1768995f35", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "92a8d3dc64a455082e9f6700a361a2637a96f2d0ffff26890e05b30dc9d052e4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "167d4f45-2bf8-4bc7-8b96-c0524396cf43", "node_type": "1", "metadata": {}, "hash": "53722f35f468f6129fa91051a822ec6822db5a9de42a4503daff12787d36368d", "class_name": "RelatedNodeInfo"}}, "text": "Subst. \n\nAbuse Treat Prev. Policy 2011 6: 33. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Behera%20D%5BAuthor%5D&cauthor=true&cauthor_uid=15515828\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Balamugesh%20T%5BAuthor%5D&cauthor=true&cauthor_uid=15515828\nhttp://www.ncbi.nlm.nih.gov/pubmed/15515828\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Noronha%20V%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Dikshit%20R%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Raut%20N%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Joshi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Pramesh%20CS%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Pramesh%20CS%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=George%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Agarwal%20JP%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Munshi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Prabhash%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22842172\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Sreeramareddy%20CT%5Bauth%5D\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Ramakrishnareddy%20N%5Bauth%5D\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Ramakrishnareddy%20N%5Bauth%5D\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Harsha%20Kumar%20H%5Bauth%5D\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Sathian%20B%5Bauth%5D\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Arokiasamy%20JT%5Bauth%5D\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    57        Nepjol.info/index.php/njb \n\n \n\n \n\n \n\n \n\n \n\n \n\nTable 1: Total cancer cases from seven reporting institute in 2003 to 2011 \n\nName of \n\nHospital \n2003 2004 2005 2006 2007 2008 2009 2010 2011 \n\nBPKMCH \n1869 \n\n(57.5%) \n\n1815 \n\n(43.2%) \n\n2154 \n\n(49.0%) \n\n2564 \n\n(52.2%) \n\n3261 \n\n(54.03%) \n\n2936 \n\n(49.4%) \n\n2772 \n\n(44.1%) \n\n3216 \n\n(47.5%) \n\n3320 \n\n(46.8%) \n\nBCH \n328 \n\n(10.0%) \n\n952 \n\n(22.7%) \n\n1008 \n\n(22.9%) \n\n1086 \n\n(22.1%) \n\n1138 \n\n(18.", "start_char_idx": 11939, "end_char_idx": 14293, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "167d4f45-2bf8-4bc7-8b96-c0524396cf43": {"__data__": {"id_": "167d4f45-2bf8-4bc7-8b96-c0524396cf43", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "368ce3de-7c28-4938-990b-85ddcca1f4c2", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "def8c730f93959725638bd52177eff96fb51680e54b8090cf9fadde020f10eac", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fdfb11c7-5ff6-460d-b235-2d04c7ada379", "node_type": "1", "metadata": {}, "hash": "feba80c61e848ce35b99cb74d67c58d63781bc9cf77e81ae938db3b6e0fe2a4b", "class_name": "RelatedNodeInfo"}}, "text": "9%) \n\n1086 \n\n(22.1%) \n\n1138 \n\n(18.85%) \n\n1253 \n\n(21.1%) \n\n1289 \n\n(20.8%) \n\n1253 \n\n(18.5%) \n\n1361 \n\n(19.2%) \n\nBPKIHS \n418 \n\n(12.9%) \n\n457 \n\n(10.9%) \n\n702 \n\n(16.0%) \n\n613 \n\n(12.5%) \n\n640 \n\n(10.6%) \n\n655 \n\n(11.0%) \n\n660 \n\n(10.6%) \n\n553 \n\n(8.2%) \n\n512 \n\n(7.2%) \n\nBH \n127 \n\n(4.0%) \n\n385 \n\n(9.2%) \n\n206 \n\n(4.7%) \n\n350 \n\n(7.1%) \n\n696 \n\n(11.53%) \n\n798 \n\n(13.4%) \n\n1228 \n\n(19.8%) \n\n1173 \n\n(17.3%) \n\n1005 \n\n(14.2%) \n\nMTH \n215 \n\n(6.6%) \n\n307 \n\n(7.3%) \n\n162 \n\n(3.7%0 \n\n172 \n\n(3.5%) \n\n192 \n\n(3.18%) \n\n153 \n\n(2.6%) \n\n79 \n\n(1.3%) \n\n77 \n\n(1.1%) \n\n160 \n\n(2.1%) \n\nTUTH \n248 \n\n(7.6%) \n\n190 \n\n(4.5%) \n\n134 \n\n(3.0%) \n\n89 \n\n(1.8%) \n\n68 \n\n(1.12%) \n\n91 \n\n(1.5%) \n\n188 \n\n(3.0%) \n\n392 \n\n(5.9%) \n\n648 \n\n(9.1%) \n\nKCH \n46 \n\n(1.4%) \n\n95 \n\n(2.3%) \n\n31 \n\n(0.7%) \n\n34 \n\n(0.7%) \n40 (0.6%) \n\n63 \n\n(1.1%) \n\n23 \n\n(0.4%) \n\n104 \n\n(1.1%) \n\n82 \n\n(1.2%) \n\nTotal 3251 4201 4397 4908 6035 5949 6199 6773 7088 \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n  \n\nTable 2: Distribution of cancer cases based on marital status in 2010 to 2011 \n\nMarital status  2010   2011  \n\n Male Female Total Male Female Total \n\nUnmarried 132 111 243 127 92 219 \n\nMarried 2678 3084 5762 2773 3243 6016 \n\nWidow 68 172 240 70 199 269 \n\nDivorced 3 4 7 1 4 5 \n\nSeparated 5 1 6 4 3 7 \n\nNot Available 106 107 213 131 135 266 \n\nNot Applicable 1 0 1 189 117 306 \n\nTotal 2993 3479 6472 3295 3793 7088 \n\nTable 3: Distribution of cancer cases based on the Religion in 2010 to 2011 \n\nReligion  2010   2011  \n\n Male Female Total Male Female Total \n\nHindu 2735 3057 5792 2683 3093 5776 \n\nBuddhist 217 254 471 301 358 659 \n\nIslam 51 53 104 57 59 116 \n\nChristian 16 32 48 24 26 50 \n\nOthers 63 85 148 160 191 351 \n\nNot \n\nAvailable 110 100 210 70 66 136 \n\nTotal 3192 3581 6773 3295 3793 7088", "start_char_idx": 14259, "end_char_idx": 15954, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fdfb11c7-5ff6-460d-b235-2d04c7ada379": {"__data__": {"id_": "fdfb11c7-5ff6-460d-b235-2d04c7ada379", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "167d4f45-2bf8-4bc7-8b96-c0524396cf43", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "ac8320d4318d70efbe6839699914d8be88865da454b7b69c7a5e67f9ffbe60e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "78002fe3-73c8-41c9-84db-5f0cb2c502c3", "node_type": "1", "metadata": {}, "hash": "a69b0babd37b3ad6536cfe2bbd86ef845c890b98ca53081a2d0e9a80cdc83d1d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    58        Nepjol.info/index.php/njb \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nTable 4: Distribution of cancer cases based on the education status in 2010 to 2011 \n\nEducation  2010   2011  \n\n Male Female Total Male Female Total \n\nLiterate 1346 1118 2464 1342 1068 2410 \n\nIlliterate 1291 1936 3227 1494 2233 3727 \n\nNot Applicable 25 17 42 397 466 863 \n\nNot Available 476 479 955 62 26 88 \n\nTotal 3138 3550 6688 3295 3793 7088 \n\nTable 5: Distribution of cancer cases based on occupational status in 2010 to 2011 \n\nOccupation  2010   2011  \n\n Male Female Total Male Female Total \n\nAgriculture 1769 1154 2923 1757 1082 2839 \n\nBusiness 172 83 255 202 182 384 \n\nHousework 206 1790 1996 265 1895 2160 \n\nOffice Work 256 77 333 251 105 356 \n\nOthers 291 158 449 328 150 478 \n\nNot Applicable 8 5 13 303 262 565 \n\nNot Available 441 363 804 189 117 306 \n\nTotal 2993 3479 6773 3295 3793 7088 \n\nTable 6: Distribution of cancer cases based on the age group in 2010 to 2011 \n\nAge Group  2010   2011  \n\n(Years) Male Female Total Male Female Total \n0-4 54 31 85 62 26 88 \n\n5-9 66 39 105 52 41 93 \n\n10-14 79 32 111 75 50 125 \n\n15-19 70 75 145 74 63 137 \n\n20-24 74 96 170 93 84 177 \n\n25-29 77 117 194 78 120 198 \n\n30-34 91 159 250 103 155 258 \n\n35-39 128 241 369 141 251 392 \n\n40-44 154 328 482 205 380 585 \n\n45-49 228 439 667 234 439 673 \n\n50-54 293 491 784 295 464 759 \n\n55-59 319 362 681 404 474 878 \n\n60-64 459 414 873 426 449 875 \n\n65-69 427 304 731 381 359 740 \n\n70-74 351 228 579 333 232 565 \n\n75-79 193 132 325 210 138 348 \n\n80 + 129 93 222 129 68 197 \n\nTotal 3192 3581 6773 3295 3793 7088", "start_char_idx": 15959, "end_char_idx": 17706, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "78002fe3-73c8-41c9-84db-5f0cb2c502c3": {"__data__": {"id_": "78002fe3-73c8-41c9-84db-5f0cb2c502c3", "embedding": null, "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-21", "node_type": "4", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "71ae8fc56073285f61afdfe6b31cfff5e0e9392cd5b9f328de8a09753f098fda", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fdfb11c7-5ff6-460d-b235-2d04c7ada379", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "356de8f35841bee942f6e4a68a8ccaff3b7068497efbf3f38c3cdcf009e351da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0d82d101-6a13-42b6-aa08-d6f592374ee0", "node_type": "1", "metadata": {}, "hash": "9c6140abae23a282c001e7551e646a9b0a0a451a5e7262557ae6a61900613ae3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    59        Nepjol.info/index.php/njb \n\n \n\nFigure 3: Showed the sex wise distribution of cancer cases in different district of Nepal in 2010 \n\nTable 7: Distribution of cancer cases based on type of diagnosis in 2010 to 2011 \n\n  2010   2011  \n\n Male Female Total Male Female Total \n\nClinical Examination 3 1 4 23 25 48 \n\nEndoscopy 36 12 48 37 17 54 \n\nBiopsy/Histology 1699 2151 3850 1648 2296 3944 \n\nCytology/Haematology 812 834 1646 860 816 1676 \n\nExploratory Surgery 2 2 4 0 0 0 \n\nBiochemical/Immunological \n\ntest 0 7 7 1 15 16 \n\nRadiology 239 225 464 300 274 574 \n\nDeath certificate 2 3 5 3 6 9 \n\nNot available 399 346  423 344 767 \n\nTotal 3192 3581 6773 3295 3793 7088 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:54-60     Kurmi et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    60        Nepjol.info/index.php/njb \n\n \n\n \n\n \n\nFigure 4: Showed the sex wise distribution of cancer cases in different district of Nepal in 2011", "start_char_idx": 17711, "end_char_idx": 18803, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0d82d101-6a13-42b6-aa08-d6f592374ee0": {"__data__": {"id_": "0d82d101-6a13-42b6-aa08-d6f592374ee0", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "78002fe3-73c8-41c9-84db-5f0cb2c502c3", "node_type": "1", "metadata": {"identifier": "njb-21", "author": "Sah, Sunil Kumar; Yadav, Naval Kishor; Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishan Das; Gupta, Birendra Prasad", "title": "Pattern of Cancer in Nepal from 2003 to 2011", "date": "2016-12-31", "file": "njb-21.pdf"}, "hash": "87ecbe6bfe3aa491320f692fa770f9d55bc9d7d82bc6d3c30e81e08bb9804f1f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6cb5dd57-cd12-418e-8918-598a509ced54", "node_type": "1", "metadata": {}, "hash": "5d94dffc465b17ab6393d550dbdf591983bc95c262abbcf530a394e1cdc037e0", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 16-19  ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal      16                                       Nepjol.info/index.php/njb \n\n \n\nCyclospora cayetanensis:  An Infestation among \nDiarrheal Children in Kathmandu Valley, Nepal \n\nBalkrishna Bhattachan1, 2*, Ganesh Rai1, Nabin Narayan Munankarmi 3,  \nJeevan Bahadur Sherchand2 \n\n1Shi-Gan International College of Science and Technology (SICOST), Kathmandu, Nepal \n2Department of Microbiology of Tribhuvan University Teaching Hospital, Kathmandu, Nepal \n\n3Biotechnology Society Nepal (BSN) Kausaltar-3, Bhaktapur, Nepal \n\n Abstract \nCyclospora cayetanensis, a coccidian parasites which is responsible for recurring diarrhea and \ngastroenteritis especially among children living under poor hygienic condition in developing \ncountry like Nepal. Aimed of this research is to find out the prevalence of intestinal parasites \nand C. cayetanensis among diarrheal children in a Pediatric Hospital in Kathmandu valley, \nNepal. Cross-sectional type of study was done. Altogether 196 stool specimens were collected \nfrom June to September 2013 among outpatient diarrheal children in Kanti Children\u2019s \nHospital.  Modified Ziehl Neelson staining method was applied for detection of oocysts of \nCyclospora after formal-ether sedimentation.    Parasites were detected in 13.7% (27/196) of \nstool samples from \u2264 15 year old diarrheal patients. C. cayetanensis was detected in 4.8% \n(8/196). In genderwise, infection rate of C. cayetanensis 4.5 % (5/112), in male were higher \nthan 3.6% (3/84) in female. In agewise, infection rate of C. cayetanensis in 5.7 % (3/52) 11-15 \nyear old were highest followed by 4.1% (3/78) in 0-5 year old and 3.0% (2/66) in 6-10 year \nold.  In seasonwise, infection rate of Cyclospora was highest in the month of August 7.4% \n(4/54) followed by 3.9% (2/51) in July, 2.3% (1/44) September and 2.1% (1/47) in June.  \nAltogether five different type of parasites were detected. Infection rate of Giardia lamblia were \nhighest 5.1 % (10/196) whereas lowest was Cryptosporidium parvum 1.0 % (2/196). Prevalence \nof C. cayetanensis is highly probable to infant, neonate, toddler and diarrheal children. \nTherefore, attention should be made in laboratory investigation of C. cayetanensis while \nsuspecting the diarrheal patients infected with other parasites.   \nKeywords:  Cyclospora, Parasites, Modified Ziehl Neelson, Children \n\n*Corresponding author \n\nEmail: balkrishna_bhattachan@hotmail.com\n\nIntroduction \nCyclospora cayetanensis, is a coccidian parasite \n\nwhich is responsible for prolonged diarrhea in \n\nboth immune-competent and immune-\n\ncompromised patients. The pathogenesis and \n\nvirulence factors phenomenon of C. cayetanensis \n\nare yet to be defined, but Inflammation and \n\njejunitis had been found out [1]. C. cayetanensis \n\ninfect human and cause of acute and chronic \n\ngastroenteritis [2]. It has been reported from \n\nvarious parts of the world in Southeast Asia [3,4].", "start_char_idx": 48, "end_char_idx": 3119, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6cb5dd57-cd12-418e-8918-598a509ced54": {"__data__": {"id_": "6cb5dd57-cd12-418e-8918-598a509ced54", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0d82d101-6a13-42b6-aa08-d6f592374ee0", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "cc0ead38d8d2a66946fdada7170d93a9dcefd4f30393a7351b4e389e4d41a166", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4faf0923-dcb2-4b43-96dd-91dd0168126b", "node_type": "1", "metadata": {}, "hash": "ff5078dded7d25576c3aeebc984235d5d6c096a97ad6566409eac69924d0b6c3", "class_name": "RelatedNodeInfo"}}, "text": "The important vehicle for infection is source of \n\nwater either by ingestion of parasite through \n\ncontaminated water directly or through \n\ncontaminated vegetable. Moreover, it has been \n\nimplicated in outbreaks developing countries \n\n(e.g. like Nepal) [5].  In the study of Sherchand \n\nand Cross, between 2001-2004 in Nepal showed \n\nthat contaminated drinking water, raw green \n\nvegetables, infected animals were the possible \n\nsources of infection with Cyclospora [6]. \n\nThe Patients suffering from cyclosporisis show \n\nthe symptoms of an abdominal cramps, diarrhea, \n\nfatigue, nausea, and vomiting, chronic watery, \n\nanorexia, and weight loss. However, the cause of \n\ndisease is still unrevealed. This study was carried \n\nout at Kanti Children\u2019s Hosptial (KCH), \n\nKathmandu Valley, aiming to find out the \n\nprevalence of parasites and C. cayetanensis \n\nespecially diarrheal children. \n\nMaterial and Methods \nStudy site:  Cross-sectional type of study was \n\nundertaken at KCH in Kathmandu Valley from \n\nJune to September 2013.  \n\nCollection of data and samples: Demographic \n\ndata were collected by using a standard \n\nquestionnaire and with proper instruction \u2264 15  \n\n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 16-19                                  Bhattachan et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal      17                                       Nepjol.info/index.php/njb \n\n \n\nyear old outpatient diarrheal children were  \n\n requested for collection of stool sample. For the \n\nnearly 30 ml of fresh stool) a clean, dry and screw \n\ncapped container were given. During collection \n\nof samples were kept in icebox by maintaining \n\ncold chain before transportation to Public Health \n\nResearch Laboratory, Institute of Medicine, and \n\nKathmandu, Nepal. \n\nMicroscopic examination  \n\nFormal-ether sedimentation Technique: Three \n\nml of stool samples was taken in test-tube and \n\nshaken well then filtered through cotton gauge. \n\nAfter that, 3 ml of ether was added and shaken \n\nwell then centrifuged. The sediment portion was \n\nexamined by microscopy after addition of saline \n\nsolution. Microscopic detection of parasites cyst \n\nwere carried out under 10X followed by 40X \n\nobjectives [7]. \n\nModified acid fast staining method: The smears \n\nwere flooded with carbol fuchsin and heated to \n\nsteaming. It was shaken with tap water, \n\ndecolorized with 5% aqueous sulfuric acid, and \n\nshake again. Flooded with methylene blue  \n\n \n\ncounterstain was added on smear formed stain, \n\nthen rinsed with tap water. Smear was drained, \n\nand air dried [8]. oocysts of C. cayetanensis were \n\nidentified having size (8-10 \u00b5m), circle shape and \n\npink color.  \n\nEthics: Written informed consent was taken from \n\nall participants and/or their parents before \n\nenrolling into the study. Institutional Review \n\nBoard, Institute of Medicine, Research \n\nDepartment, Kathmandu, Nepal was approved \n\nthis research.  Win-pepi statistical software was \n\nused for data calculation where p value of data   \n\n<0.05 considered as statistically significant. \n\nResults  \nParasites were detected in 13.7% (27/196) of stool \n\nsamples from \u2264 15 year diarrheal patients. C. \n\ncayetanensis was detected in 4.8% (8/196). \n\nInfection rate of C. cayetanensis in 5.7 % (3/52) 11-\n\n15 years old were highest followed by 4.1% \n\n(3/78) in 0-5 years old and 3.0% (2/66) in 6-10 \n\nyears old [Table 1].  \n\nIn gender, ratio between male and female were \n\n1.3:1, infection rate of C. cayetanensis 4.5 % \n\n(5/112) in male were higher than C. cayetanensis \n\n3.6% (3/84)  in female [Table 2].", "start_char_idx": 3124, "end_char_idx": 6701, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4faf0923-dcb2-4b43-96dd-91dd0168126b": {"__data__": {"id_": "4faf0923-dcb2-4b43-96dd-91dd0168126b", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6cb5dd57-cd12-418e-8918-598a509ced54", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "e3acfb8425f9bee6b845869b87bbce68e82a6730f78d6c6582c355e441c563c5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6446478d-6aad-4b3b-9f4f-01ab9e4ef507", "node_type": "1", "metadata": {}, "hash": "d375d6725f1eb0e209b0e8d37f962323f975e3b7743d73fe0d24f125acebef9c", "class_name": "RelatedNodeInfo"}}, "text": "Table 1.  Distribution of age and intestinal parasites among diarrheal children from outpatients in Kanti Children\u2019s \nHospital  \n\nCharacteristics  Total  no. of children Any parasite \ndetected \n\nP-value C. cayetanensis P- value \n\n  No No.(%)  No. (%)  \nAge (year) 0-5 78 11 (14.1) 0.902 3 (4.1) 0.768 \n 6-10 66 8  (12.1)  2 (3.0)  \n\n  11-15 52 8  (15.4)  3 (5.7)  \n\nTotal   196 27(13.8)  8 (4.1)  \n\nTable 2.  Distribution of sex and   intestinal parasites among diarrheal children from outpatients in Kanti Children\u2019s \nHospital  \n\nCharacteristics       Total  no. of children  Any parasite detected   p-value  C. cayetanensis P- value  \n\n  No No.(%)      No. (%)  \n\nGender Male 112 16 (31.3) 0.835 5 (4.5) 0.764 \n\n Female  84 11 (13.1)  3 (3.6)  \n\nTotal   196 27 (13.8)  8 (4.1)  \n\nTable 4: Number of positive and percentage rate of \n\nintestinal parasites among diarrheal children were \n\nshown in Table 4.   \n\nName of parasites Number (%) \n\nGiardia lamblia  10 (5.1) \n\nCyclospora cayetanensis  8 (4.8) \n\nEntamoeba histolytica  4(2.5) \n\nEntamoeba coli  3(1.5) \n\nCryptosporidium parvum  2(1.0) \n\n*None of multiparasites was detected. \n\nTable 3: Monthwise- distribution of Cyclospora \ncayetanensis and total parasites in proportion in \nstool samples \n\n Cyclospora \n\ncayetanensis (%) \n\nTotal \n\nparasites (%) \n\nMonth   \n\nJune 2.1 12.9 \n\nJuly 3.9 9.8 \n\nAugust  7.4 18.8 \n\nSeptember  2.3 15 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 16-19                                  Bhattachan et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal      18                                       Nepjol.info/index.php/njb \n\n \n\nIn seasonwiser, infection rate of C. cayetanensis \n\nwas highest in the month of August 7.4% (4/54) \n\nfollowed by 3.9% (2/51) in July, 2.3% (1/44) \n\nSeptember and 2.1% (1/47) in June [Figure 1].  \n\nAltogether 6 different types of parasites were \n\ndetected. Infection rate of Giardia lamblia were \n\nhighest 5.1 % (10/196) whereas lowest was \n\nCryptosporidium parvum 1.0 % (2/196) [Figure 2]. \n\n \n\nPhotograph 1: Oocyst of Cyclospora cayetanensis (Ziehl- \nNeelsen, 100X)  \n\nDiscussion  \nPrevalence of C. cayetanensis infections among \n\n\u226415 year outpatient diarrheal children in KCH, \n\nKathmandu, Nepal was conducted in this study. \n\nIntestinal parasitosis is detected to be highly \n\nprevalent in Nepal, small developing country \n\nlocated   in south Asia [9].    Infection rate of C. \n\ncayetanensis and other parasites was higher \n\nbetween 11- 15 years old in diarrheal children. \n\nAltogether five species of parasites were detected \n\nin 13.7% of stool samples from \u2264 15 year old \n\ndiarrheal patients. Among them, most of \n\nprevalence in G. lamblia was detected.", "start_char_idx": 6705, "end_char_idx": 9394, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6446478d-6aad-4b3b-9f4f-01ab9e4ef507": {"__data__": {"id_": "6446478d-6aad-4b3b-9f4f-01ab9e4ef507", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4faf0923-dcb2-4b43-96dd-91dd0168126b", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "ad88710f1d4b4e2b03fde30853236ccd3ad22c25d0dba2d83d03c35bfa933bf1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "612a5f91-8cdc-449f-9de2-293defbd3445", "node_type": "1", "metadata": {}, "hash": "70db66f27f28c3db7cb267e15b82267788c44b578130065d2d6181818268a9da", "class_name": "RelatedNodeInfo"}}, "text": "Among them, most of \n\nprevalence in G. lamblia was detected.  In Nepal, \n\nC. cayetanensis was detected in 4.8% of patients. \n\nIn previous study, 7.9 % were found to be \n\npositive for C. cayetanensis in diarrheal Children \n\nin Nepal [10].   \n\nIn this study, we could not detect significant \n\ndifferences in prevalence of C. cayetanensis among \n\n< 15 year age groups and in gender. In gender, \n\nprevalence of C. cayetanensis 4.5% in male was \n\nhigher than in female 3.6%. In previous study, \n\nprevalence of C. cayetanensis in female (53.4%) \n\nwas higher than male (46.6%) in Nepal [10]. Total \n\nof the 60 patients infected with Cyclospora \n\nidentified in this study, 63.4% were male and \n\n36.6% were female in Mexico [11]. \n\nIn age, prevalence of C. cayetanensis in 5.7 % 11-\n\n15 years were highest. In previous study, the  \n\nmost of  the infection rate  of C. cayetanensis \n\ninfection  (50.7 %) was found between 2-5 year \n\nold of age group whereas the lowest prevalence \n\n(4.1%) were above 28 years of age in Nepal [10].  \n\nCyclospora was most frequently identified in \n\nboys of school age (36.7%) in Mexico [11]. The \n\nhighest attack rates occur among children older \n\nthan 18 months where as in our study, however,  \n\nall age groups may acquire the disease,  the \n\nhighest attack rates was detected  among \n\nchildren age between 2 to 5 years [12, 13]. There \n\nis no apparent immunity to infection, and \n\nreinfection can occur at all ages [14, 15]. \n\nPrevalence of C. cayetanensis was most in the \n\nmonth of August. The higher distribution of \n\nCyclospora infection in Nepal occurs during \n\nsummer and rainy season [10]. Their infections \n\nwere relevantly identified in the rainy season \n\n(June\u2013August) in children of Mexico [11]. \n\nCyclosporiasis occurs with high incidence during \n\nthe rainy seasons from April to June in Peru and \n\nMay to September in Nepal [1, 2]. \n\nIn Kanti Children\u2019s Hospital, C. cayetanensis \n\ninfection with diarrheal illness patients is not \n\nroutinely diagnosed. Thus, adequate diagnosis \n\nand treatment are not always conducted \n\npromptly. In addition, Cyclospora makes it \n\ndifficult to include its differential diagnosis due \n\nto lack of epidemiologic information. Therefore, \n\nour study suggests that there is need of highly \n\nspecific assays to diagnose Cyclospora infections. \n\nConclusions \nPrevalence of C. cayetanensis is highly probable in \n\ninfant, neonate, toddler, and children and is one \n\nof the important etiologic agents of diarrheal \n\nillness among children.  It is also equally \n\nprobable infection in male and female as well as \n\nin rainy season. Therefore, attention should be \n\nmade for laboratory investigation of C. \n\ncayetanensis while suspecting the diarrheal \n\npatients infected with other parasites.    \n\n   \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 16-19                                  Bhattachan et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal      19                                       Nepjol.info/index.php/njb \n\n \n\nAuthor Contributions  \nBB and GR design the proposal and format of \n\nresearch. BB collects the stool sample from \n\nhospital and proceeding in Lab of TUTH. JBS and \n\nBB and NNM write the article. All authors \n\nrevised and finalized the draft. \n\nAcknowledgements  \nThe authors would express sincere thanks to Mrs. \n\nSarmila Tandukar Mr. Kalyan Subedi, Ms. \n\nAnisha Shrestha.  We are indebted to hospital\u2019s \n\nstaffs and guardian of children who support \n\ncontinually.", "start_char_idx": 9334, "end_char_idx": 12810, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "612a5f91-8cdc-449f-9de2-293defbd3445": {"__data__": {"id_": "612a5f91-8cdc-449f-9de2-293defbd3445", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6446478d-6aad-4b3b-9f4f-01ab9e4ef507", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "ef04323951b6bb9f18a30f2a8aead6874ea29a634fbf1055be373367c88d3be9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9197ec7e-fb22-4d58-a378-76485bd3f955", "node_type": "1", "metadata": {}, "hash": "b68486f4fd7bae474cc7f3469b1234125b1888beb034ff137fd4d2bc0430261d", "class_name": "RelatedNodeInfo"}}, "text": "We are indebted to hospital\u2019s \n\nstaffs and guardian of children who support \n\ncontinually.   \n\nConflict of interest:  \nNone declared   \n\nReferences  \n1. Ortega YR, Roxas CR, Gilman RH, Miller NJ, \n\nCabrera L, Taquiri C, Sterling CR: Isolation of \nCryptosporidium parvum and Cyclospora \ncayetanensis from vegetables collected in \nmarkets of an endemic region in Peru. \nAmerican Journal of Tropical Medicine and Hygiene. \n1997 57:683-686. \n\n2. Ortega YR., Sterling CR, Gilman RH, Cama VA, \nDiaz F.:  Cyclospora species\u2014a new protozoan \npathogen of humans. N Engl J Med. 1993 \n328:1308\u20131312. \n\n3. Soave R: Cyclospora: an overview. Clin Infect \nDis. 1996,23: 329-378. \n\n4. Hart AS, Ridinger MT, Soundarajan R, Peters CS, \nSwiatlo AL, Kocka FE: Novel organism \nassociated with diarrhoea in AIDS. Lancet. 1990 \n335: 69. \n\n5. Rabold JG, Hoge CW, Shlim DR: Cyclospora \noutbreak associated with chlorinated drinking \nwater (letter). Lancet. 1994 344: 1360- 1361.  \n\n6. Sherchand JB, Cross JH, Jimba M, Sherchand S, \nShrestha MP: Study of Cyclospora cayetanensis \nin health care facilities, sewage water and \ngreen leafy vegetables in Nepal. Southeast \nAsian Journal Trop Med Public Health. 1999; 30: \n58-63 \n\n7. Bhattachan B, Panta YB, Tiwari S, et al: Intestinal \nParasitic Infection Among School children In \n\nChitwan District Of Nepal. J Inst Med. 2015 \n37(2): 79-84. \n\n8. Garcia LS, Bruckner DA, Brewer TC, et al: \nTechniques for the recovery and identification \nof Cryptosporidium oocysts from stool \nspecimens. J Clin Microbiol. 1983; 18:185-190.  \n\n9. Rai SK, Hirai K, Abe A, et al: Intestinal \nparasitosis among school children in a rural \nhilly area of Dhading District, Nepal. Nepal \nMed Coll J. 2002 4: 54-58 \n\n10. Sherchand JB, Sherchand JB, Cross JH. An \nepidemiological study of Cyclospora \ncayetanensis in Nepalese people. J  Inst Med. \n\n2007  29 (1) :8\u201313. \n11. Orozco-Mosqueda GE, Mart\u0131\u00b4nez-Loya OA, and \n\nOrtega YR: Cyclospora cayetanensis in a \nPediatric Hospital in Morelia, Mexico. Am J \nTrop Med Hyg. 2014 91(3): 537\u2013540, \ndoi:10.4269/ajtmh.13-0535. \n\n12. Hoge CW, Shlim DR, Ghimire M, Rabold JG, \nPandey P, Walch A Rajah JG, Guadio P & \nEcheverria P: Placebo controlled trial of co-\ntrimoxazole for Cyclospora infection among \ntravelers and foreign residents in Nepal. \nLancet. 1995, 345: 691-693. \n\n 13. Schubach TM, Neves ES, Leite AC, Araujo \nAQC & de Mouta H: Cyclospora cayetaensis \nin an asymptomatic patient infected with \nHIV and HTLV- 1. Transaction of the Royal \nSociety of Tropical Medicine. 1997, 91: 175. \n\n 14. Connor BA & Shlim DR: Food borne \ntransmission of Cyclospora. Lancet.", "start_char_idx": 12720, "end_char_idx": 15328, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9197ec7e-fb22-4d58-a378-76485bd3f955": {"__data__": {"id_": "9197ec7e-fb22-4d58-a378-76485bd3f955", "embedding": null, "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-212", "node_type": "4", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "a4986feff6e6444ab1517262b6eec2806e4f8992cc28fb6e9cb108d1554de667", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "612a5f91-8cdc-449f-9de2-293defbd3445", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "fea0915e0e0178161a75602ec3fc8e00551d19aa83b19d5632e22fd25f2cba4f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "faf97926-e690-4b99-9971-952dee2d37f5", "node_type": "1", "metadata": {}, "hash": "121f85635f31fd7edb6671688762a480ddaf08a9efeb2527bfedd33e0737d2ec", "class_name": "RelatedNodeInfo"}}, "text": "Lancet. 1995 346: \n1634. \n\n 15. Caramello P, Brancale T, Forno B, Lucchini A, \nMacor A, Mazzucco G, Tettoni C & Ullio A: \nClinical and diagnostic aspects of travelers \ndiarrhea due to Cyclospora organisms. Journal \n\nof Travel Medicine. 1995 2: 232-234.", "start_char_idx": 15321, "end_char_idx": 15573, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "faf97926-e690-4b99-9971-952dee2d37f5": {"__data__": {"id_": "faf97926-e690-4b99-9971-952dee2d37f5", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9197ec7e-fb22-4d58-a378-76485bd3f955", "node_type": "1", "metadata": {"identifier": "njb-212", "author": "Bhattachan, Balkrishna; Rai, Ganesh; Munankarmi, Nabin Narayan; Sherchand, Jeevan Bahadur", "title": "Cyclospora cayetanensis: An Infestation among Diarrheal Children in Kathmandu Valley, Nepal", "date": "2019-01-15", "file": "njb-212.pdf"}, "hash": "6595f66505f63142b26d2136dc19ecd375335c632b2c8d4327e658e62e2c6021", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "298938c4-bbfa-4480-84fb-ae9feea85841", "node_type": "1", "metadata": {}, "hash": "6c936e3e05ca03b6aceb4e34cb5a95d2705a55d83e6c27ff8439c632d90d0461", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\n ORIGINAL RESEARCH ARTICLE \n \n\n \u00a9NJB, Biotechnology Society of Nepal                   1                                                Nepjol.info/index.php/njb \n\nComparative Antioxidant, Antimicrobial and Phytochemical \nAssesments of Leaves of Desmostachya  bipinnata  L. Stapf, \n\nHordeum vulgare L. and Drepanostachyum  falcatum (Nees) \nKeng f. \n\nPragya Nepal, Minu Singh, Amina Baniya, Sushma Singh, Hari Krishna Sainju, Rajani Shrestha \nDepartment of Biotechnology, Asian Institute of Technology and Management (AITM), Khumaltar, \n\nLalitpur, Nepal \n\nAbstract \nNepal is rich in varieties of religious plants. The locally used religious plants also carry medicinal \nimportance. Desmostachya  bipinnata L. Stapf, Hordeum  vulgare L. and Drepanostachyum  falcatum (Nees) \nKeng f. are three plants belonging to the family Poaceae having religious significance in different \npractices of Hinduism. They were also used as traditional medicines by our ancestors but nowadays \nthey are underutilized. In this research, our core objective was to validate the traditional assumption of \nuse of these plants in medicinal purposes by carrying out the assessments like antimicrobial \nassessment, antioxidative assessment and phytochemical assessment. Methanolic extracts produced \nfrom leaves of all three plants were examined for antimicrobial activities through agar well diffusion \nmethod. The same extracts were also assessed for determining their antioxidative potentials with the \nuse of DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical scavenging assay followed by qualitative \nphytochemical analysis and GCMS (Gas Chromatography Mass Spectroscopy). Most promising \nantimicrobial activity was shown by Desmostachya bipinnata L. against Salmonella typhimurium and \nStaphylococcus aureus, Drepanostachyum falcatum (Nees) Keng f. against Salmonella typhimurium and \nKlebsiella pneumoniae and Hordeum vulgare L. against Salmonella typhmurium and Staphylococcus aureus. \nThe antioxidant activity of the plant extracts were observed in descending order of Hordeum vulgare \nL.>Desmotachya bipinnata L. > Drepanostachyum falcatum (Nees) keng f. and phytochemical assessment \nof the extracts indicated the presence of alkaloids, glycosides, sterols, Triterpenes, Saponins, \nFlavonoids, Coumarins, Phlobatanin and reducing sugars. Through this project, we can clarify that the \nabove mentioned plants have bioactive compounds which contributed for the presence of \nantimicrobial and antioxidative property in the plants. \nKey words: Antimicrobial, Antioxidant, Desmostachya bipinnata L. Stapf, Drepanostachyum falcatum \n\n(Nees) Keng f., Phytochemical screening, Hordeum vulgare. \n\n*Corresponding Author \n\nEmail: Pragya.nepal07@gmail.com \n\nIntroduction \nReligions have created major linkage between \n\nhuman and nature by bounding human beings \n\nto several aspects of natural systems. All Hindu \n\nfamilies in Nepal and India \n\nperform pujas (religious rituals) on certain \n\noccasions. There is no religious ritual, which \n\ndoes not require plants and their products [1]. \n\nMost of these plant species also have important \n\nmedicinal value.", "start_char_idx": 47, "end_char_idx": 3297, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "298938c4-bbfa-4480-84fb-ae9feea85841": {"__data__": {"id_": "298938c4-bbfa-4480-84fb-ae9feea85841", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "faf97926-e690-4b99-9971-952dee2d37f5", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "13f6db5fab7d3857a1a298af5527be7dc27c90b7501ec04de8d6384a6d3ced26", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "304e1a47-fdea-4e51-9b14-22c9b2f02d70", "node_type": "1", "metadata": {}, "hash": "41cbf271f4948995e66b33202ad71afd64b39dae94fe5e3cd17aadea36cc3297", "class_name": "RelatedNodeInfo"}}, "text": "Most of these plant species also have important \n\nmedicinal value. For example; Ocimum \n\ntenuiflorum locally known as Tulasi is a holy \n\nplant having many religious significances and \n\non the other hand, it has been used as a \n\ntraditional medicine to cure common cold and \n\nflu. These kind of religious plants were \n\ninvolved by our ancestors in rituals to create a \n\nremark upon their importance as medicine or \n\nas a valuable product [2]. Now, we are \n\nunaware about the main reasons of use of these \n\nplants but we are still using them in religious \n\npurposes. Since, their uses have only been \n\nlimited to religious practices; we tried to \n\ndisclose the hidden medicinal importance of \n\nthese plants by conducting different \n\nassessments on them.  It is also reported that \n\nedible wild plants carrying religious \n\nimportance such as, Ocimum tenuiflorum and \n\nDesmostachya bipinnata are the important source \n\nof fibre, vitamins, minerals and other nutrients \n\nwhich are used for the therapy of various \n\ndiseases [3]. \n\nDesmostachya bipinnata L., belonging to family \n\nPoaceae is a perennial grass abundantly \n\ndistributed in India. This plant is an important \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   2                                                Nepjol.info/index.php/njb \n\ningredient while performing \u2018hom\u2019, which is a \n\nsacred practice in Hindu rituals. Its root stock \n\nand leaves are used in Indian traditional \n\nsystem of medicine. Many medicinal properties \n\nand use of Desmostachya bipinnata L. have been \n\nreported in reference literatures [4]. \n\nHordeum vulgare (Barley) is an annual grass \n\nfrom the Poaceae family and it is stated to be \n\none of the earliest known crops to human kind. \n\nBarley seedling\u2019s leaves are locally called as \n\nJamara and it is one of the most important \n\nplants in our greatest festival, Vijaya Dashami \n\nmeanwhile, it is also traditionally used \n\nextensively to cure jaundice and gall bladder\u2019s \n\nstones. Barley is consumed for curing digestive \n\ndisorders such as; diarrhoea, stomach pain and \n\ninflammatory bowel conditions. \n\nDrepanostachyum falcatum (Arundinaria falcata \n\nNees) is a common species of hill bamboo and \n\nis locally called as Nigalo. It is found in the \n\nhomesteads in an abundant amount. This plant \n\nis used as an effective soil stabilizer for farming \n\nin Nepal and is used in religious practices like; \n\nHindu marriage, house warming rituals etc.  \n\nMaterials and Methods \nPlant materials \nLeaves of Desmostachya bipinnata (Kush), \n\nHordeum vulgare L.(Jamara) and \n\nDrepanostachyum falcatum (Nees) Keng f. \n\n(Nigalo) were collected from Birgunj (located at \n\nParsa District, Narayani zone), Kupandole \n\n(Located at Lalitpur district, Bagmati zone) and \n\nBalaju (Located at Kathmandu district, Bagmati \n\nzone) respectively during October of 2016.  \n\nExtraction \nThe collected leaves were first washed then \n\ndried in hot air oven at 40\u00baC-70\u00baC followed by \n\ngrinding. Sample were dissolved in parts of \n\n80% methanol in 1:3 ratios and placed in hot \n\nwater bath shaker for 16 to 18 hours followed \n\nby filtration using muslin cloth [5]. The filtrate \n\nwas stored and the residue was re-extracted \n\nunder the same. The extracts obtained were \n\npooled and filtered. The combined methanol \n\nspecimen was evaporated to dryness using a \n\nvacuum rotary evaporator at 65\u00baC.", "start_char_idx": 3231, "end_char_idx": 6680, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "304e1a47-fdea-4e51-9b14-22c9b2f02d70": {"__data__": {"id_": "304e1a47-fdea-4e51-9b14-22c9b2f02d70", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "298938c4-bbfa-4480-84fb-ae9feea85841", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "6ac0b841936cc90598893588af636db524752cb699be8bbfcdcf6f668cc58a61", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e7eb4f38-4f23-46d2-b657-e9edfaef3a49", "node_type": "1", "metadata": {}, "hash": "7958a07b178ef7a6d1cdc8e084b5e04f979aaf7aa31640e90b08fd2b5a1fe9b4", "class_name": "RelatedNodeInfo"}}, "text": "GC-MS Analysis of Compounds present \nin plant extracts \nGas Chromatography-Mass Spectrometry (GC-\n\nMS) is a method, which combines the features \n\nof gas liquid chromatography and mass \n\nspectrometry to identify different substances \n\npresent in a test sample [6].The process of GC-\n\nMS was operated on GC-MS equipment i.e. \n\nThermo GC-TRACE ultra ver: 5.0 and Thermo \n\nMS DSQ II. Features of the GC-MS system \n\nwere; TR 5-MS capillary non-polar column \n\nhaving 35 Mts dimension and thickness of film \n\nused in the process was 0.35\u03bcm.Using Helium \n\nas the carrier gas, flow rate of the mobile phase \n\nwas set at 1.0 ml/min. In the gas- \n\nchromatography section, prior temperature \n\nprogramme of 45\u00b0C was elevated to 255\u00b0C at \n\n5\u00b0C/min and1 \u03bcl sample was injected. Samples \n\nwere first dissolved in chloroform to form a \n\nsolution and were finally run at a range of 50-\n\n655 m/z. The final results were compared by \n\nthe use of Wiley Spectral library search \n\nprogramme. \n\nPhytochemical Screenings \nQualitative phytochemical screening was \n\ncarried out by using following standard \n\nprotocols [7]. \n\nTest for basic alkaloids (Dragondroff's test): \n\n2 mg of extract was dissolved in 3 ml of 2 % \n\n(v/v) HCL .To the solution, Dragondroff's \n\nreagent was added thoroughly. Appearance of \n\norange brown precipitate indicated the \n\npresence of basic alkaloids. \n\nTest for glycosides: \n\n100\u00b5l of glacial acetic acid was added to about \n\n2 mg of extract, followed by addition of few \n\ndrops of 5% ferric chloride (FeCl3). To the \n\nsolution, few drop of H2SO4 was added until \n\nthe formation of blue colour, which then, \n\nconfirmed presence of glycosides. \n\nTest of Sterols and Triterpenes (Salkwoski's \n\ntest): \n\n2 mg of extract was dissolved in 1ml of \n\nChloroform. Later, 1 ml conc. H2SO4 was added \n\nto the solution. Eventually, appearance of \n\nbrown ring between junctions of two liquids \n\nhttp://www.tipdisease.com/2013/11/diarrhea-causes-symptoms-treatment.html\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   3                                                Nepjol.info/index.php/njb \n\nindicated the presence of sterols and the brown \n\nring with upper layer green indicated the \n\npresence of triterpenes. \n\nTest for Tannins and Polyphenol: \n\n2 mg extract was dissolved thoroughly in 1 ml \n\nof water. To the solution, 3 drops of 1% (w/v) \n\nferric chloride (FeCl3) was added. Appearance \n\nof blue black/ violet colour indicated the \n\npresence of Tannins and Polyphenols. \n\nTest for Saponins: \n\n2 mg extract was dissolved in 2 ml of water. \n\nThe solution was shaken vigorously for 30 \n\nminutes. Persistence of thick froth (about 1 cm) \n\nheight even after 30 minutes indicated the \n\npresence of saponins. \n\nTest for Flavonoids: \n\nTo 2 mg of extract, 5ml of 10% (v/v) \n\nAmmonium hydroxide (NH4OH) was added \n\nand to the solution, 1 ml of conc. H2SO4 was \n\nadded. Disappearance of yellow coloration \n\nindicated the presence of flavonoids. \n\nTest for Coumarins: \n\n2 mg extract was thoroughly dissolved in hot \n\nwater of 2ml. After cooling, the solutions were \n\ndivided into two test tubes.", "start_char_idx": 6683, "end_char_idx": 9867, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e7eb4f38-4f23-46d2-b657-e9edfaef3a49": {"__data__": {"id_": "e7eb4f38-4f23-46d2-b657-e9edfaef3a49", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "304e1a47-fdea-4e51-9b14-22c9b2f02d70", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "648a51a3b098d371d6a28c4cce3754f703953f58b569154b2f226fa47da52b36", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4e6d9f80-9bc1-48d9-b0e6-b2a61f8946c2", "node_type": "1", "metadata": {}, "hash": "e22f0fc799796b0f3eab37f6f544c8204232d4c4d140befb6b93f68ebba0ea4c", "class_name": "RelatedNodeInfo"}}, "text": "After cooling, the solutions were \n\ndivided into two test tubes.  To one of the test \n\ntubes, 10% (v/v) Ammonium hydroxide \n\n(NH4OH) was added until the solution became \n\nbasic. The other test tube was used as control. \n\nThe test tubes were kept under UV light. \n\nFluorescence indicated the presence of \n\nCoumarins. \n\nTest for Phlobatanin: \n\n1mg extract solution was boiled with 2ml of 1% \n\nHCL until the appearance of red precipitate \n\nwas visible, which then, indicated the presence \n\nof Phlobatanin. \n\nTest for reducing sugars (Fehling's test): \n\nAbout 1 mg of extract was mixed with 1 ml of \n\nwater.  To this solution, 1 ml of Fehling's \n\nreagent (1:1 mixture of Fehling's reagent A and \n\nB) was added. Appearance of red precipitate \n\nindicated the presence of Phlobatatin. \n\nAntimicrobial Screening \nAntimicrobial screening was conducted by \n\nAgar well diffusion method. Sample was \n\nprepared by adding 25 mg extract to 500\u00b5l of \n\n100% Dimethyl sulfoxide (DMSO) followed by \n\n10-15 minutes of vortexing. 200\u00b5l of \n\naforementioned DMSO solution was added to \n\n200\u00b5l of double distilled water and was termed \n\nas 'Pure solution'. Again, pure solution was \n\nadded to DMSO in 1:1 and 2:1 ratios. For \n\npositive control, antibiotics; Chloramphenicol, \n\nTetracycline and Cefoxitin were used whereas, \n\nImidazole prepared in DMSO (0.1g/ml) was \n\nused as antifungal agent. Similarly, for negative \n\ncontrol 100% DMSO was used. \n\nBacterial inoculums were prepared in Nutrient \n\nBroth by taking a loop full of pure culture of \n\nmicroorganism and incubating them at 37\u00baC for \n\n3 hours. Turbidity of the broth was compared \n\nto Mcfarland turbidity standard number 0.5. \n\nMicroorganisms used \n\nA total of seven reference microbial strains \n\n(three Gram negative bacteria; Escherichia coli \n\nATCC 25922, Klebsiella pneunomiae ATCC \n\n700603, Salmonella typhimurium  ATCC \n\n14028,one Gram positive bacteria; \n\nStaphylococcus aureus ATCC 25923 and four \n\nfungi; Fusarium spp., Trichoderma spp., \n\nAspergillus flavus , Aspergillus niger) were used \n\nas the test organisms for the anti-microbial \n\nscreening. \n\nAntibacterial Screening \nMHA (Mueller Hinton Agar) plates were \n\nswabbed with 3 hours broth culture using \n\ncotton swab. Wells of 6mm were bored using \n\nsterile borer and 50\u00b5l of extract solution was \n\npoured using micropipette. The plates were \n\nkept for half an hour for diffusion in room \n\ntemperature and incubated at 37\u00baC for 24 \n\nhours. Anti-bacterial activity of each extract \n\nwas expressed in terms of diameter of zone of \n\ninhibition (mm) produced by respective extract \n\nagainst microorganisms after incubation. The \n\nprocedure was done in replicate manner. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   4                                                Nepjol.info/index.php/njb \n\nAntifungal Screening \nFungal inoculums were prepared in PDB \n\n(Potato Dextrose Broth) by taking a loop full of \n\npure culture of microorganisms and incubating \n\nthem at 28\u00baC for 24 hours. Petri plates \n\ncontaining PDA (Potato Dextrose Agar) were \n\nswabbed with 24 hours broth culture using \n\ncotton swab.", "start_char_idx": 9803, "end_char_idx": 13004, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4e6d9f80-9bc1-48d9-b0e6-b2a61f8946c2": {"__data__": {"id_": "4e6d9f80-9bc1-48d9-b0e6-b2a61f8946c2", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e7eb4f38-4f23-46d2-b657-e9edfaef3a49", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "48ff220ebdde6f6b3519b011bf5123201a7483203337a5f15609316abf85181b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "099b76dc-e6a3-4ff5-af3f-80e5a55145bd", "node_type": "1", "metadata": {}, "hash": "05a3c471a43c5b1db2580200fa3f565bf0e9c106ea016852ded2f1472b8f2af6", "class_name": "RelatedNodeInfo"}}, "text": "Wells of 6mm were bored using \n\nsterile borer and 50\u00b5l of extract solution was \n\npoured in them using micropipette. The plates \n\nwere kept for half an hour at room temperature \n\nfor diffusion and incubated at 28\u00baC for \n\nincubation. The antifungal activity of each \n\nextract was expressed in terms of diameter of \n\nzone of inhibition (mm) produced by respective \n\nextract against fungus after incubation. \n\nAntioxidant assay \nThe antioxidant potentials of different extracts \n\nwere determined using DPPH radical \n\nscavenging assay. Dry extract was dissolved in \n\n1ml methanol at different concentrations \n\n(100\u00b5g/ml, 200\u00b5g/ml, 300\u00b5g/ml, 400\u00b5g/ml \n\nand 500\u00b5g/ml) and was added to 4ml of \n\n0.004% methanol solution of DPPH. After 30 \n\nminutes incubation at room temperature, the \n\nabsorbance was read against a blank at 517nm \n\nin spectrophotometer (Cary 60 UV-Vis) from \n\nAgilent technologies at NAST. Inhibition of free \n\nradical by DPPH in percent (%) was calculated \n\nby using equation as: \n\nI (%) = [(A blank-A sample)/A blank] \u00d7100, where A \n\nblank is the absorbance of control reaction \n\n(containing all reagents except the test \n\ncompound and A sample is the absorbance of the \n\ntest sample.  Extract concentration providing \n\n50% Inhibition (IC50) was calculated from the \n\ngraph plotted for inhibition percentage against \n\nextract concentration. \n\nPreparation of Ascorbic acid \n\nAscorbic acid solution of 100 \u00b5g/ml, 200 \n\n\u00b5g/ml, 300 \u00b5g/ml, 400 \u00b5g/ml and 500 \u00b5g/ml \n\nwere prepared in methanol. \n\nScreening of Anti-oxidant activity \n\n1000 \u00b5l of extracts solutions in methanol (100 \n\n\u00b5g/ml, 200 \u00b5g/ml, 300 \u00b5g/ml, 400 \u00b5g/ml and \n\n500 \u00b5g/ml) was added to 4ml of 0.004% \n\nmethanol solution of DPPH. After 30 minutes \n\nincubation at room temperature, the \n\nabsorbance was read against a blank at 517nm \n\nin spectrophotometer (Cary 60 UV-Vis) from \n\nAgilent technologies. Inhibition of free radical \n\nby DPPH in percent (%) was calculated by \n\nusing equation as: I (%) = [(Ablank - Asample)/A \n\nblank] \u00d7100, where Ablank is the absorbance of \n\ncontrol reaction (containing all reagents except \n\nthe test compound and Asample is the absorbance \n\nof the test sample.  Extract concentration \n\nproviding 50% Inhibition (IC50) was calculated \n\nfrom the graph plotted for inhibition \n\npercentage against extract concentration. \n\nStatistical Analysis \nData are expressed as means and statistical \n\nanalysis was performed with Microsoft excel \n\n2010 and IBM SPSS Statistics 23. Difference on \n\nstatistical analysis of data was considered \n\nsignificant at P < 0.05. \n\nResults \nAnalysis of GC-MS of Plant extracts \nThe graph generated by Gas Chromatography \n\nshowed composition of the extracts and the \n\ngraph showed by Mass Spectrophotometer \n\ngave the percentage of each component   \n\n(Figures 1, 2 and 3). The horizontal axis \n\nrepresented percentage area whereas the \n\nvertical axis represented the retention time of \n\nseveral components found in each extract. \n\nThere were variations in retention time and \n\npercentage area of the three different extracts. \n\nTotal of 10, 32 and 28 compounds were \n\nidentified through GC-MS from Desmostachya \n\nbipinnata L., Hordeum vulgare L. and \n\nDrepanostachyum falcatum (Nees) Keng f. \n\nrespectively (Figures 1, 2 and 3).", "start_char_idx": 13005, "end_char_idx": 16258, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "099b76dc-e6a3-4ff5-af3f-80e5a55145bd": {"__data__": {"id_": "099b76dc-e6a3-4ff5-af3f-80e5a55145bd", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4e6d9f80-9bc1-48d9-b0e6-b2a61f8946c2", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "40bdb827bf05c2089d6c4196a0596d9d728a80156cd1a9099ec9b28d6a1ed923", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f107e2c1-4e57-4d09-ab30-4d6744089b09", "node_type": "1", "metadata": {}, "hash": "fff0c702612a95a96e8fff216a1f638864ea9371c746e830ec50e1041bb67c1a", "class_name": "RelatedNodeInfo"}}, "text": "The major \n\ncompounds found in Desmostachya bipinnata L. \n\nwere 2,3 benzofuran dihydro and Octasiloxane, \n\n1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15,-\n\nhexacamethyl  (Table 1) and the major \n\ncompounds found in Drepanostachyum falcatum \n\n(Nees) Keng f. were Phenol 2, 6-dimethoxy, \n\nBenzoic acid and n-Hexadecanoic acid (Table \n\n1). Similarly, compounds like Quinoline, \n\nPthalic anhydride, 1, 2-Benzenedicarboxylic \n\nacid and Indole (Table 1) were identified in \n\nHordeum vulgare. L.. The compounds were \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   5                                                Nepjol.info/index.php/njb \n\nFigure 1: GC-MS graph for Drepanostachyum falcatum (Nees) Keng f \n\n \nFigure 2: GC-MS graph for Hordeum  vulgare  L. \n\n \nFigure 3: GC-MS graph for Desmostachya bipinnata L. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   6                                                Nepjol.info/index.php/njb \n\nTable 1: Major compounds present in three plants extracts observed through GC-MS. \n\nS.N Extract Name of compound Retention factor Peak area \n1 \n \n\nDrepanostachyum \nfalcatum (Nees) Keng \nf. \n\nPhenol, 2, 6-dimethoxy \n \n\n10.823 95 \n\nBenzoic acid, 4-ethoxy, ethyl ester 13.649 93 \n\nn-Hexadecanoic acid. 19.478 98 \n\n2 Hordeum vulgare L. Phthalic anhydride 10.372 95 \n1H-Indole, 4-methyl 11.325 94 \nQuinoline, 2-ethyl  14.624 81 \n\n3 Drepanostachyum  \nfalcatum (Nees) Keng \nf \n \n\nBenzofuran 2,3- dihydro 8.934 56 \n\nOctasiloxane, \n1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15,-\nhexacamethyl \n\n19.731 72 \n\n \n\nidentified with the database of National \n\nInstitute Standard and Technology. Majority of \n\nthe compounds extracted from all three extracts \n\nbelonged to hydrocarbon class. GC-MS results \n\nof Drepanostachyum falcatum (Nees) Keng f. \n\nshowed presence of wide range of compounds \n\nlike saturated fatty acids like n-Hexadecanoic \n\nacid, unsaturated cyclic chemical compound \n\nlike pyran-4-one, Aromatic organic compound \n\nlike Syringol and Indene, ranges of Aldehydes \n\nlike Vanillin and Benzaldehydes. \n\nSimilarly, the GC-MS result of Hordeum vulgare \n\nL. also showed the presence of Aromatic \n\nHeterocyclic organic compounds like Indole \n\nand Indolizine, Anhydrides of Thalic acid, \n\nheterocyclic aromatic organic compounds like; \n\nQuinoline and Pyrrole and other organic \n\ncompounds like;  Acetamide, Guanidine etc.", "start_char_idx": 16259, "end_char_idx": 18802, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f107e2c1-4e57-4d09-ab30-4d6744089b09": {"__data__": {"id_": "f107e2c1-4e57-4d09-ab30-4d6744089b09", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "099b76dc-e6a3-4ff5-af3f-80e5a55145bd", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "5f4b1499ee4890f77b2981abc8756749e854692eb0e8ccc323424da8768bd188", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c0d0427a-f4fb-4170-9818-c4232f4f44a0", "node_type": "1", "metadata": {}, "hash": "2feca48a9ef38601b6a6161734e20217d1844890624b380c78a7489d9fcf3053", "class_name": "RelatedNodeInfo"}}, "text": "Phytochemical Screening \nPhytochemical screening (Table 2) of \n\nDrepanostachyum  falcatum (Nees) Keng f. \n\nshowed presence of Alkaloids, Glycosides, \n\nSterols, Saponin, Coumarins and reducing \n\nsugar in good amount and Triterpenes in fair \n\namount. Whereas, Hordeum   vulgare  L. extract \n\ncontained phytochemicals like Alkaloid, \n\nSterols, Saponins, flavonoids, Coumarins, \n\nPhlobatanin and reducing sugar in fair amount \n\n(Table 2). Similarly, Desmostachya bipinnata L. \n\nextract showed presence of Glycosides and \n\nFlavonoids in fair amount. From Table 2, \n\npresence of various phytochemicals in various \n\namounts in the three plant extracts could be \n\nobserved. Drepanostachyum falcatum (Nees) \n\nKeng f. showed presence of most of the \n\nphytochemicals in comparatively good amount \n\nthan Hordeum vulgare L. and Drepanostachyum \n\nfalcatum (Nees) Keng f. \n\nAntibacterial assay \n The antibacterial test showed a very promising \n\nresult as all the extracts showed zone of \n\ninhibition against both gram positive and gram \n\nnegative bacteria which was significantly \n\ncomparable to the action of antibiotics against\n\nTable 2: Phytochemical assessment of all three plant extracts \n\nS.N Tests for Phytochemicals \nPlant extracts \n\nDesmostachya bipinnata L. Hordeum vulgare L. Drepanostachyum \nfalcatum (Nees) Keng f \n\n1. Basic Alkaloids + ++ +++ \n2. Glycosides  ++ - +++ \n3. Sterols + ++ +++ \n\n4. Triterpenes + - ++ \n5. Tannis and polyphenols - - + \n6. Saponins + ++ +++ \n7. Flavonoids ++ ++ - \n8. Coumarins + ++ +++ \n9. Phlobatanin  - ++ - \n10. Reducing sugar + ++ +++ \n\nIndex: (+++) strongly positive, (++) medium positive, (+) weak positive and (-) negative \n\nhttps://en.wikipedia.org/wiki/Saturation_(chemistry)\nhttps://en.wikipedia.org/wiki/Heterocyclic\nhttps://en.wikipedia.org/wiki/Aromaticity\nhttps://en.wikipedia.org/wiki/Organic_compound\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   7                                                Nepjol.info/index.php/njb \n\nFigure 4: ZOI shown by various concentrations \nof the three extracts against Salmonella \ntyphimurium \n\n \nFigure 5:  ZOI shown by various concentrations \nof the three extracts against Staphylococcus \naureus. \n\n \nFigure 6:  ZOI shown by various concentrations \nof the three extracts against Klebsiella \npneumoniae. \n\nbacteria. Most promising antimicrobial activity \n\nwas shown by Desmostachya bipinnata L with \n\nzone of inhibition of 21.9mm against Salmonella \n\ntyphimurium and 19.65 mm against S. aureus \n\n(Figure 4 and 5). Hordeum vulgare showed zone \n\nof inhibition of 20.5 mm L. against Salmonella \n\ntyphi and of 24.45 mm against S. aureus (Figure \n\n4 and 5). Drepanostachyum falcatum (Nees) Keng  \n\n \nFigure 7: ZOI shown by various concentrations \n\nof the three extracts against E. coli.  \n\n \nFigure 8: ZOI shown by Antibiotics against \n\nbacteria. \n\nf. showed zone of inhibition of 22.85 mm \n\nagainst Salmonella typhi and 22.5 mm against \n\nklebsiella  pneumonia  (Figure 4 and 6).", "start_char_idx": 18805, "end_char_idx": 21861, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c0d0427a-f4fb-4170-9818-c4232f4f44a0": {"__data__": {"id_": "c0d0427a-f4fb-4170-9818-c4232f4f44a0", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f107e2c1-4e57-4d09-ab30-4d6744089b09", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "339986f2b1ecd4af05551b5be18a35bcb887652767f2e14ddd76ad911dea9b39", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2adf60c0-8ab1-4765-9226-4f197276f436", "node_type": "1", "metadata": {}, "hash": "46eb25659bf4744a43620d65df8c7903af5c169ce13ecf4512845cff0094d0bf", "class_name": "RelatedNodeInfo"}}, "text": "Hordeum \nvulgare showed zone of inhibition of 22.5 mm \n\nagainst E. coli (Figure 7).  The zone of \n\ninhibitions shown by the extracts solution was \n\ncompared to the zone inhibition showed by the \n\nantibiotics (Figure 8) Although, all three plants \n\nbelonged to the same family Poaceae, the \n\npresence of biomolecules in each plant varied \n\nfrom another yet, all three plants showed a \n\nappreciable result against both gram-positive \n\nbacteria and gram negative bacteria.  \n\nAntifungal assay \nIn the case of Antifungal assessment, widest \n\nzone of inhibition of 15mm was shown by \n\nsample 2:1 of Hordeum vulgare against \n\nTrichoderma spp. (Figure 9) and pure sample of \n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\n30\n\npure 2:01 1:01\n\nZ\no\n\nn\ne\n\n o\nf \n\nIn\nh\n\nib\nit\n\nio\nn\n\n(i\nn\n\n m\nm\n\n) \n\nPlant extracts used in different \u2026 \n\nH.vulgare\nD.bipinnata\nD.falcatum\n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\nPure 2:01 1:01Z\no\n\nn\ne\n\n o\nf \n\nIn\nh\n\nib\nit\n\nio\nn\n\n (\nin\n\n m\nm\n\n) \n\nPlant extracts used in different \nconcentrations \n\nH.vulgare\n\nD.bipinnata\n\nD.falcatum\n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\npure 2:01 1:01\n\nZ\no\n\nn\ne\n\n o\nf \n\nIn\nh\n\nib\nit\n\nio\nn\n\n(i\nn\n\nm\nm\n\n) \n\nPlant extracts used in different \nconcentrations \n\nH.vulgare\nD.bipinnata\nD.falcatum\n\n0\n5\n\n10\n15\n20\n25\n30\n35\n40\n45\n\nZ\no\n\nn\ne\n\n o\nf \n\nin\nh\n\nib\nit\n\nio\nn\n\n (\nin\n\nm\nm\n\n) \n\nBacteria used \n\nchloraphenicol\nCefoxitin\nTetracycline\n\nB \nC \n\nD \n\nA \n\nE \n\nB \nA \n\nD \n\nC \n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\nPure 2:01 1:01\n\nZ\no\n\nn\ne\n\n o\nf \n\nin\nh\n\nib\nit\n\nio\nn\n\n (\nin\n\nm\nm\n\n) \n\n \nPlant extracts used in various \u2026 \n\nH.vulgare\n\nD.bipinnata\n\nD.falcatum", "start_char_idx": 21862, "end_char_idx": 23378, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2adf60c0-8ab1-4765-9226-4f197276f436": {"__data__": {"id_": "2adf60c0-8ab1-4765-9226-4f197276f436", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c0d0427a-f4fb-4170-9818-c4232f4f44a0", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "621ff7353993fa3b083601487cc5342a6ac6ff974c3f4f057c7d4abf061473e8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d8bc12a0-e2d8-40c1-96a5-f6e1042719dd", "node_type": "1", "metadata": {}, "hash": "519c7e9438a08fefa81583862b4b8b0fba47788f4669a8e11d0603bb067e14fb", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   8                                                Nepjol.info/index.php/njb \n\nDrepanostachyum falcatum against Fusarium spp. \n\n(Figure 10). \n\n \nFigure 9: ZOI shown by Hordeum vulgare \nagainst fungus \n\n \nFigure 10: ZOI shown by Drepanostachyum \nfalcatum against fungus \n\nDPPH radical Scavenging assay \nTable 3: IC50 values of Desmostachya bipinnata L., \nHordeum  vulgare  L. and Drepanostachyum  \nfalcatum  (Nees) Keng f. \n\nS.N Plant extracts IC50 \n\n1. Desmostachya  bipinnata L. 143.36 \n\n2. Hordeum  vulgare  L. 135.625 \n\n3. Drepanostachyum  falcatum \n(Nees) Keng f \n\n590 \n\n4. Abscorbic Acid 86.20 \n\nIn the DPPH method, the antiradical activity \n\nwas evaluated by the capacity of antioxidant \n\ncompound to reduce the DPPH radical as \n\nindicated by the decrease in its absorbance at \n\n517 nm until the reaction reached a plateau. \n\nThe antiradical activity was defined as the \n\namount of antioxidant necessary to decrease \n\nthe initial DPPH concentration by 50% [8].  The \n\nIC50 values of all the plant extracts and ascorbic \n\nacid (Table 3) illustrates that the antioxidant \n\nactivity of the three plants extracts are in the \n\ndescending order of Hordeum vulgare \n\nL.>Desmotachya bipinnata L. > Drepanostachyum \n\nfalcatum (Nees) keng f. DPPH radical \n\nscavenging assay of the three plants are \n\ngraphically represented from figure 11-14. \n\nHordeum vulgare L. and   Desmotachya bipinnata \n\nL. were able to inhibit 50% concentration of \n\nDPPH radicals at 135.625\u00b5g/ml and \n\n143.36\u00b5g/ml (Table 3) against, IC50 of the \n\nascorbic acid's which was at 86.20\u00b5g/ml (Table \n\n3). \n\n \nFigure 11: DPPH radical Scavenging assay of \nDesmostachya bipinnata L. \n\nFigure 12: DPPH radical Scavenging assay of \nHordeum vulgare L. \n\n \nFigure 13: DPPH radical Scavenging assay of \nDrepanostachyum falcatum (Nees) Keng f. \n\n0\n\n5\n\n10\n\n15\n\n20\n\npure\n\n2:01\n\nC(+)\n\nC(-)\n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\npure\n\n2:01\n\nC(+)\n\nC(-)\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\n100 200 300 400 500\n%\n\n I\nn\n\nh\nib\n\nit\nio\n\nn\n \n\nConcentration of extract(\u00b5g/ml) \n\n0\n\n10\n\n20\n\n30\n\n40\n\n50\n\n100 200 300 400 500\n\n%\n I\n\nn\nh\n\nib\nit\n\nio\nn\n\n \n\nconcentration of extract(\u00b5g/ml) \n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n100 200 300 400 500\n\n%\n i\n\nn\nh\n\nib\nit\n\nio\nn\n\n \n\nConcentration of Abscorbic acid(\u00b5g/ml) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   9                                                Nepjol.info/index.php/njb \n\n \nFigure 14: DPPH radical Scavenging assay of \nAscorbic acid.", "start_char_idx": 23382, "end_char_idx": 26039, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d8bc12a0-e2d8-40c1-96a5-f6e1042719dd": {"__data__": {"id_": "d8bc12a0-e2d8-40c1-96a5-f6e1042719dd", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2adf60c0-8ab1-4765-9226-4f197276f436", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "f311e3b70b442e2dbf917893f94b071549d39b819009eee5ea6c435063ffadda", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3bc0ccc2-abf8-48b6-b396-b0ece1e09b4d", "node_type": "1", "metadata": {}, "hash": "187d445e34c3ef602f2e72de498077abe32d906797d5b69b46628c129321f820", "class_name": "RelatedNodeInfo"}}, "text": "Discussion \nThe benzoic acid present in Drepanostachyum   \n\nfalcatum (Nees) Keng f.is a key component in \n\nprevention of infection caused by several \n\nbacteria. Compounds present in Desmostachya \n\nbipinnata L. such as, 4-hydroxynorephedrine is \n\nan active sympathomimetic metabolite \n\nof amphetamine in humans [9]. Amphetamine \n\nis a  potent central nervous \n\nsystem (CNS) stimulant which have been used \n\nfor treatment of attention deficit hyperactivity \n\ndisorder (ADHD) [10].  \n\nUnlike our test sample, the result on essential \n\noil of   GC-MS of Desmostachya bipinnata L., \n\nconducted by Kumar & Patel [10] showed the \n\npresence of 16 compounds with the similarity \n\nvalues ranging from 97% to 100%  whereas, in \n\nour project only 10 compounds with much \n\nlesser similarity values (approx. 60%) were \n\nobserved. The reasons for these differences \n\nmight be due to several variations in samples \n\n.As, our solution was in crude form whereas \n\nthe other one was the essential oil. There is also \n\na difference in extraction procedures and it \n\nmay also be due to the differences in the \n\ngeographical inheritance of the plants.  \n\nAs a major class of natural products, alkaloids \n\nhave been used in various parts of the world as \n\na source of remedies to treat a wide variety of \n\nillnesses yet they are underrepresented in the \n\ncontext of newly introduced medicines [11]. \n\nWhereas, another group of glycosides i.e. \n\nSaponins are used widely for their effects \n\non ammonia emissions in animal feeding and \n\nSaponins have also been used \n\nas adjuvants in vaccines e.g. Quil A; \n\ncomponent QS-21, isolated from the bark \n\nof Quillaja saponaria Molina, to stimulate both \n\nthe Th1 immune response and the production \n\nof cytotoxic T-lymphocytes (CTLs) against \n\nexogenous antigens [12].Phytochemical \n\nanalysis showed presence of Coumarin in all \n\nthree plant extracts where, Drepanostachyum  \n\nfalcatum (Nees) Keng f. showed strongly \n\npositive result.  \n\nIn a research done by Kumar [13] for \n\nantimicrobial assay  in essential oil of \n\nDesmostachya bipinnata ,the diameter of the \n\ninhibition zone of oil of Desmostachya  bipinnata \n\nvaried from 15.2 to 56.2 mm where the largest \n\nzone of inhibition was obtained for S. \n\nepidermidis (56.2mm) and lowest for S. aureus \n\n(33.9mm) which is opposite to ours and the \n\nreason behind this could be dissimilarities in \n\npresence of phytochemicals due to  condition \n\nfrom where the plant had originated including \n\nthe difference in the temperature, humidity and \n\nother environmental stresses. Madineni [14] \n\nfound that Thaumatin like proteins was \n\nextracted from barley have antimicrobial \n\nactivity against Candida albicans, Bacillus subtilis, \n\nE. coli, Saccharomyces cerevisiae. Those proteins \n\ncould be present in our   samples too as we had \n\ngood result upon E.coli. Antimicrobial assay \n\nverified that the plants rich in phytochemicals \n\nlike saponins, coumarins, sterols, alkaloids and \n\nreducing sugars showed more promising \n\nresults. \n\nFrom this research, we figured out Antifungal \n\nproperties of these plant extracts aren\u2019t as \n\npromising as Antibacterial properties. \n\n.Desmostachya bipinnata L. didn't show zone of \n\ninhibition against Aspergillus  flavus and \n\nAspergillus niger at all , although different \n\nconcentration of plant extracts showed zone of \n\ninhibition against different fungus (table: 2).  \n\nOur research has also focused on the use of \n\nantioxidants, particularly, on the importance of \n\nnaturally derived antioxidants, which may \n\nreduce free radicals, ROS production and may \n\nexhibit defensive property.", "start_char_idx": 26042, "end_char_idx": 29637, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3bc0ccc2-abf8-48b6-b396-b0ece1e09b4d": {"__data__": {"id_": "3bc0ccc2-abf8-48b6-b396-b0ece1e09b4d", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d8bc12a0-e2d8-40c1-96a5-f6e1042719dd", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "9a582fe7114a87a6b7a2efef79d89e3ae22225b1645ff37bbc78e3c9743d8fa3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c54bd289-f113-4dbf-94bc-242fbf798cf8", "node_type": "1", "metadata": {}, "hash": "c5a27f46badb05eb9ba6f30f68f47580f08fe14a06bb647a3aed1350117ac206", "class_name": "RelatedNodeInfo"}}, "text": "The antioxidant \n\nactivity of the three extracts were in the order \n\nof Hordeum vulgare L.>Desmotachya bipinnata L. \n\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\n\n100 200 300 400 500\n\n%\n I\n\nn\nh\n\nib\nit\n\nio\nn\n\n \n\nConcentration of extract(\u00b5g/ml) \n\nhttps://en.wikipedia.org/wiki/Potency_(pharmacology)\nhttps://en.wikipedia.org/wiki/Central_nervous_system\nhttps://en.wikipedia.org/wiki/Central_nervous_system\nhttps://en.wikipedia.org/wiki/Stimulant\nhttps://en.wikipedia.org/wiki/Attention_deficit_hyperactivity_disorder\nhttps://en.wikipedia.org/wiki/Attention_deficit_hyperactivity_disorder\nhttps://en.wikipedia.org/wiki/Adjuvant\nhttps://en.wikipedia.org/wiki/Vaccine\nhttps://en.wikipedia.org/wiki/Quil_A\nhttps://en.wikipedia.org/wiki/QS-21\nhttps://en.wikipedia.org/wiki/Quillaja_saponaria\nhttps://en.wikipedia.org/wiki/Cytotoxic_T-lymphocytes\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 1-10    Nepal et al. \n\n \n\n \n\n \u00a9NJB, Biotechnology Society of Nepal                   10                                                Nepjol.info/index.php/njb \n\n> Drepanostachyum falcatum (Nees) keng f. The \n\nantioxidative effect was mainly due to phenolic \n\ncomponents, such as phenolic acids and \n\nphenolic diterpenes [15]. Phenolic compounds \n\nhave been proved to be responsible for the \n\nantioxidant activity in many medicinal plants. \n\nConclusion \nThe importance of religious plants is still \n\nprevailed in our society although scientific \n\nvalidation on use of these plants has not been \n\ndone much. There was an urge to identify the \n\nbio-active compounds present in those plants \n\nfor which we planned to conduct this project \n\non religious plants. \n\nDifferent bioactive compounds such as \n\nalkaloids, saponins, steroids, reducing sugars \n\nand glycosides were present in the plants. Plant \n\nwhich showed most promising result in \n\nphytochemical screening is Drepanostachyum \n\nfalcatum (Nees) Keng f. Similarly, these plants \n\nalso showed significant antioxidant property \n\nthrough DPPH radical scavenging assay. Since, \n\nthe plants were rich in various phytochemicals; \n\nthey had a moderate to significant antibacterial \n\nproperty but they showed quite weaker \n\nantifungal property which may be due to \n\nabsence of some other bioactive compounds \n\nwhich were required to inhibit growth or kill \n\nthe fungus. It can be concluded that the \n\nreligious plants that we use in our daily life \n\nrequires scientific validation for being use \n\nwhich can only done with research. These \n\nplants shouldn't only be limited to its religious \n\npractices, instead it could be used in \n\nmultidisciplinary approaches. As we have \n\nshown in this project, the plants of religious \n\nvalue have important phytochemicals, \n\nantioxidative capacity and antimicrobial \n\nproperty, these plants could be also used in \n\nmedicinal and therapeutic fields \n\nReferences \n1. Niroula G: Religion and Conservation: A \n\nReview of Use and Protection of Sacred. \nJournal of Institute of Science and Technology, \n2015, 20(2): 61-66, IOST, Tribhuvan University. \n\n2. Sapkota PP: Religious Culture and Medicinal \nPlants: An Anthropological Study. Dhaulagiri \nJournal of Sociology and Anthropology. 2013 7:197. \n\n3. Dandapat S, Kumar M, Kumar A, Sinha MP: \nTherapeutic efficacy and nutritional \npotentiality of indian bay leaf (cinnamomum \ntamala buch. - hem.). Intnl J Pharm. (2013), 6. \n\n4.", "start_char_idx": 29639, "end_char_idx": 32990, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c54bd289-f113-4dbf-94bc-242fbf798cf8": {"__data__": {"id_": "c54bd289-f113-4dbf-94bc-242fbf798cf8", "embedding": null, "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-213", "node_type": "4", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "d621675507c8d3a278eef290098fb884068563e8eb53aeb3e1dd361f69b5279a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3bc0ccc2-abf8-48b6-b396-b0ece1e09b4d", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "edfb5ab7f412e404c5683e58dd06d1147fc0c1d0e38cf5e02334356a583a3830", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "66d2b0bc-afc1-446b-b100-58f3fa5f46fd", "node_type": "1", "metadata": {}, "hash": "f9146f5b2dfc9e1a640565c4021ad05fda0e77b964a5103315baf907ad7b48db", "class_name": "RelatedNodeInfo"}}, "text": "Intnl J Pharm. (2013), 6. \n\n4. Santhanam R, Okoro CK, Rong X, Huang Y, \nBull AT, Andrews BA, Asenjo JA, Weon HY, \nGoodfellow M: Streptomyces deserti sp. Nov., \nisolated from hyper-arid Atacama desert soil. \nAntonie Van Leeuwenhowk, 2012  8 \n\n5. Chang-Geun K, Dae-Sik H, Young-Hwan K, \nEuikyung K, Jong-Shu K: Evaluation of \nAntimicrobial Activity of the Methanol \nExtracts from 8 Traditional Medicinal Plants. \nToxicol Res. 2011 27(1)31-32. \n\n6. kumar J, Kamajaj M, Nandagopalan V, \nAnburaja, V., & Thiruvengadam, M. A Study \nof Phytochemical Constituents in Caralluma \n\nUmbellata. International Journal of \nPharmaceutical Science Invention 2013 37. \n\n7. Chhetri H, Yogol NS, Sherchan J, KC A, \nMansoor S, & Thapa P: Phytochemical and \nAntimicrobial Evaluations of some Medicinal \nPlants of Nepal. Kathmandu University Journal \nof Science, Engineering and Technology. \n2008 4(1):49-54. \nhttps://doi.org/10.3126/kuset.v4i1.2883. \n\n8. Lahouar L. Phytochemical content and \nantioxidant properties of diverse varieties. \nFood Chem. 2014 581. \n\n9. Santagati NA, Marrazzo A, Ronsisvalle G: \nSimultaneous determination of amphetamine \nand one of its metabolites by HPLC with \n\nelectrochemical detection. J Pharm Biomed \nAnal. 2002 30(2):247-55. \n\n10. Kumar A and Patel J: Chemical composition \nand antimicrobial activity of the essential oil \n\nof Coriandrum sativum. International Journal of \nPhytomedicine. 2010 436-439. \n\n11. Vafa Amirkia MH: Alkaloids as drug leads - A \npredictive structural and biodiversity-based \n\nAnalysis. Elsevier Phytochemistry letters, 2014 5-\n6. \n\n12. Patrick H Demana, C. F. Effect of \nincorporation of the adjuvant Quil A on \nstructure and immune stimulatory capacity of \nliposomes. Immuno.  Cell Biol., 2004 5. \n\n13. Kumar AK, Sharvanee S, Patel J, Choudhary \nRK: Chemical composition and antimicrobial \nactivity of the essential oil of Desmostachya \nbipinnata Linn. Int J Phytomed. 2010 4. \n\n14. Jebor MA: Characterization and antimicrobial \nactivity of barley grain. Int J curr microbiol Appl \nSci 2013. 47. \n\n15. Fereidoon SJP: Phenolic antioxidants CRC \nCritical Rev. Food Sci Nutr. 1992. 2:67-103.", "start_char_idx": 32960, "end_char_idx": 35087, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "66d2b0bc-afc1-446b-b100-58f3fa5f46fd": {"__data__": {"id_": "66d2b0bc-afc1-446b-b100-58f3fa5f46fd", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c54bd289-f113-4dbf-94bc-242fbf798cf8", "node_type": "1", "metadata": {"identifier": "njb-213", "author": "Nepal, Pragya; Singh, Minu; Baniya, Amina; Singh, Sushma; Sainju, Hari Krishna; Shrestha, Rajani", "title": "Comparative Antioxidant, Antimicrobial and Phytochemical Assesments of Leaves of Desmostachya bipinnata L. Stapf, Hordeum vulgare L. and Drepanostachyum falcatum (Nees) Keng f.", "date": "2019-01-15", "file": "njb-213.pdf"}, "hash": "785310a7dbe636e836c09a895cb306492a7d532f04142203f2ca7e310f7112fc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5362892d-159c-43cf-a368-2f283f711c3f", "node_type": "1", "metadata": {}, "hash": "da53b28dca9b3cb4c4f5ba9cae5c03fb9565efd48972a6ca0d529b6108315abe", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1: 11-15                                   ISSN 2091-1130 (Print)/ISSN 2467-9319 (online) \n\n ORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal                              11                    Nepjol.info/index.php/njb \n \n\nComparative Study of Growth Statistics of Two Species of \nPaulownia and Optimization of Rooting Methods \n\nHari Krishna Saiju*, Abhishesh Bajracharya, Brishav Rajbahak, Stuti Ghimire \n\nDepartment of Biotechnology, Asian Institute of Technology and Management (AITM) Lalitpur, Nepal \n\nAbstract \nPaulownia is a fast-growing woody tree, native to the forests of China. It belongs to the family \n\nScrophulariaceae and is mainly used as a source of wood for furniture and musical instruments. \n\nDue to its fast-growing nature and high-quality of wood, there has been growing interest in \n\ncultivation and research of Paulownia in Nepal. Growth comparison was performed by measuring \n\nshoot length in in vitro condition. Among two species of Paulownia - Paulownia tomentosa (Thunb.) \n\nSteud and Paulownia fortuneii (Seem.) Hemsl., the growth rate of P. tomentosa was found to be 0.355 \n\ncm/week while that of P. fortuneii was found to be 0.637 cm/week in in-vitro conditions in MS \n\nmedium supplemented with  0.1 mg/l NAA and 1mg/l BAP. Optimization of rooting methods was \n\nalso performed, in which, sand rooting was found to be easier and more effective than in-vitro \n\nrooting. Dipping the plantlets in 1 mg/l of NAA was found to produce longer and denser roots \n\nthan lower or higher concentrations during sand rooting. \n\nKeywords: Paulownia tomentosa, Paulownia fortuneii, growth comparison, in-vitro rooting, sand-\nrooting, nodal culture \n*Corresponding Author \n\nEmail: hk.saiju@aitm.edu.np \n\nIntroduction \nPaulownia is a fast-growing, woody tree native to \n\nthe forests of China [1]. It is a deciduous tree but \n\nbecomes evergreen in the tropics [2].  It belongs to \n\nthe family Scrophulariaceae and is majorly used as \n\na source of wood for furniture and musical \n\ninstruments [3]. It has a very low thermal \n\nconductivity making it ideal for construction of \n\ninsulative structures [4]. Each tree can produce 44 \n\ncubic feet of wood in average and can be harvested \n\nafter 8-10 years of plantation. It is also tolerant to \n\npollutants and can grow in many types of soils. Its \n\nleaves and flowers show medicinal properties and \n\ncan also be used as fodder and fertilizers due to \n\ntheir high nitrogen concentration [5]. Its tolerance to \n\ndrought and soil extremes makes it commercially \n\nimportant for use in the reclamation of surface-\n\nmined land [6]. It is also a suitable raw material for \n\npyrolysis conversion into liquid and gaseous \n\nproducts [7]. The generic name, Paulownia, honors \n\nAnna Pavlovna of Russia [8].  \n\nPaulownia tomentosa was first introduced in Nepal in \n\n1988 AD by International Centre for Integrated \n\nMountain Development (ICIMOD) in Godavari. It \n\nwas normally propagated through seeds but due to \n\nseed dormancy and slow seedling growth, tissue \n\nculture products have been used. Among the \n\nseveral species of Paulownia, Paulownia tomentosa \n\n(Thunberg) Steudel and Paulownia fortuneii \n\n(Seemann) Hemsley are the most widely used \n\nspecies in the context of Nepal. The former is \n\nusually found below an altitude of 1800m while the \n\nlatter is usually found below an altitude of 2000m \n\n[1, 9]. \n\nDue to the booming market for Paulownia plants in \n\nNepal, the plantlets have to be manufactured in \n\nhuge amounts which can be performed by tissue \n\nculture.", "start_char_idx": 48, "end_char_idx": 3640, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5362892d-159c-43cf-a368-2f283f711c3f": {"__data__": {"id_": "5362892d-159c-43cf-a368-2f283f711c3f", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "66d2b0bc-afc1-446b-b100-58f3fa5f46fd", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "c1531977f597d8e48154394120c742fcfc0b11199d7fe8f7ab26490d015b6565", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "439356cd-4636-4651-9b12-994ee4fdb26c", "node_type": "1", "metadata": {}, "hash": "25883657220301ae3cf1fad393fbfae596ad01d089937aeb864dc686c583c3c8", "class_name": "RelatedNodeInfo"}}, "text": "But there have been no publications of \n\nresearch about the in-vitro growth statistics and \n\nvery few data regarding lab-to-land techniques like \n\nacclimatization and rooting methods. In this study, \n\nwe aim to compare the in-vitro growth statistics of \n\nthe two species to shed light on their growth \n\npatterns. Furthermore, we have extrapolated the \n\ntype and concentrations of hormones and the \n\nmethod of rooting required for optimal root \n\ninitiation. \n\nMaterials and Methods \nSample and Material Collection \nTwo different species of Paulownia, namely, \n\nPaulownia tomentosa and Paulownia fortuneii were \n\nused in this experiment. Paulownia fortuneii samples \n\nwere brought in sterile culture jars from the \n\nDepartment of Plant Resources (DPR), Thapathali. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:  11-15  Saiju et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal                              12                    Nepjol.info/index.php/njb \n \n\nPaulownia tomentosa samples available in the Plant \n\nTissue Culture laboratory, Himalayan White House \n\nInternational College, Khumaltar were used for the \n\nproject. \n\nMultiplication  \nDue to the requirement of a high number of plants, \n\nthe samples were mass propagated in MS medium \n\nsupplemented with hormone concentrations of 0.1 \n\nmg/l Naphthalene Acetic Acid (NAA - \n\nmanufactured by Sigma Chemical Co.) and 1 mg/l \n\nBenzyl Amino Purine (BAP - manufactured by S.D. \n\nFine-Chem Limited) in 150 ml culture vessels. The \n\ngrowth medium and culture equipment were \n\nautoclaved (manufactured by Life Steriware) at \n\n121\u00b0C temperature and 15 lb./sq. inch pressure and \n\nnodal culture of the samples was performed in a \n\nLaminar Air Flow Hood (manufactured by Amar \n\nChand & Company (ACCO). As a result, we \n\nperformed subcultures twice and prepared a total \n\nof 30 vessels for each sample, with an average of 5 \n\nexplants per vessel, which were stored in an \n\nincubation room, under fluorescent tube-lights \n\n(2000 lux) at a constant temperature of 25\u00b0C [1]. \n\nShoot Growth Comparison  \nAfter two subsequent subcultures, we met the \n\nrequired explant number which was estimated to be \n\n250 explants. Following this, nodal cultures were \n\nperformed using MS medium with 0.1 mg/l NAA \n\nand 1 mg/l BAP, with only one explant per vessel, \n\nfor ease of measurement. 20 vessels were produced \n\nfor each plant species, which were also stored in the \n\nincubation room, under 2000 lux fluorescent tube \n\nlights at 25\u00b0C temperature. Culture vessels were \n\nrecorded alongside a scale every week for 7 \n\nconsecutive weeks [1]. \n\nRooting Optimization \nSand Rooting  \nIn this experiment, a total of 30 culture vessels were \n\nremoved from the incubation room and exposed to \n\nindirect sunlight at room temperature for 10 days \n\nfor acclimatization process. Nodal cuttings of the in-\n\nvitro plants, including at least one leaf, were \n\nprepared and placed into solutions of differing \n\nconcentrations of NAA (0.5 mg/l, 1 mg/l and 1.5 \n\nmg/l) for about 10 minutes. The cuttings were then \n\ntransplanted into rooting trays packed with \n\nautoclave-sterilized wet sand and placed into a \n\npolythene chamber. After a week of incubation \n\n(with water spraying twice a day) at room \n\ntemperature, liquid MS media was added to the \n\nsand twice a week. The plantlets were removed \n\nfrom the sand after 4 weeks of incubation, for the \n\nmeasurement of root density and length [10-12] \n\nIn-vitro Rooting  \nIn this experiment, a total of 30 plantlets from \n\nculture vessels were transferred into MS media \n\nwith differing NAA concentrations (0.5 mg/l, 1 \n\nmg/l, and 1.5 mg/l). These vessels were stored in \n\nthe incubation room under 2000 lux fluorescent \n\nlights at 25\u00b0C temperatures for two months.", "start_char_idx": 3641, "end_char_idx": 7390, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "439356cd-4636-4651-9b12-994ee4fdb26c": {"__data__": {"id_": "439356cd-4636-4651-9b12-994ee4fdb26c", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5362892d-159c-43cf-a368-2f283f711c3f", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "7d6d600684586b92e5aa8e1eecf0735612bc8aa3d2681cccc89d1a5f78fb9761", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a4fac723-5445-4a47-87cc-45248310d89e", "node_type": "1", "metadata": {}, "hash": "534564b039fc3adf6a4f1523b2d9929b0bc857df59ae27fea6bb949b3d1d9dc5", "class_name": "RelatedNodeInfo"}}, "text": "Plants \n\nwere carefully extracted from the media using \n\nforceps for measurement of root density and length \n\n[13, 15]. \n\nResults \nIn-vitro Growth Statistics \nAccording to the data in Table 1, P. fortuneii was \n\nfound to have a higher average growth rate of 0.637 \n\ncm (SD=0.22) per week in comparison to 0.355 cm \n\n(SD=0.12) per week growth rate of P. tomentosa. \n\nThere was a significant difference in the growth \n\nrates of P. tomentosa and P. fortuneii; t (6)=5.150, p = \n\n0.002. \n\nBoth the line graphs represented peaks followed by \n\na gradual decline in growth rate. In-vitro growth \n\nwas found to peak during the 3rd week for P. \n\ntomentosa (0.543 cm) and during the 4th week for P. \n\nfortuneii (0.727 cm). In the following weeks, the \n\ngrowth receded slowly for the rest of the culture \n\nperiod. Within the 7th week, plant height reached to \n\nan average of 5.188 cm (n=16; SD=0.98) for P. \n\ntomentosa and 7.197 cm (n=16; SD=1.68) for P. \n\nfortuneii.  \n\nRooting Optimization \nSand rooting: Comparative study of root \n\ndevelopment between two species  \n\nOut of 64 explants, 35% survived after 4 weeks of \n\nincubation in the polythene chamber. Among those, \n\n55% were P. fortuneii samples and 45% were P. \n\ntomentosa samples. \n\nAs shown in Table 2, the average root length of P. \n\ntomentosa samples was estimated to be 3.72 cm \n\n(n=11; SD=1.32) and the average root number was \n\ncalculated to be 10.22 (n=11; SD=5.64). For P. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:  11-15  Saiju et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal                              13                    Nepjol.info/index.php/njb \n \n\nfortuneii samples, the average root length was \n\nestimated to be 3.04 cm (n=12; SD=1.72) and the \n\naverage root number was calculated to be 7.5 (n=12; \n\nSD= 7.38). \n\nTable 2: Rooting Optimization with different concentrations of phytohormone NAA, performed in sand and in-\nvitro medium \n\nType of \nRooting \n\nSpecies Hormone \nConcentration \n\n(mg/l) \n\nSample \nSize (n) \n\nAvg. Root Length (cm) Avg.", "start_char_idx": 7392, "end_char_idx": 9432, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a4fac723-5445-4a47-87cc-45248310d89e": {"__data__": {"id_": "a4fac723-5445-4a47-87cc-45248310d89e", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "439356cd-4636-4651-9b12-994ee4fdb26c", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "4b05321da1d8d32cd2e25bc628b1202f2c961d4397e4854ac4b91722d4bc12e5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "eb70ab44-26be-4903-a1e3-5745b74b0729", "node_type": "1", "metadata": {}, "hash": "438cf0b71cd384f13198bdf4f641b594626de00a6cab0136841a6ea03792cd18", "class_name": "RelatedNodeInfo"}}, "text": "Root Length (cm) Avg. Root Number \n\nSR PT 0.5 3 3.33\u00b11.44 7.50\u00b15.00 \n\n1 8 3.87\u00b11.35 11.25\u00b15.82 \n\n1.5 0 NA NA \n\nTotal 11 3.72\u00b11.32 10.22\u00b15.64 \n\nPF 0.5 3 4\u00b11.32 5.83\u00b12.88 \n\n1 5 3.4\u00b12.16 11.50\u00b110.24 \n\n1.5 4 1.87\u00b10.75 3.75\u00b12.50 \n\nTotal 12 3.04\u00b11.72 7.50\u00b17.38 \n\nSum Total 23 3.36\u00b11.55 8.80\u00b16.60 \n\nIVR PT 0.5 1 2.60\u00b10 3.30\u00b10 \n\n1 2 1.61\u00b10.43 1.37\u00b10.53 \n\n1.5 2 2.13\u00b10.38 2.35\u00b11.20 \n\nTotal 5 1.96\u00b10.44 2.15\u00b11.04 \n\nPF 0.5 4 3.91\u00b11.03 2.92\u00b11.40 \n\n1 3 2.36\u00b10.11 2.51\u00b10.72 \n\n1.5 1 4.40\u00b10 4.80\u00b10 \n\nTotal 8 3.39\u00b11.09 3.00\u00b11.25 \n\nSum Total 13 2.84\u00b11.13 2.67\u00b11.20 \n\nAbbreviations: SR - Sand Rooting, IVR - In-Vitro Rooting, PT -  Paulownia tomentosa, PF - Paulownia \nfortuneii, NA \u2013 Not Applicable \n\nFigure 1: Line graph showing the growth trend of the \ntwo species in in-vitro conditions (n=16). Plants grown \nin MS medium with supplementation of 0.1 mg/l NAA \nand 1 mg/l BAP. Peaks in growth rate can be seen in \nWeek 3 for P. tomentosa (0.543 cm/week) and in Week 4 \nfor P. fortuneii (0.908 cm/week) \n \n \n\n \nFigure 2: Figure represents a sample of P. tomentosa \n\nundergoing in-vitro growth for 7 weeks. The gradual in-\n\nvitro growth in plant height as well as biomass can be \n\nclearly visualized from this figure. \n\n \nFigure 3: Figure represents a sample of P. fortuneii \n\nundergoing in-vitro growth for 7 weeks. The gradual in-\n\nvitro growth in plant height as well as biomass can be \n\nclearly visualized from this figure. \n\n  \n\n0\n\n0.2\n\n0.4\n\n0.6\n\n0.8\n\n1\n\nP\nla\n\nn\nt \n\nG\nro\n\nw\nth\n\n (\nin\n\n c\nm\n\n) \n\nGrowth Trend of two species of Paulownia \n(in-vitro) \n\nP. fortuneii\n\nP. tomentosa\n\nTable 1: Average height growth rate (in cm/week) of both species of plants for 7 weeks (n=16). The average in-vitro \ngrowth rate of P. fortuneii was found to be higher than that of P. tomentosa. \n\nSpecies\\Time Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 \nAvg. Net \n\nGrowth Rate \n\nP. fortuneii 0.186 0.547 0.789 0.908 0.72 0.65 0.662 0.637 \n\nP. tomentosa 0.142 0.331 0.543 0.397 0.359 0.301 0.411 0.355 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:  11-15  Saiju et al.", "start_char_idx": 9411, "end_char_idx": 11473, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "eb70ab44-26be-4903-a1e3-5745b74b0729": {"__data__": {"id_": "eb70ab44-26be-4903-a1e3-5745b74b0729", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a4fac723-5445-4a47-87cc-45248310d89e", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "33d5d8a0cf51e00be1393de4c243d932f1ee66ce841b091129269c9d7bd6e899", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "93248e78-1d8e-49b3-9c46-5e2ebc43a8fc", "node_type": "1", "metadata": {}, "hash": "88266fbc8169954e9a7516b96731b4358869ed5e90ce782f5d873090b6067a2b", "class_name": "RelatedNodeInfo"}}, "text": "1:  11-15  Saiju et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal                              14                    Nepjol.info/index.php/njb \n \n\nSand rooting: Comparative study of effect \nof varying concentrations of auxin (NAA) \ntreatment in root development \nAs shown in Table 2, among the surviving samples, \n\n26% had been treated with 0.5 mg/l NAA, 56% had \n\nbeen treated with 1mg/l NAA and 17% had been \n\ntreated with 1.5 mg/l NAA. \n\nSamples treated with 1mg/l of NAA showed \n\nhighest root density in both P. tomentosa and P. \n\nfortuneii. None of the P. tomentosa plants treated \n\nwith 1.5mg/l NAA survived the sand rooting, but \n\nboth the average root length and root number from \n\nP. fortuneii plants treated with 1.5 mg/l were found \n\nto be low. P. fortuneii plants dipped in 0.5 mg/l \n\nNAA showed the highest average root length of \n\n4cm (n=3; SD=1.32). \n\nIn-vitro rooting: Comparative study of \nroot development between two species  \nCallus formation was observed in all the in-vitro \n\nrooting samples. Root initiation was observed after \n\n3 weeks of sub-culture. Out of 13 viable samples, \n\n38% were P. tomentosa samples and 61% were P. \n\nfortuneii samples. \n\nAs shown in Table 2, the average root length of P. \n\ntomentosa samples were observed to be 1.96 cm \n\n(n=5; SD=0.44) and the average root number was \n\ncalculated to be 2.15 (n=5; SD=1.04). For P. fortuneii \n\nsamples, the average root length was observed to be \n\n3.39 cm (n=8; SD=1.09) and the average root \n\nnumber was calculated to be 3 (n=8; SD= 1.25). \n\nIn-vitro rooting: Comparative study of \neffect of varying concentrations of auxin \n(NAA) treatment in root development \nAs shown in Table 2, among the viable samples, \n\n38% had been treated with 0.5 mg/l NAA, 38% had \n\nbeen treated with 1 mg/l NAA and 23% had been \n\ntreated with 1.5 mg/l NAA. \n\nSamples treated with 0.5mg/l NAA showed the \n\nhighest average root length and density for P. \n\ntomentosa, while samples treated with 1.5mg/l \n\nshowed the highest average root length and density \n\nfor P. fortuneii.  \n\nComparison of root length and root \nnumber between in-vitro rooting samples \nand sand rooting samples \nAs shown in Table 2, while comparing average root \n\nlength and root number between different rooting \n\ntechniques, sand rooting was clearly better suited \n\nfor root development than in-vitro rooting \n\ntechnique. \n\nIn case of sand rooting samples, their average root \n\nlength was estimated to be 3.36 cm (n=23; SD=1.55) \n\nand the average root number was calculated to be \n\n8.80 (n=23; SD=6.60). For in-vitro rooting samples, \n\nthe average root length was estimated to be 2.84 cm \n\n(n=13; SD=1.13) and the average root number was \n\ncalculated to be 2.67 (n=15; SD= 1.20). \n\nDiscussion \nMass propagation of Paulownia plants by tissue \n\nculture is gaining popularity in Nepal. Due to this, \n\nmany research works are being carried out on \n\ndifferent species of Paulownia both in and outside \n\nNepal.  \n\nP. fortuneii was found to have higher average \n\ngrowth rate among the two species. This result \n\ncould be interpreted as P. fortuneii having a greater \n\nmetabolic ability to utilize the energy and nutrients \n\nfrom MS medium than that of P. tomentosa.  \n\nDespite the lower average growth rate in in-vitro \n\ncondition, P. tomentosa overcame its flaws by \n\ndemonstrating superiority during sand rooting.", "start_char_idx": 11450, "end_char_idx": 14796, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "93248e78-1d8e-49b3-9c46-5e2ebc43a8fc": {"__data__": {"id_": "93248e78-1d8e-49b3-9c46-5e2ebc43a8fc", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "eb70ab44-26be-4903-a1e3-5745b74b0729", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "117dca0b40f63ae86c1f14333201e31322100ec1dd6a18895bde4e10cc01a0b5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1df716aa-d2e7-408f-a613-67f7c0a333d7", "node_type": "1", "metadata": {}, "hash": "38d76396c81dd06799f0e0b850fab8fad78a3dc9f9db42222b06501cb8f55193", "class_name": "RelatedNodeInfo"}}, "text": "P. \n\ntomentosa samples were found to have higher \n\naverage root length as well as higher root density \n\nthan that of P. fortuneii. Optimization test of \n\nhormone pre-treatment showed that the samples \n\ntreated with 0.5 and 1 mg/l NAA had greater \n\naverage root length than those treated with \n\n1.5mg/l.  Out of 10 P. tomentosa samples dipped in \n\n1.5mg/l NAA, none of the samples survived the \n\nsand rooting process. P. fortuneii samples dipped in \n\n1.5mg/l NAA also resulted in shorter and sparser \n\nroots. It was also observed that samples treated \n\nwith 1mg/l NAA had higher average root density. \n\nThese results suggest that concentrations of 0.5 and \n\n1mg/l are optimum for treatment before transfer to \n\nsand.  \n\nCallus formation was observed in in-vitro rooting \n\nsamples which are probably induced by mechanical \n\ndamage to the nodal cutting and presence of auxin \n\n(NAA) during the culture process. P. fortuneii \n\ndemonstrated higher densities and lengths of roots \n\nin in-vitro rooting. This result also supports our \n\ninterpretation that P. fortuneii may have a greater \n\nability to utilize energy and nutrients from MS \n\nmedium. Conversely, root length and density were \n\nfound to be higher in in-vitro media having \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 8  Vol. 6, No. 1:  11-15  Saiju et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal                              15                    Nepjol.info/index.php/njb \n \n\nhormone concentration of 0.5 mg/l and 1.5 mg/l. \n\nThis inconsistency in the result may have stemmed \n\nfrom inadequate sample size, so further studies \n\nmust be conducted.  \n\nWhile comparing the results from sand and in-vitro \n\nrooting techniques, we found sand rooting \n\ntechnique resulted in longer and denser roots. Due \n\nto the high availability of water and nutrients \n\nduring in-vitro conditions, smaller and sparse roots \n\nmay have been enough to sustain the plant whereas \n\nthe scarcity of water and nutrients during sand \n\nrooting may have promoted higher root growth. A \n\nstudy performed by Rodrigues et al. in 1995 \n\ndemonstrated a significant increase in root density \n\nduring water deficit, which is similar to our \n\nfindings [14-16]. \n\nConclusion \nIn the comparison of in-vitro growth statistics of P. \n\ntomentosa and P. fortuneii, the latter was found to \n\nhave higher average growth rate. Growth curves \n\nwere found to peak at 3rd and 4th weeks respectively \n\nfor the two species. \n\nIn the study to optimize rooting, P. tomentosa was \n\nfound to have higher root length as well as root \n\ndensity during sand rooting, but P. fortuneii showed \n\nbetter root development in in-vitro conditions. \n\nAmong the different concentrations of NAA, 0.5 \n\nand 1 mg/l were found to bear the best results \n\nduring sand rooting. In case of in-vitro rooting, 0.5 \n\nand 1.5 mg/l concentrations of NAA were found to \n\ngive better roots. \n\nComparison between the two rooting techniques \n\nshowed that sand-rooting is the better method for \n\nroot induction in case of Paulownia plants as both P. \n\ntomentosa and P. fortuneii demonstrated better \n\nresults during sand rooting. \n\nReferences \n1. Rajbahak S, et al: Clonal Propagation of Paulownia \n\ntomentosa Steud. for Commercial Production. Bull. \nDept. Pl. Res. 2014, 36:56-60. \n\n2. Siebold and Zuccarini Fl: Paulownia. Flora of China \n1998, 18:8-10. \n\n3. Melhuish JH, Gentry CE and Beckjord, P R: Paulownia \ntomentosa Seedling growth at Differing Levels of \n\npH, Nitrogen, and Phosphorous. J Environ Hort. 1990, \n8:205-207. \n\n4.", "start_char_idx": 14797, "end_char_idx": 18311, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1df716aa-d2e7-408f-a613-67f7c0a333d7": {"__data__": {"id_": "1df716aa-d2e7-408f-a613-67f7c0a333d7", "embedding": null, "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-214", "node_type": "4", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "55e0c52f64f6191bc925a9fd4b626b47089ca542d443a957443fabc304acee70", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "93248e78-1d8e-49b3-9c46-5e2ebc43a8fc", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "215cc556c468d9e38da1754895cd12d38989e56807f1c7e746c65f334dc859be", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "277055a3-7e73-4333-ab54-d56f8ccf5125", "node_type": "1", "metadata": {}, "hash": "14be179ae107975294e1cdeafcab6e2568cde25fe29ef12fc0138329ca6d3de7", "class_name": "RelatedNodeInfo"}}, "text": "J Environ Hort. 1990, \n8:205-207. \n\n4. Akyildiz MH and Kol HS: Some Technological \nProperties and Uses of Paulownia tomentosa Steud. \nwood. J Environmental Biology 2010, 31:351-355. \n\n5. Zhu ZH, Chao CJ, Lu XY, Xiong YG: Paulownia in \nChina: Cultivation and Utilization. Asian Network of \nBiological Sciences.  Edi- A.N. Rao et al. 1986, 1-65. \n\n6. Tang, RC, et al: Paulownia - A crop tree for wood \nproducts and reclamation of surface-mined land. \nSouth J Appl For. 1980, 4:19-24. \n\n7. Yorgun S and Yildiz D: Slow pyrolysis of Paulownia \nwood: Effects of Pyrolysis Parameters on Product \nYields and Bio-oil Characterization. J. Anal. Appl. \nPyrolysis. 2015, 114:68-78. \n\n8. Coombes AJ: The A to Z of Plant Names. USA Timber \nPress 2012, 312. \n\n9. Zima A, Hosek J, Treml J, Musel\u00edk J, Such\u00fd, P et al: \nAntiradical and Cytoprotective Activities of Several \nC-Geranyl-substututed Flavonones from Paulownia \ntomentosa Fruit. Molecules. 2010 15:6035-6049. \n\n10. Rajkarnikar KM and Rajbahak S: In-vitro \nMultiplication of Paulownia fortuneii (Seem.) \nHemsl. through Seed Culture. Bull Dept Pl Res. \n2015 37:63-65. \n\n11. Kumar PP, Rao CD, Rajaseger G, Rao AN: Seed \nSurface Architecture and Random Amplified \nPolymorphic DNA Profiles of Paulownia fortunei, \nPaulownia tomentosa and their Hybrid. Annals of \nBotany 1999, 83:103-107. \n\n12. Rajbhandary SB and Bajaj YPS: Rooting of in vitro \nproduced shoots in nonsterile sand - An inexpensive \nand efficient technique for enmasse \nmicropropagation. Biotechnology in Agriculture and \nForestry 1991, 17:262-268. \n\n13. Venkateswarlu B, Mukhopadhyay J, Sreenivasan E, \nKumar VM: Micropropagation of Paulownia \nfortuneii through in vitro axillary shoot \nproliferation. Indian J Exp Biol 2001, 39:594-599. \n\n14. Magar LB, Shrestha N, Khadka S, Joshi, J., Acharya, J., \nGyanwali, G., Marasini, B., Rajbahak, S., & Parajuli, N: \nChallenges and Opportunity of in vitro propagation \nof Paulownia tomentosa Steud for Commercial \nProduction in Nepal. Int J Appl Sci Biotechnol 2016, \n4:155-160. \n\n15. Bergmann BA and Whetten R: In vitro rooting and \nearly greenhouse growth of micropropagated \n\nPaulownia elongata shoots. New Forests 1997, 15:127-\n138. \n\n16. Rodrigues ML, Pacheco CMA and Chaves MM: Soil-\nplant water relations, root distribution and biomass \npartitioning in Lupinus albus L. under drought \nconditions. J Exp Bot 1995, 46:947-956", "start_char_idx": 18273, "end_char_idx": 20649, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "277055a3-7e73-4333-ab54-d56f8ccf5125": {"__data__": {"id_": "277055a3-7e73-4333-ab54-d56f8ccf5125", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1df716aa-d2e7-408f-a613-67f7c0a333d7", "node_type": "1", "metadata": {"identifier": "njb-214", "author": "Saiju, Hari Krishna; Bajracharya, Abhishesh; Rajbahak, Brishav; Ghimire, Stuti", "title": "Comparative Study of Growth Statistics of Two Species of Paulownia and Optimization of Rooting Methods", "date": "2019-01-15", "file": "njb-214.pdf"}, "hash": "0b2e40676485d0f85ab0ac3aeb7af0134f43411be11cbe4b07ead3bb13fde3a0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "48760fec-04da-4eef-954d-79355b79bf89", "node_type": "1", "metadata": {}, "hash": "599192093d47d715a540a9ad364d72341bbacf73f0d760cc38dfcf904fd90a53", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53    ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    47        Nepjol.info/index.php/njb \n\n \n\nStudy of Phytochemical, Antioxidant and Antimicrobial \nActivity of Artocarpus heterophyllus \n\nNita Thapa, Pratiksha Thapa, Jay Bhandari, Prasodhan Niraula, Nikita Shrestha, Bhupal G. Shrestha* \n\nDepartment of Biotechnology, Kathmandu University, Dhulikhel, Nepal. \n\nABSTRACT \nIn today\u2019s world, search for natural medicines is increasing as a result of drug resistance of pathogens and also \n\ndue to negative consequences of antibiotic. Presence of phytochemicals, antioxidant potential and antimicrobial \n\nactivity of Artocarpus heterophyllus was carried out in this study. Leaf of this plant was subjected to warm \n\nextraction with three different solvents namely methanol, aqueous methanol and ethyl acetate. Leaf extract \n\nshowed the presence of coumarin, alkaloid, terpenoid in methanol solvent; tannin, coumarin, saponin in aqueous \n\nmethanol extract and coumarin, terpenoids in ethyl acetate solvent. Further, antimicrobial activity was assessed \n\nthrough disc diffusion method with six pathological bacteria and two fungi strains in four different \n\nconcentrations of plant extract. Largest ZOI of 16mm was obtained against B. subtilis in 200mg/ml concentration \n\nfor ethyl acetate extract. Antioxidant potential was measured by DPPH (Diphenyl-2-picrylhydrazyl) assay. DPPH \n\nfree radical Scavenging Activity was expressed in % inhibition with L Ascorbic acid as standard and leaf extract in \n\nmethanol showed the best activity. \n\nKeywords:  Phytochemical, Antioxidant, Antimicrobial, IC50, ZOI (Zone of Inhibition). \n\n*Corresponding Author \n\nEmail: bgs@ku.edu.np \n\nIntroduction \nIn the written record, the study of herbs dates back \n\nover 5,000 years to the Sumerians, who created clay \n\ntablets with lists of hundreds of medicinal plants \n\nsuch as myrrh and opium [1]. The traditional use of \n\nherbs to prevent, treat and even cure various \n\nillnesses and diseases has largely been replaced by \n\nmodern medicine. However, it has been estimated by \n\nWorld Health Organization (WHO) that 80 percent \n\nof the population of some Asian and African \n\ncountries presently depend on traditional herbal \n\nmedicine for their most basic health care needs. Also \n\nWHO notes that between 25-40% of pharmaceuticals \n\ndrugs are derived from plants. It is also noted that \n\n40-50% of medicines are direct or synthetic copies of \n\nplant ingredients. The use of medicinal plants offers \n\npoorer populations the ability to fight diseases at low \n\ncosts. The use of traditional medicine and medicinal \n\nplants in most developing countries, as a normative \n\nbasis for the maintenance of good health, has been \n\nwidely observed [2]. Herbal medicine is still the \n\nmainstay of about 75 - 80% of the whole population, \n\nand the major part of traditional therapy involves the \n\nuse of plant extract and their active constituents [3]. \n\nIn recent years, antimicrobials derived from the \n\nplants have been receiving increasing attention as \n\nsynthetic antibiotics have shown ineffectiveness \n\nagainst several pathogenic organisms due to \n\nincreasing drug resistance [4]. Nepal is botanically \n\nrich in all the three levels of biodiversity which are \n\nspecies diversity, genetic diversity and habitat \n\ndiversity. In Nepal, there are various plants with \n\npotent medicinal properties and have been used \n\nsince ancient ages.", "start_char_idx": 47, "end_char_idx": 3588, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "48760fec-04da-4eef-954d-79355b79bf89": {"__data__": {"id_": "48760fec-04da-4eef-954d-79355b79bf89", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "277055a3-7e73-4333-ab54-d56f8ccf5125", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "418460e8d665d24ddd629c9e5f3e0e9397a4547402d9f3aa716ac20c0304ea3a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9ea30508-f0c6-45e8-9e95-2cd9be9dd441", "node_type": "1", "metadata": {}, "hash": "405eac1834475764d37382840980aef53b9e785fcd5e6eccb0a5271bac6d19e2", "class_name": "RelatedNodeInfo"}}, "text": "Among the 7,000 species of \n\nmedicinal plants recognized all over the world, more \n\nthan 900 types of precious medicinal plants are said \n\nto be found in Nepal [5]. \n\nA. heterophyllus bark and fruit are medicinally used \n\nto treat sprains, fractures, diabetes and are also used \n\nfor laxative effect of abdomen and to increase the \n\nbreast milk production in nursing mothers [6]. In \n\naddition to the antimicrobial activity of A. \n\nheterophyllus, anti-inflammatory, antioxidant, anti-\n\ncholinergic, anti-diabetic, immune modulatory effect, \n\ninhibition of protease, oestrogen regulation and \n\ninhibition of melanin biosynthesis have also been \n\nreported through several pharmacological research \n\ninvestigations of the plant parts [7]. A. heterophyllus is \n\na plant belonging to Moraceae family and is \n\ncommonly referred to as \u2018Jackfruit\u2019 in English and \n\n\u2018Katahar\u2019 in Nepali. It is found to originate from \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    48        Nepjol.info/index.php/njb \n\n \n\nSoutheast Asia and is widely cultivated in tropical \n\nlowlands and are found to have distinct aroma. The \n\nbark, roots, leaves and fruit are found to contain \n\nmedicinal properties and are widely used in various \n\ntraditional and folk systems of medicine in order to \n\ntreat a range of ailments. \n\nMaterials and Methods \nSample preparation \nA. heterophyllus leaves were collected from Pachkhal, \n\nNepal. The leaves were washed thoroughly under \n\ntap water and shade dried at room temperature (24\u2013\n\n26\u00b0C) and then pulverized by a mechanical grinder. \n\nThe powder was then passed through a 40-mesh \n\nsieve and stored in a well closed container before its \n\nuse. \n\nSolvent Extraction \nWarm extraction was done using Soxhlet apparatus \n\nusing methanol and ethyl acetate as solvent. 6gm of \n\nthe powdered sample was packed into the filter \n\npaper and were placed into the thimble of the \n\nSoxhlet apparatus. 250ml of methanol /ethyl \n\nacetate/aqueous methanol was added to the thimble. \n\nThe apparatus was operated continuously for 3 days \n\nmonitoring the circulation of water into the \n\ncondenser. The soxhlet extract was then treated with \n\nhexane in order to remove chlorophyll pigment by \n\nthe help of the separating funnel. The process was \n\nrepeated until the chlorophyll pigment was \n\ncompletely extracted in hexane. Then the final extract \n\nwas collected in Eppendorf tube after the extract was  \n\ndried of solvent on water bath at 50\u02daC [8]. Various \n\nconcentrations were then made by dissolving in \n\nDimethyl Sulfoxide (DMSO). \n\nPhytochemical screening \nPresence of phytochemicals were analyzed by \n\nfollowing standard procedure which are as follows: \n\nA. Test for Basic Alkaloids (Mayer\u2019s Test) \n5ml of extract was concentrated to yield a residue. \n\nResidue was dissolved in 3ml of 2% (v/v) HCL. 3 \n\ndrops of Mayer\u2019s reagent were added. Appearance of \n\nthe dull white precipitate indicated the presence of \n\nbasic alkaloids. \n\nB. Test for coumarin \n4ml extract solution was taken. 1-2 drops of water \n\n(hot) was added. Volume was made half (for UV \n\nfluorescence). 10% NH4OH was added to another \n\nhalf volume (for strong fluorescence). Presence of \n\ngreen fluorescence indicated the presence of \n\ncoumarin. \n\nC. Test for Saponins \n2ml extract was shaken vigorously for 30 seconds in \n\na test tube. Persistence of thick forth even after 30 \n\nminutes indicated the presence of saponins. \n\nD. Test for Glycosides \n2ml of extract was dried till 1ml.1-2ml NH4OH was \n\nadded and shaken.", "start_char_idx": 3589, "end_char_idx": 7206, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9ea30508-f0c6-45e8-9e95-2cd9be9dd441": {"__data__": {"id_": "9ea30508-f0c6-45e8-9e95-2cd9be9dd441", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "48760fec-04da-4eef-954d-79355b79bf89", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "692a62ebd12e1ea521a008a277ff4df7ea08cc869e1b3a2f2e4b1a45063f2293", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7e27f1bc-e582-46f5-848b-3e65c292b6d8", "node_type": "1", "metadata": {}, "hash": "7186dfa16f9f538738f7a7a81d00eb05d9f844266c0bfe720dafaf5e196a08c7", "class_name": "RelatedNodeInfo"}}, "text": "Appearance of cherish red color \n\nindicated the presence of glycosides. \n\nE. Test for Reducing Sugar (Fehling\u2019s Test) \n1ml of extract was taken.1ml distill water was \n\nadded.5-8 drops of Fehling\u2019s solution (hot) was \n\nadded. Presence of brick red precipitation indicated \n\nthe presence of reducing sugar. \n\nF. Test for steroids \n1ml extract was dissolved in 10 ml chloroform. Equal \n\nvolume of conc. H2SO4 was added by the side of test \n\ntube. Upper layer turned red and sulphuric acid \n\nlayer turned yellow with green fluorescence. This \n\nindicated the presence of steroids. \n\nG. Test for Quinone \n ml of extract was taken.1ml of conc. H2SO4 was \n\nadded. Formation of red color indicated the presence \n\nof quinone. \n\nH. Test for Terpenoids \n1 ml of extract was mixed with 2 ml of chloroform. \n\n3ml of conc. H2SO4 was added to form a layer. \n\nReddish brown precipitate coloration at the interface \n\nformed indicated the presence of terpenoid. \n\nAntioxidant Assay \nDiphenyl-2-picrylhydrazyl (DPPH) radical \n\nscavenging activity was done for determining \n\nantioxidant activity. \n\nPreparation of DPPH solution \nDPPH solution of 100\u00b5M was prepared by dissolving \n\n3.94mg of DPPH in 100ml of methanol. It was \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    49        Nepjol.info/index.php/njb \n\n \n\nprotected from light by covering the bottle with \n\naluminum foil. \n\nPreparation of standard solution \n10mg/ml stock solution of Ascorbic acid was \n\nprepared and test solution of 1, 2, 3, 4, 5 and 10 \n\n\u00b5g/ml of ascorbic acid was prepared from stock \n\nsolution by dilution. \n\nPreparation of Test sample \n10mg of plant extract was dissolved in 1ml of \n\nmethanol to prepare stock solution of 10mg/ml. Test \n\nsolution 1, 2, 3, 4, 5 and 10 \u00b5g/ml was prepared from \n\nstock solution by dilution. Experiments were done in \n\ntriplicate. \n\nEstimation of DPPH scavenging activity \nFor this, 1 ml methanol and 1 ml DPPH solution \n\nwere mixed as control. 2ml methanol was taken in \n\nEppendorf tube as blank. 1ml ascorbic acid was \n\nmixed with 1 ml DPPH as standard and 1ml plant \n\nextract was mixed with 1ml DPPH as sample. All \n\nthese mixtures were immediately kept in dark to \n\nprevent from light. After 30 minutes, absorbance was \n\ntaken in 517nm. \n\nAntimicrobial Screening \nAgar Disc Diffusion Assay was performed to test \n\nantibacterial and antifungal activities [9]. Fungal \n\nstrains Rhizopus, Aspergillus flavus and bacterial \n\nstrains Pseudomonas aeruginosa, Bacillus subtilis, \n\nBacillus thuringiensis, Escherichia coli, Proteus mirabilis \n\nwere subjected to sensitivity test. Whatman Filter 1 \n\ndisc (6mm) was autoclaved and dipped in extract of \n\nvarious concentrations and introduced on the upper \n\nlayer of agar plate earlier swabbed with microbial \n\nconcentration with the help of inoculating loop. \n\nStandard antibiotic discs Chloramphenicol 30 \u00b5g(C \n\n30) , Gentamicin 10 \u00b5g (GEN 10), Ciprofloxacin 30 \u00b5g \n\n(CF 30), Cefotaxime 30 \u00b5g (CTX 30) and Tetracycline \n\n30 \u00b5g (TE 30) were used. The plates were incubated \n\novernight at 37oC.", "start_char_idx": 7207, "end_char_idx": 10367, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7e27f1bc-e582-46f5-848b-3e65c292b6d8": {"__data__": {"id_": "7e27f1bc-e582-46f5-848b-3e65c292b6d8", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9ea30508-f0c6-45e8-9e95-2cd9be9dd441", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "b357213dabdea954b063d480593486cdee42cfe7d398b207fa67099fe7ec5625", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b4752ac3-6336-4ccd-abdd-2da10c70b9e7", "node_type": "1", "metadata": {}, "hash": "f2ea71f8e0e31d85f38b1b49cf1afd63ff6fcbc54eac551ee6ab49815db49c30", "class_name": "RelatedNodeInfo"}}, "text": "The plates were incubated \n\novernight at 37oC. Microbial growth inhibition was \n\ndetermined by measuring the diameter of zone of \n\ninhibition (ZOI). Results of antimicrobial activity are \n\nexpressed as average of triplicates. \n\nRESULTS \nIn our study, when six gram of dried sample was \n\nused for extraction, aqueous methanol exhibited \n\nhighest yield percentage i.e. 10.98% and least yield \n\nwas given by methanol solvent i.e. 0.22%. Ethyl \n\nacetate solvent gave yield of 4.68% when used as \n\nshown in Table 1. \n\nTable 1: Yield Value of Artocarpus heterophyllus \n\nS.N. Solvent sample \nloaded \n(gm) \n\nYield \namount \n(gm) \n\nYield \nPercentage \n(%) \n\n1 Ethyl \nAcetate \n\n6 0.28 4.68 \n\n2 Aqueous \nMethanol \n\n6 0.66 10.98 \n\n3 Methanol 6 0.013 0.22 \n\n \n\nExtraction and characterization of several active \n\nphytocompounds from these green factories have \n\ngiven birth to some high activity profile drugs [10]. \n\nSecondary metabolites of plants serve as defense \n\nmechanisms against predation by many \n\nmicroorganisms, insects and herbivores [11]. \n\nMethanol extract showed presence of coumarin, \n\nsteroids; ethyl acetate showed presence of coumarin, \n\nterpenoid whereas aqueous methanol showed \n\npresence of tannin, coumarin and saponin as shown \n\nin Table 2.  \n\nTable 2:  Phytochemical screening \n\nPhytochemicals Methanol Ethyl \nAcetate \n\nAqueous \nMethanol \n\nAlkaloids + - - \n\nTannins - - + \n\nReducing \nsugar \n\n- - - \n\nCoumarin + + + \n\nGlycosides - - - \n\nQuinone - - - \n\nSteroids - - - \n\nTerpenoids + + - \n\nSaponin - - + \n\n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    50        Nepjol.info/index.php/njb \n\n \n\n \n \n\nThis finding is similar to the findings of Rawat et al \n\n[12]. Coumarin was present in all three extracts \n\ntested and it can be said that this plant\u2019s leaf exhibit \n\nblood-thinning, anti-fungicidal and anti-tumor \n\nactivities. Presence of terpenoid is responsible for its \n\naromatic properties whereas it also possesses \n\nantibacterial and anti-cancer properties. This plant \n\ncan also reduce problem of cholesterol as presence of \n\nsteroid is noted. Saponin is only present in aqueous \n\nmethanol extract and it can also contribute to \n\nlowering of blood cholesterol and inhibition of  \n\ncancer cell growth. Saponin containing plants are \n\nused as traditional medicines, especially in Asia and \n\nare intensively used in food, veterinary and \n\nmedicinal industries [13]. Tannin is found to present \n\nin aqueous methanol extract which is naturally \n\noccurring plant polyphenols that have a \n\ncharacteristic binding and precipitating proteins. \n\nPhytochemical screening from other research \n\nconfirmed the presence of phytosterols, \n\nanthraquinone, terpenoids, phenols, glycosides, \n\nflavonoids and diterpenes in both of the trees i.e. \n\nArtocarpus heterophyllus and Artocarpus altilis [14]. \n\nThe antioxidant activity of tannins results from their \n\nfree radical and reactive oxygen species-scavenging \n\nproperties, as well as the chelation of transition metal \n\nions that modify the oxidation process [15].", "start_char_idx": 10321, "end_char_idx": 13471, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b4752ac3-6336-4ccd-abdd-2da10c70b9e7": {"__data__": {"id_": "b4752ac3-6336-4ccd-abdd-2da10c70b9e7", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7e27f1bc-e582-46f5-848b-3e65c292b6d8", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "7d0e293b9652264d1ec4152cc01003d3b1f3996f851c2f835af768831225fa19", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8725d393-4bc8-4c26-99a9-aeaf3383c3f8", "node_type": "1", "metadata": {}, "hash": "9ed49ed37b1bfa9c53e233832b8780c1f2ee0a6c2b197f5737eb3a818baef527", "class_name": "RelatedNodeInfo"}}, "text": "Table 3: Antimicrobial Assay \n\nSolvent Microorganism Extract Concentration (in mg/ml) and Zone of Inhibition (in mm) \n\n200mg/ml 100mg/ml 50mg/ml 25mg/ml \n\nMethanol \n\nB. cereus 11 9 7 7 \nB. thuringiensis 11 10 7 6.5 \n\nE. coli 6.5 6 6 6 \nP. aeruginosa 7 7 6.5 6.5 \nP. mirabilis 7.5 7 6..5 6.5 \nRhizopus 9 8 7 6.5 \nA. flavus 9 9 7.5 7 \nB. subtilis 9 7.5 7 6.5 \n\n     \n\nEthyl \nAcetate \n\nB. cereus 11 7 6.5 6.5 \nB. thuringiensis 7 6.5 6.5 6.5 \n\nE. coli 8 7.5 7.5 6.5 \nP. aeruginosa 12 11 8 7 \nP. mirabilis 9 7.5 7 6.5 \nRhizopus 8 7 6.5 6.5 \nA. flavus 9 8 7.5 7 \nB. subtilis 16 13 11 10 \n\n     \n\nAqueous \nMethanol \n\nB. cereus 10 7.5 7.5 7.5 \nB. thuringiensis 6.5 6 6 6 \n\nE. coli 7.5 6.5 6.5 6.5 \nP. aeruginosa 6.5 6.5 6.5 6 \nP. mirabilis 7.5 6.5 6 6 \nRhizopus 9 8 7 6.5 \nA. flavus 8 7 7 5.5 \nB. subtilis 9 7.5 6.5 6.5 \n\nTable 4: Zone size interpretative standards for selected antimicrobial discs (CTX30, GEN10, CF30, C30 TE30) and their \nobserved ZOI during experiment \n\nAntibiotics ZONE OF INHIBITION (mm) \n\nB. cereus E. coli B. thuringiensis P. mirabilis Rhizopus A. flavus P. aeruginosa B. sub tilis \n\nCTX30 18 20 11 20 23 26 34 34 \nGEN10 24 25 20 14 26 23 34 22 \nCF30 30 25 23 20 32 30 47 32 \nC30 23 29 24 12 33 36 30 24 \nTE30 31 24 25 11 25 26 34 31", "start_char_idx": 13475, "end_char_idx": 14724, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8725d393-4bc8-4c26-99a9-aeaf3383c3f8": {"__data__": {"id_": "8725d393-4bc8-4c26-99a9-aeaf3383c3f8", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b4752ac3-6336-4ccd-abdd-2da10c70b9e7", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "4e040ade257e2962cb6c3611748474b74b0665c9522d3423ca2c3d033f822ba8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6d737e44-6cbc-4c88-8617-3081cc46d86c", "node_type": "1", "metadata": {}, "hash": "1c27863f93b68bb8c4c66f1ea9f64301fbd2bbe282f83b19f7514129977db7ae", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    51        Nepjol.info/index.php/njb \n\n \n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\nZ\nO\n\nI \n(m\n\nm\n) \n\nAntimicrobial Activity of Aqueous Methanol \nsolvent \n\n200mg/ml 100mg/ml 50mg/ml 25mg/ml\n\n \n\nFigure 1: A, B and C are antimicrobial activity of extract \nprepared in methanol, aqueous methanol and ethyl \nacetate. Figure D represent antimicrobial activity of the \nstandard antibiotics with 8 \n\nAntimicrobial assay was performed by agar disc \n\ndiffusion method by using six bacterial strains and \n\ntwo fungal strains. This activity was assessed by \n\nmeasuring the diameter of ZOI of four \n\nconcentrations used as shown in Table 3. The \n\nobtained ZOI of standards for selected antimicrobial \n\ndiscs is shown in Table 4. Largest ZOI of 16mm was \n\nobtained against B. subtilis in ethyl acetate extract, \n\nwhereas 11mm ZOI was largest in methanol extract \n\nobtained against B. cereus. Antimicrobial activity was \n\ncomparatively better in ethyl acetate extract in \n\ncomparison to methanol and aqueous methanol \n\nextract.  \n\nBesides this, antibacterial activity was higher than \n\nantifungal activity as largest ZOI of 9mm is obtained \n\nagainst fungi Rhizopus and A. flavus. Also ZOI was \n\nlargest in 200mg/ml and least in 25mg/ml \n\nconcentration, showing a dose dependent response. \n\nClinical microbiologists are showing interest in the  \n\nTable 5: Antioxidant Activity \n\nConcentration \n(\u00b5g/ml) \n\n% Scavenging Activity \n\nMethanol Ethyl Acetate Aqueous Methanol \n\n Ascorbic acid Leaf extract Ascorbic acid Leaf extract Ascorbic acid Leaf extract \n\n1 8.07 6.63 14.37 3.26 11.38 10.41 \n\n2 18.45 11.93 17.69 6.52 15.78 14.13 \n\n3 25.81 15.25 22.54 8.01 31.67 18.15 \n\n4 35.46 20.68 28.72 10.95 41.42 22.76 \n\n5 44.25 28.03 34.66 14.43 44.19 26.63 \n\n10 89.25 61.84 38.45 17.86 94.04 47.17 \n\n0\n5\n\n10\n15\n20\n25\n30\n35\n40\n45\n50\n\nZ\nO\n\nI \n(m\n\nm\n) \n\nAntimicrobial Activity of \nStandard Antibiotics\n\nCTX30 GEN10 CF30 C30 TE30\n\nC \n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\nZ\nO\n\nI \n(m\n\nm\n) \n\nAntimicrobial Activity of Methanol solvent \n\n200mg/ml 100mg/ml 50mg/ml 25mg/ml\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\nZ\nO\n\nI \n(m\n\nm\n) Antimicrobial Activity of Ethyl Acetate solvent \n\n200mg/ml 100mg/ml 50mg/ml 25mg/ml\n\nC", "start_char_idx": 14729, "end_char_idx": 17039, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6d737e44-6cbc-4c88-8617-3081cc46d86c": {"__data__": {"id_": "6d737e44-6cbc-4c88-8617-3081cc46d86c", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8725d393-4bc8-4c26-99a9-aeaf3383c3f8", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "571b4ae0900edbdbc345b7f4ff3e082e230f1a0f1eaca076ba282a3316fb110d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bb944cf6-3033-4dc6-b07d-38b617b2c217", "node_type": "1", "metadata": {}, "hash": "97475a7df3dca43f83543965fbc716f896be4e7d48b1507ec7db30fed258ee37", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    52        Nepjol.info/index.php/njb \n\n \n\nIC50 value of ascorbic acid = 5.62\u00b5g/ml \n\nIC50 value of Artocarpus heterophyllus = 8.33\u00b5g/ml \n\n \nIC50 value of ascorbic acid = 12.96\u00b5g/ml \n\nIC50 value of Artocarpus heterophyllus = 29.29\u00b5g/ml \n\n \nIC50 value of Ascorbic acid=5.27\u00b5g/ml \n\nIC50 value of Artocarpus heterophyllus=10.69\u00b5g/ml \n\nFigure 2: A, B and C represents antioxidant activity of \n\nmethanol, ethyl acetate, aqueous methanol respectively. \n\ntopic of antimicrobial plant extracts as they believe \n\nthat these phytochemicals will find their way as \n\nalternate to antimicrobial drugs as those prescribed \n\nby physicians. Also several of these plant extracts are \n\nalready being tested in humans and this is probably \n\nbecause of increased public awareness of problems \n\nwith the over prescription and misuse of traditional \n\nantibiotics. Also nowadays multiple drug resistance \n\nhas developed due to the indiscriminate use of \n\ncommercial antimicrobial drugs commonly used in \n\nthe treatment of infectious disease [16]. \n\nDPPH stable free radical method is an easy, rapid \n\nand sensitive way to survey the antioxidant activity \n\nof a specific compound or plant extracts [17]. In our \n\nstudy, effective antioxidant activity was shown by \n\nmethanol extract where IC50 value for ascorbic acid \n\nwas 5.62\u00b5g/ml and leaf extract showed value of 8.33 \n\n\u00b5g/ml as shown in Table 5. Similarly, IC50 value for \n\nascorbic acid was 12.96\u00b5g/ml and leaf extract \n\nshowed value of 29.29 \u00b5g/ml when ethyl acetate was \n\nused as solvent. IC50 value for ascorbic acid was \n\n5.27\u00b5g/ml and leaf extract showed value of \n\n10.69\u00b5g/ml when aqueous methanol was used as \n\nsolvent. Among three solvents used for the study, \n\nmethanol extract showed best the antioxidant \n\nactivity. So, the leaf extract of the plant could have \n\nsome anti-cancer activity as has been earlier reported \n\nfor Ashwagandha [18, 19]. \n\nConclusion \nWe have been able to show that the leaf of A. \n\nheterophyllus contains presence of different \n\nphytochemicals that have potential health benefits. \n\nExtraction efficiency of leaf extract was higher in \n\naqueous methanol solvent. Preliminary study \n\nindicated the presence of phytochemicals which can \n\nfurther be studied to explore medicinal properties. \n\nThe plant extract also showed more antibacterial \n\nactivity than antifungal activity against major \n\npathogens. Methanol extract showed effective \n\nantioxidant activity in comparison to other solvent \n\nused in this study. Further, these can be subjected to \n\nisolation of the therapeutic antimicrobials which can \n\nbe beneficial to mankind.  \n\nAcknowledgement \nWe would like to express our deep gratitude to \n\nKathmandu University Biotechnology Department \n\nfor providing us work space and necessary guidance \n\nfor the successful completion of this research work. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 47-53                                            Thapa et al.\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    53        Nepjol.info/index.php/njb \n\n \n\nReferences \n1. Sumner J: The natural history of medicinal plants. \n\nTimber press. 2000. \n\n2. Culture and Health, Orientation Texts \u2013 World \nDecade for Cultural Development 1988 \u2013 1997, \nDocument CLT/DEC/PRO \u2013 1996, Paris, France, pg. \n129.  \n\n3. Akerele O: Summary of WHO Guidelines for the \nAssessment of Herbal Medicines Herbal Gram. \n1993, 22: 13-28.  \n\n4.", "start_char_idx": 17044, "end_char_idx": 20624, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bb944cf6-3033-4dc6-b07d-38b617b2c217": {"__data__": {"id_": "bb944cf6-3033-4dc6-b07d-38b617b2c217", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6d737e44-6cbc-4c88-8617-3081cc46d86c", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "f4645e7b68ecd0e4c6ca043aa4261abafefb81436653db16165899a9dd7d802a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "364d2a14-5390-4d49-a832-7711b435b0c1", "node_type": "1", "metadata": {}, "hash": "43d09f0e57d34be60889df9bf56b70f0c2e36fd5e009555c6db95f163c6a6c6f", "class_name": "RelatedNodeInfo"}}, "text": "1993, 22: 13-28.  \n\n4. Akgul C, Saglikoglu G: Antibacterial activity of crude \nmethanolic extract and its fractions of aerial parts of \nAnthemis tinctoria. Ind J Biochem Biophy. 2005, 42: \n395-397.  \n\n5. Manandhar NP: Plants and People of Nepal. Timber \nPress. USA. 2000: 50p.  \n\n6. Fernando S:  Herbal food and medicines in Sri \nLanka. New Delhi. (Original work published in \n1982).2003. \n\n7. Jitendra R, Kalpana S, Shweta S, Kumar M S, Manish \nB: Artocarpus heterophyllus (jackfruit) potential \nunexplored in dentistry- an overview. UJP. 2014, \n3(1): 50-55.  \n\n8. Shrestha P et al: Phytochemical screening, \nantimicrobial activity and cytotoxicity of Nepalese \nmedicinal plants Swertia chirayita and \nDendrobium amoenum. NJB 3.1. 2015 : 48-57.  \n\n9. Lamichhane  B, Adhikari S, Shrestha P,  Shrestha B G: \nStudy of phytochemical, antioxidant, antimicrobial \nand anticancer activity of Berberis Aristata. J Trop \nLife Sci. 2014, 4(1): 01-07.  \n\n10. Mandal V, Mohan Y, Hemalatha S: Microwave \nassisted extraction\u2014an innovative and promising \nextraction tool for medicinal plant research. \nPharmacognosy Reviews. 2007, 1(1):7-18.  \n\n11. Lutterodt G D, Ismail A, Basheer R H,  Baharudin H \nM: Antimicrobial effects of Psidium guajava \nextracts as one mechanism of its antidiarrhoeal \naction. Malaysian J Med Sci. 1999, 6(2): 17-20.  \n\n12. Rawat A, Mahajan S, Gupta A, Agnihotri R K, Wahi \nN, Sharma R: Detection of toxigenic fungi and \nmycotoxins in some stored medicinal plant \nsamples. IJASBT. 2014, 2(2): 211-216.  \n\n13. Hostettmann K, Marston A: Chemistry and \nPharmacology of Natural Products, Saponin. 1995.   \n\n14. Suman R, Arti T: Evaluation of phytochemical and \nantimicrobial effect of Artocarpus heterophyllus \nleaves extracts. J Pharm Sci. 2014. \n\n15. Serrano J, Puupponen-Pimia R, Dauer A, Aura A, \nSaura-Calixto F: Tannins: current knowledge of \nfood sources, intake, bioavailability and biological \neffects. Mol Nutr Food Res. 2009, 53:310\u2013329.  \n\n16. Joshi B, Lekhak S, Sharma A: Antibacterial property \nof different medicinal plants: Ocimum sanctum, \n\nCinnamomum zeylanicum, Xanthoxylum armatum \nand Origanum majorana. Kathmandu university \njournal of science, engineering and technology. 2009, \n5(1): 143-150.  \n\n17. Koleva I I, van Beek T A, Linssen J P, Groot A D, \nEvstatieva L N: Screening of plant extracts for \nantioxidant activity: a comparative study on three \ntesting methods. Phytochem anal. 2002, 13(1): 8-17.  \n\n18.", "start_char_idx": 20602, "end_char_idx": 23044, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "364d2a14-5390-4d49-a832-7711b435b0c1": {"__data__": {"id_": "364d2a14-5390-4d49-a832-7711b435b0c1", "embedding": null, "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-22", "node_type": "4", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "2fe0802ff448d2aaa6e3f08584675d7e2e7f5508d90ca2d3ed025fc406c7d6d1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bb944cf6-3033-4dc6-b07d-38b617b2c217", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "fea065868b8da8089a8e7194d810022d4c15d82e92e6a760b344e69fa1d8c57c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3a559783-fa75-49f7-826e-6070be01229b", "node_type": "1", "metadata": {}, "hash": "4f0f2ede0b6e348a03a43c2af75d5d2dd95e0b4821598cf00272f992c524a77c", "class_name": "RelatedNodeInfo"}}, "text": "2002, 13(1): 8-17.  \n\n18. Widodo N , Kaur K, Shrestha B G, Takagi Y, Ishii T, \nWadhwa R , Kaul S C:  Selective Killing of Cancer \nCells by Leaf Extract of Ashwagandha: \nIdentification of a Tumor-Inhibitory Factor and the \nFirst Molecular Insights to Its Effect. Clin Cancer Res. \n2007, 13(7): 2298-2306.  \n\n19. Widodo N, Takagi Y, Shrestha B G, Ishii I, Kaul S C, \nWadhwa R: Selective killing of cancer cells by leaf \nextract of Ashwagandha: Components, activity and \npathway analyses. Cancer Letters. 2008, 262: 37\u201347.", "start_char_idx": 23019, "end_char_idx": 23538, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3a559783-fa75-49f7-826e-6070be01229b": {"__data__": {"id_": "3a559783-fa75-49f7-826e-6070be01229b", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "364d2a14-5390-4d49-a832-7711b435b0c1", "node_type": "1", "metadata": {"identifier": "njb-22", "author": "Thapa, Nita; Thapa, Pratiksha; Bhandari, Jay; Niraula, Prasodhan; Shrestha, Nikita; Shrestha, Bhupal G", "title": "Study of Phytochemical, Antioxidant and Antimicrobial Activity of Artocarpus heterophyllus", "date": "2016-12-31", "file": "njb-22.pdf"}, "hash": "4851bcafc0bd256f62d2e5575aa90e9f809621c952f47223bab427842cab59f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9fed29a8-d859-4cdf-9df6-feb77f106d56", "node_type": "1", "metadata": {}, "hash": "811f8a370b037cf2b6b01574d1fdda83b30506e718af2a5bdef27ea980a41a48", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 1-6   Research article   DOI: https://doi.org/10.54796/njb.v10i1.224  \n \n\n  \n\n\u00a9NJB, BSN   1 \n\nDevelopment of Effective Protocol for four Varieties of large Cardamom  \nAnna Balzer1, Sujit Shah1,2 , Yam Kumar Ale1, Dipti Adhikari1, Sanjit Niroula1, Jawaharlal Mandal1, Amar \n\nBahadur Pun1, Krishna Poudel1, Parashuram Bhantana1, Govinda Timsina1, Ravindra Karn1, Sujwal karki1 \n\n1Agricultural Research Station, Pakhribas, Dhankuta, Nepal \n2Daffodil Agro Biological Research Center, Lalitpur, Nepal   \n\nReceived: 24 Mar 2021; Revised: 9 Jan 2022; Accepted: 16 Jan 2022; Published online: 30 Jul 2022 \n\nAbstract \n\nLarge cardamom is one of the most important spices that can significantly contribute to the economical farming in the \ncountry of Nepal.  It is grown in Nepal and north-eastern states of India which provide suitable agroclimatic growing \nconditions of high humidity, ambient temperature and high rainfall. Meeting the demand for high quality plants and \nyield of cardamom is challenging with traditional methods of propagation. The present study has used the plant tissue \nculture technique to produce high quality plants. In this regard, MS media with three different hormonal combinations \nwere used for the development protocol for 8 weeks. Shoot length, root length, shoot number and root number were \nassessed at intervals.  The best protocol for growth was MS media with 1 mg/L BAP + 0.5 mg/L IBA for the Ramsey \nvariety, with no significant difference for Golsai, Dambarsai, or Sikkimae varieties. Similarly, the acclimatization and \nfield transfer study was done. The use of any substrate composition in ratio of coco peat: soil 1:2; moss: coco peat 1:2 \nand sawdust: coco peat 1:2 enables transfer of healthy plants to the field. The results indicate that the varieties respond \ndifferently to the micropropagation process and to hormone concentrations indicated by differing root and shoot \nproduction. The protocol of 1mg/L BAP and 0.5mg/L IBA could be used for the Ramsai while optimal shoot \nproduction for Golsai and Sikkimae should be at 0.5mg/L and 5mg/L for shoot production. All varieties showed \noptimal root production at 0mg/L BAP and 0.5mg/L IBA. This study sheds light on the different responsiveness of \nvarieties to tissue culture and hormone concentrations for both root and shoot development in micropropagation.  \n\nKeywords: Cardamom, 6-benzylaminopurine, micropropagation, Amomum subulatum, variety, tissue culture \n\n Corresponding author, email: sujitaug16shah@gmail.com \n\nIntroduction \n\nLarge cardamom, Amomum subulatum Roxb. belongs to \n\nthe family Zingiberaceae and its cultivation is confined to \n\nthe sub-Himalayan range of eastern Nepal, northern \n\nIndia (Sikkim and West Bengal) and Bhutan [1]. Nepal is \n\nthe world's largest producer of large cardamom \n\nsupplying close to 50% of the world's market demand \n\nwith 14200 ha cultivated [1], these are concentrated in \n\nTaplejung, Sankhuwasabha, Panchthar, and Ilam \n\ncomprosing 80% of Nepal\u2019s production [2]. The annual \n\nproduction is 6026 mt from productive area of 11665 \n\nhectares and supports the livelihood of more than 70,000 \n\nfamilies directly and indirectly [3].", "start_char_idx": 48, "end_char_idx": 3261, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9fed29a8-d859-4cdf-9df6-feb77f106d56": {"__data__": {"id_": "9fed29a8-d859-4cdf-9df6-feb77f106d56", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3a559783-fa75-49f7-826e-6070be01229b", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "b43f09236742ac7d6926cfbcbd5b3af6ce937badc896a9ae61f0ddc3a16e0c28", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9900e708-cef3-4e65-ab5b-04b2e66738b6", "node_type": "1", "metadata": {}, "hash": "7a00fa2f324d26d4edd84cfc1682ae5721c93b9f19b8eb0fe379a4d3075ab001", "class_name": "RelatedNodeInfo"}}, "text": "It has been regarded as \n\none of the important spices upon which the livelihood of \n\nmost farmers depends.  \n\nHowever, the quality and quantity of the cardamom have \n\ndeclined over the years to disease associated with it [4]. \n\nCommon cardamom diseases include foorkey \n\n(nanovirus) [5], and chirke diseases (macluravirus) [6], \n\nand phoma leaf spot (Phoma hedericola) [7]. These have \n\ndirectly or indirectly affected the stakeholders and the \n\neconomy of the country. The demand and the price have \n\nbeen declining over the years because of low quality and \n\nfor this reason many farmers have stopped cultivating \n\nlarge cardamom.  Large cardamom has a high price \n\nvolatility which poses a risk to long term farming, and \n\nreduced production is caused by aging orchards, disease, \n\npoor management, and lack of new planting material [8, \n\n9]. These problems pose potential risks to the large \n\ncardamom industry in Nepal.  \n\nA plant tissue culture technique (shoot tip culture) \n\nprovides an alternate way to meet the quality and \n\nquantity of the cardamom, producing vigorous disease-\n\nfree plantlets in large numbers. The techniques will help \n\nto restore the high quality of plantlets and fulfill the \n\ndemand of disease-free plants in the cardamom pocket \n\nzone of Nepal [10]. This will in turn help to reestablish \n\nthe ability to supply the regional and international \n\ncardamom markets. \n\nIn this regard, present investigation has developed an \n\neffective protocol for producing highly demanded large \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nmailto:sujitaug16shah@gmail.com\nhttps://orcid.org/0000-0002-6175-5242\nmailto:sujitaug16shah@gmail.com\nmailto:sujitaug16shah@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 - 6      Balzer et al.  \n\n\u00a9NJB, BSN   2 \n\ncardamom varieties such as Ramsai, Dambarsai, Golsey, \n\nand Sikkimme. \n\nMaterial and methods \n\nExplants were collected from the nursery of NARC Sub-\n\nStation Pakhribas. The freshly collected sucker (explants) \n\nwere washed under running tap water for at least 30 \n\nminutes to remove soil and other external particles \n\nattached on their surface. Then, the explants were dipped \n\nin water containing Tween 20 (0.1%) for 15-20 minutes, \n\nshaken well and again washed in running tap water until \n\nall the detergents washed off clearly and rinsed with \n\ndistilled water. Thereafter, dipped into 1% sodium \n\nhypochlorite solution for 15 minutes and followed \n\nsubsequently by with 70% ethyl alcohol for 2 minutes \n\n[11].  Finally, explants were rinsed thoroughly with \n\nsterile water for 5 times and ready for cut after drying in \n\nfilter paper. Multiplications were made from sterile \n\nmother stock which had been established through several \n\nrounds of subculturing with the aim to minimize the \n\nneed for rhizome collection and allow year-round \n\nmultiplication. Plants were assessed daily to monitor \n\ngrowth and contamination, and were sub-cultured every \n\n7-21 days.  \n\nPlant growth assay \nThe protocorms produce from the explants were used for \n\nthe plant growth assay. MS (Murashige & Skoog) media \n\nwas prepared [12].", "start_char_idx": 3262, "end_char_idx": 6505, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9900e708-cef3-4e65-ab5b-04b2e66738b6": {"__data__": {"id_": "9900e708-cef3-4e65-ab5b-04b2e66738b6", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9fed29a8-d859-4cdf-9df6-feb77f106d56", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "b5be2c07617c4811fd1cd6d44ef2afc039e2612b19cc6d705ef741da561c8473", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "27d22a24-2edf-4bd7-960e-b46cfb1c9740", "node_type": "1", "metadata": {}, "hash": "eff79093a973c40665a4948214af62c32ca4bbff702a7b4d65ad039d7545bf71", "class_name": "RelatedNodeInfo"}}, "text": "MS (Murashige & Skoog) media \n\nwas prepared [12]. The different hormonal compositions \n\n(MS + 0mg/L BAP + 0.5mg/L IBA; MS+0.5 mg/L BAP + \n\n0.5 mg/L IBA; MS + 1mg/L BAP + 0.5 mg/L IBA; MS + \n\n5mg/L BAP + 0.5mg/L IBA) were used for the plant \n\ngrowth assay for all the varieties Ramsey, Golsey, \n\nDambarsai, and Sikkimme. The plantlets were grown in \n\naseptic conditions under a 16 h photoperiod, at 25\u00b1 2\u00baC. \n\nGrowth pattern of the plantlets were recorded after 60\u00b15 \n\ndays. Shoots were then excised and placed on fresh \n\nmedia to grow until field transfer.  \n\nAcclimatization and field transfer \nThe in-vitro plantlets were acclimatized using different \n\nsubstrate compositions. The substrates used were \n\nsterilized cocopeat, moss, sawdust and soil. The ratio of \n\nsubstrate cocopeat:soil 1:2; moss: cocopeat 1:2 and \n\nsawdust:cocopeat 1:2. The trial was kept under \n\nobservation for 4 weeks in a greenhouse. The plant height \n\nwas measured at weekly intervals.  \n\nStatistical analysis & growth ranking \nPlant growth assays were performed independently for a \n\nminimum of eight replicates to measure the shoot and \n\nroot length and number. Both were tested by ANOVA \n\nwith the alpha error level set at p\u22650.05 (GraphPad Prism). \n\nNormal distribution was tested on each data set \n\n(D\u2019Agostino & Pearson), with 22% of data sets being \n\nconsidered normally distributed. Non-Gaussian \n\ndistribution was therefore assumed for ANOVA. The \n\ncontrol was considered to be 0mg/L BAP media. A \n\ngrowth ranking system was implemented to identify \n\noptimal hormone concentrations within the study due to \n\nhigh variability and low sample sizes. Growth rankings \n\nwere determined by firstly normalizing each parameter \n\nto the highest average value. Shoot or root growth \n\nranking was then a multiplicative product (unitless) of \n\nthe two parameters (length and number).  \n\nResults \n\nPlant growth assay \n\n \n\n   \n\nFigure 1. The plant growth assay perfomerd for 6o days \nin-vitro condition for all four varieties of cardamom \nDambarsai (a), Golsai (b), Sikkimae (c), and Ramsai (d) \nwith MS+1mg/L BAP +0.5mg/L IBA \n\nThe in-vitro plant growth assay performed for four \n\nvarieties of cardamom to investigate the optimum \n\nconcentration of hormone required of the plant growth \n\n(Figure 1). Length and numbers of shoots and roots are \n\nindicated in Table 1. Shoot number was significantly \n\nhigher in the Sikkimae variety on 5mg/L BAP producing \n\n8.4\u00b10.9 shoots per cutting. Root number was significantly \n\nlower in the Ramsai variety at 5mg/L BAP producing \n\n1.5\u00b10.4 roots per cutting.  \n\na b \n\nc d \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 - 6      Balzer et al.  \n\n\u00a9NJB, BSN   3 \n\nTable 1: Growth patterns of Dambarsai, Golsai, Sikkimae, and Ramsai cardamom varieties in response to differing cytokinin \nconcentration in micropropagation. \n\nTable 2: Growth ranking for optimal shooting and rooting of Dambarsai, Golsai, Sikkimae, and Ramsai cardamom varieties in \nresponse to differing cytokinin concentration in micropropagation. Optimal raking for the trail for shoot and root responses are \nshown in bold\n\n.", "start_char_idx": 6456, "end_char_idx": 9560, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "27d22a24-2edf-4bd7-960e-b46cfb1c9740": {"__data__": {"id_": "27d22a24-2edf-4bd7-960e-b46cfb1c9740", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9900e708-cef3-4e65-ab5b-04b2e66738b6", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "510f744e75dec3f3e2dc76568cb4dae35fca8f4c9c28b6144ce3b2de338cf02f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "424d23ab-336b-4003-a467-e86425db3015", "node_type": "1", "metadata": {}, "hash": "60497a290cdfa8e9c0992aaf480ecff1f26ff4bb28638e8a6a66dc5fee670c29", "class_name": "RelatedNodeInfo"}}, "text": "Optimal raking for the trail for shoot and root responses are \nshown in bold\n\n.  \n\nIn Ramsai this concentration also produced a lower root \n\nnumber and length than all other treatments with a \n\nlength of 27.5\u00b16.8 mm length. Shoot number was highest \n\nin the 0.5mg/L BAP media for Golsai at 7.8\u00b11.0 shoots \n\nper cutting. No other measurements were significantly \n\ndifferent (Table 1). The number of shoots and roots \n\nproduced differed widely between varieties. The \n\nmaximum number of shoots produced in a media protocol \n\nper variety from 2.6 for Ramsey to 8.4 for the Sikkimae \n\nvariety.  Root number varied from 3.1 (Golasi) to 5.4 \n\n(Sikkimae).  \n\nTable 2 indicates growth ranking for best shoot and root \n\ndevelopment. Ranking based on growth of shoots and \n\nroots indicated that shoot and root optimization differed \n\nwithin each variety. The best ranking media to produce \n\nroot for all varieties was the 0.5mg/L IBA media with no \n\nexogenous BAP (Table 2).  Highest ranking media for \n\nshoot production was 1mg/L BAP for Dambarsai, \n\n0.5mg/L BAP for Golsai, 5mg/L BAP for Sikkimae and \n\n1mg/L BAP for Ramsai (Table 2).  \n\n \n\n \n\nValues are indicated as mean \u00b1 SEM. * indicates \n\nsignificant difference at the end of the trial period from \n\nthe control BAP concentration of 0mg/L.  \n\nAcclimatization and field transfer \nThe plants that were grown for 65 days were taken for \n\nacclimatization and field transfer (Figure 2). The plant \n\nheight in all three different substrate compositions \n\n(cocopeat: soil 1:2; Moss: cocopeat 1:2 and sawdust: \n\ncocopeat 1:2) was recorded at 15 and 30 days. The total \n\ngrowth accumulated from day 0 (transfer date) is shown \n\nin Figure 2. The growth of plants in each substrate \n\ncomposition did not differ between substrates. Survival \n\nand health of plants did not differ in the 30 day trial \n\n(Figure 2 a-c).  \n\nDiscussion \n\nThe previous study has shown the use of 0.5mg BAP + \n\n1.0mg IBA in one liter MS media as a standard protocol \n\nfor the single Ramsey variety [12]. Similarly, use of MS+ \n\nsucrose 40 g + BAP 3mg/L + 0.5 NAA + 2mg/L IBA was \n\nDambarsai\n\nShoot number 2.5 \u00b1 0.5 2.9 \u00b1 0.5 4.3 \u00b1 0.8 3.4 \u00b1 0.7\n\nShoot length (mm) 36.3 \u00b1 15.7 41.3 \u00b1 13.6 42.7 \u00b1 10.3 37.9 \u00b1 10.0\n\nRoot number 3.0 \u00b1 1.0 2.6 \u00b1 1.2 3.7 \u00b1 1.0 0.9 \u00b1 0.7\n\nRoot length (mm) 100.0 \u00b1 52.9 68.8 \u00b1 44.8 71.7 \u00b1 23.1 12.9 \u00b1 12.0\n\nGolsai\n\nShoot number 3.8 \u00b1 1.1 7.8 \u00b1 1.0* 4.8 \u00b1 1.0 6.0 \u00b1 1.3\n\nShoot length (mm) 53.0 \u00b1 17.2 38.5 \u00b1 6.4 33.3 \u00b1 5.8 32.1 \u00b1 7.6\n\nRoot number 3.1 \u00b1 1.1 0.7 \u00b1 0.4 0.9 \u00b1 0.5 0.1 \u00b1 0.1\n\nRoot length (mm) 51.0 \u00b1 20.9 6.0 \u00b1 4.", "start_char_idx": 9481, "end_char_idx": 12042, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "424d23ab-336b-4003-a467-e86425db3015": {"__data__": {"id_": "424d23ab-336b-4003-a467-e86425db3015", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "27d22a24-2edf-4bd7-960e-b46cfb1c9740", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "24417d5e476ef304a4cc37fac8e99434b7de14887917e002dc4d6975fb1086f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ea04964f-78ed-429b-a38a-1a27e518f3e8", "node_type": "1", "metadata": {}, "hash": "69fc6f59b181076a4a357d4428ea6592efc91a2c4c95ef7c6c7d8d31cb9f3101", "class_name": "RelatedNodeInfo"}}, "text": "0 \u00b1 20.9 6.0 \u00b1 4.1 19.6 \u00b1 14.6 0.5 \u00b1 0.5\n\nSikkimae\n\nShoot number 4.4 \u00b1 0.6 3.2 \u00b1 1.5 3.7 \u00b1 1.1 8.4 \u00b1 0.9*\n\nShoot length (mm) 48.1 \u00b1 20.8 35.0 \u00b1 19.7 42.2 \u00b1 7.1 82.3 \u00b1 13.1\n\nRoot number 4.9 \u00b1 1.8 5.4 \u00b1 2.0 3.3 \u00b1 1.0 2.3 \u00b1 0.8\n\nRoot length (mm) 101.3 \u00b1 47.0 63.8 \u00b1 40.4 93.3 \u00b1 32.6 20.5 \u00b1 10.5\n\nRamsai\n\nShoot number 2.2 \u00b1 0.5 2.3 \u00b1 0.4 2.6 \u00b1 0.4 1.5 \u00b1 0.2\n\nShoot length (mm) 61.7 \u00b1 16.0 47.1 \u00b1 12.2 57.5 \u00b1 10.8 35.0 \u00b1 3.6\n\nRoot number 5.2 \u00b1 1.1 2.3 \u00b1 0.8 2.7 \u00b1 0.7 1.5 \u00b1 0.4*\n\nRoot length (mm) 112.8 \u00b1 29.3 96.3 \u00b1 31.9 111.5 \u00b1 38.4 27.5 \u00b1 6.8*\n\n0mg/L BAP + 0.5mg/L IBA 0.5mg/L BAP + 0.5mg/L IBA 1mg/L BAP + 0.5mg/L IBA 5mg/L BAP + 0.5 mg/L IBA\n\n0mg/L BAP + 0.5mg/L IBA 0.5mg/L BAP + 0.5mg/L IBA 1mg/L BAP + 0.5mg/L IBA 5mg/L BAP + 0.5 mg/L IBA\n\nDambarsai\n\nShoot 0.49                                     0.64                                        1.00                                     0.70                                      \n\nRoot 0.82                                     0.49                                        0.72                                     0.03                                      \n\nGolsai\n\nShoot 0.48                                     0.73                                        0.39                                     0.47                                      \n\nRoot 1.00                                     0.03                                        0.11                                     0.00                                      \n\nSikkimae\n\nShoot 0.31                                     0.16                                        0.22                                     1.00                                      \n\nRoot 0.90                                     0.63                                        0.57                                     0.09                                      \n\nRamsai\n\nShoot 0.86                                     0.68                                        0.93                                     0.32                                      \n\nRoot 1.00                                     0.37                                        0.51                                     0.07", "start_char_idx": 12025, "end_char_idx": 14169, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ea04964f-78ed-429b-a38a-1a27e518f3e8": {"__data__": {"id_": "ea04964f-78ed-429b-a38a-1a27e518f3e8", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "424d23ab-336b-4003-a467-e86425db3015", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "ee7358ca398bbccae3742b38c0ab7e9fdb89b04fbd6d6d11fb7c15849d40ad55", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6e6e2248-0641-493f-9e5e-65eeb3e8bc27", "node_type": "1", "metadata": {}, "hash": "acf5c06f470aa387f56975ff8639f2e24a74b08504efbfb59900f6b636468c2d", "class_name": "RelatedNodeInfo"}}, "text": "00                                     0.37                                        0.51                                     0.07                                      \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 - 6      Balzer et al.  \n\n\u00a9NJB, BSN   4 \n\ndemonstrated [13]. Revisiting the investigation, we have \n\ndemonstrated the use of 1mg/L BAP + 0.5mg/L IBA is \n\nfar better for the overall in-vitro plant growth in the \n\nRamsai variety. We also show that the optimal \n\nconcentration of BAP with 0.5mg/L IBA, based on this \n\nstudy, for shoot number production in the Golsai and \n\nSikkimae varieties are 0.5mg/L and 5mg/L respectively. \n\nWhile it is useful to have high shoot numbers, the shoots \n\nthemselves must also show sufficient development in \n\nsize. Therefore, we implemented a ranking system to look \n\nat both number and length of shoots and roots. The \n\noptimal media for shoot growth differed by variety, \n\nhowever for root growth the optimal media was that \n\nwhich contained no exogenous BAP.  \n\n \n\n \n\n \n\nFigure 2. The plants after 65 days of growth were transfer to \nthree different sterile substrate composition; (Sawdust: coco \npeat 1:2 Fig. (a); Coco peat: Soil 1:2 fig (b); Moss: coco peat 1:2 \nfig(c)) for 30 days. Growth measured intern of plant height for \n4 weeks showing no significant difference in height during the \ntrail at level of p>0.05. \n\nMost significantly, root initiation is higher in the presence \n\nof a lower cytokinin concentration.  The role of \n\ncytokinin\u2019s in root formation is complex and depends on \n\nmany interacting factors, and affects both the type and \n\namount of root growth [14-16]. In all varieties, the \n\noptimal root production as a rank was best in a media \n\ncontaining no exogenous cytokinin with 0.5mg/L IBA. \n\nPrevious studies have shown variability across cytokinin \n\nconcentrations with respect to root number formation in \n\nZingeriberaceae. No clear trend between BAP \n\nconcentration and root growth was shown in Kaempferia \n\nparviflora tissue culture studies with 0, 1.5 and 2.5mg/L \n\nproducing the same number of roots [17]. Similarly \n\nmicropropagation experiments using BAP and NAA \n\nshowed no consistent correlation with cytokinin \n\nconcentration and root number in Amomum subulatum, \n\nwhereas increasing cytokinin resulted in increasing shoot \n\nnumber up to 4\u00b5M BAP concentration [13].  \n\nIt may be beneficial to implement 2 stage in vitro \n\npropagation systems for cardamom whereby shoot \n\nmultiplication is carried out on 0.5mg/L BAP + 0.5mg/L \n\nIBA for Golsai, 1mg/L BAP + 0.5mg/L IBA for \n\nDambarsai and Ramsai, and 5mg/L BAP + 0.5mg/L IBA \n\nSikkimae. For root development all should be transferred \n\nto media containing nil exogenous cytokinin.  \n\nAlthough not the primary aim of this study, it has become \n\napparent that there are different responses of varieties to \n\ntissue culture. When looking at the number of shoots and \n\nroots produced per variety, these varied widely per \n\nvariety. This outcome suggests that the Sikkimae variety \n\nis more responsive to the micropropagation process than \n\nothers. This is not unexpected and has been observed in \n\nother species including oil palm (Elaeis guineensis) [18], \n\npotato (Solanum tuberosum) [19], gerbera (Gerbera \n\njamesonii) [20], and grapes (Vitis vinifera) [21], among \n\na \n\nb \n\nc \n\n\n\nNepal J Biotechnol.", "start_char_idx": 14041, "end_char_idx": 17380, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6e6e2248-0641-493f-9e5e-65eeb3e8bc27": {"__data__": {"id_": "6e6e2248-0641-493f-9e5e-65eeb3e8bc27", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ea04964f-78ed-429b-a38a-1a27e518f3e8", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "da813d2aba68e66ff5a013a08a611f32114e60b77d5cf7bcfa0ddccce99fd6d7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0027f0a7-4a99-44c6-8b27-c9bb404f28bd", "node_type": "1", "metadata": {}, "hash": "06ea24215b41df56aaffcb31ccd436de08b2cb4f186e84307534288138c4381a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 - 6      Balzer et al.  \n\n\u00a9NJB, BSN   5 \n\nmany others. Further studies may establish this so we \n\nsuggest additional research is undertaken. This outcome \n\npresents an opportunity to focus production on varieties \n\nwho respond better to tissue culture, provided they also \n\nhave desirable field characteristics. Varieties with lesser \n\nresponses may need to be the focus of additional research \n\nto optimize and meet demands.  \n\nThe acclimatization and field transfer are important steps \n\nfor the mass propagation. In this regard, the all three-\n\nsubstrate combination showed uniform growth and \n\nsurvival rate for plants growth. Any of these substrate \n\ncombination (cocopeat: soil 1:2; Moss: cocopeat 1:2 and \n\nsawdust: cocopeat 1:2) can be used for acclimatization \n\nbased on the availability which allows for greater \n\nproduction flexibility. Other studies have shown optimal \n\nhardening of Zingiberaceae species cow dung, coir \n\ndust,soil:sand and cow dung mixes all with high survival \n\nrates of 90-100% [22]. Studies using peatmoss, sand, \n\nvermiculite and perlite mixtures found optimal survival \n\nof 100% on peatmoss alone [23]. Further work may be \n\ncarried out to determine whether Golsai, Sikkimae, \n\nDambarsai, and Ramsai varieties respond differently to \n\nother auxins and cytokinins to optimize the protocol \n\nfurther. Establishments of in vitro mother stock healthy \n\nplantlets is the major outcome of the present study. This \n\nwill allow for ongoing propagation of clones year-round \n\nand will no longer be dependent on rhizome formation. \n\nThis will also reduce the risk of disease contamination \n\nbeing introduced by continued field collection and allow \n\nclonal propagation every 60 days which will increase \n\nproductivity to meet grower demand.  \n\nConclusion \n\nThe present investigation revisited the protocol that was \n\nalready established to optimize the hormonal \n\nconcentrations for maximum plant growth.  In this \n\nregard, the present study has developed the protocol \n\nwith the use of a lower concentration of hormone for the \n\nfour varieties of plant. The MS media composition with \n\n1mg/L BAP + 0.5mg/L IBA can be recommended as \n\nstandard protocol for cardamom at this stage. Similarly, \n\nsubstrate (cocopeat: soil 1:2; moss: cocopeat 1:2 and \n\nsawdust: cocopeat 1:2) depending upon the availability \n\ncan used for acclimatization process. Further research \n\nmay elucidate better protocols including the use of \n\ndifferent plant growth regulators, carbon sources, and \n\nnutrient sources. Similarly, a two-stage process should be \n\nstudied; where shoot production is carried out on media \n\ncontaining optimal BAP concentrations per variety, and \n\ntransferred to cytokinin free media for root development.   \n\nAuthor Contributions \n\nAuthor 1 and 2 designed the research experiment and \n\nperformed the entire research. Author 3 established the \n\nin-vitro plantlet. Author 2 wrote the manuscript. Authors \n\n1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12 revised the manuscript. \n\nAuthor 6 supervised the entire research work.  \n\nCompeting Interests \n\nThe authors declare no competing interests.  \n\nFunding \n\nThis study was funded by the Nepal Agricultural \n\nResearch Council.  \n\nAcknowledgements \n\nWe would like to acknowledge the support and \n\ncommitment of all staff involved in the cardamom \n\nproduction program at the Pakhribas Agricultural \n\nResearch Station.  \n\nData availability \n\nPlease contact the corresponding author for access to the \n\ndata.  \n\nEthical Approval \n\nNo ethical approval was required, sought or granted for \n\nthis study.  \n\nReferences \n1.", "start_char_idx": 17361, "end_char_idx": 20990, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0027f0a7-4a99-44c6-8b27-c9bb404f28bd": {"__data__": {"id_": "0027f0a7-4a99-44c6-8b27-c9bb404f28bd", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6e6e2248-0641-493f-9e5e-65eeb3e8bc27", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "f4e8fe086a61d47f17ac10e4374f98cbefb6eacafd9c10e08e2e5e47f900ba33", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b738f19f-36b8-4c8e-803b-ccc09ae1fc2f", "node_type": "1", "metadata": {}, "hash": "e1eba5c9527814d73c6c3347615968e49551985a2da370f72645df9b2a19d165", "class_name": "RelatedNodeInfo"}}, "text": "References \n1. Gautam N, et al., Technology, chemistry and bioactive properties of \n\nlarge cardamom (amomum subulatum roxb.): an overview. Int. J Appl. \nSci. Biotech.2016. 4(2): p. 139-149. http://dx.doi.org/ \n10.3126/ijasbt.v4i2.15104 \n\n2. Khatiwada A, Subedi A, Dangol R. A Review on Status of \nProduction of Large Cardamom in Nepal and its Marketing in \nNational and Global Scenario. Malay. J Halal Res. 2019. 2(1): p. 16-\n21.http://dx.doi.org/10.2478/mjhr-2019-0004 \n\n3. Rao, Y., et al., Large cardamom (Amomum subulatum Roxb.)-a \nreview. 1993 \n\n4. Deka T,  Sudharshan M,  Saju K.  New record of bumble bee, \nBombus breviceps Smith as a pollinator of large cardamom. Current \nSci. 2011. 100(6): p. 926-928 \n\n5. Mandal B, Shilpi S, Barman AR, Mandal S, Varma A. Nine novel \nDNA components associated with the foorkey disease of large \ncardamom: evidence of a distinct babuvirus species in \nNanoviridae. Virus Res. 2013;178(2):297-305. doi:10.1016/ \nj.virusres.2013.09.027 \n\n6. Mandal B, Vijayanandraj S, Shilpi S, Pun KB, Singh V, Pant RP, Jain \nRK, Varandarasan S, Varma A. Disease distribution and \ncharacterisation of a new macluravirus associated with chirke \ndisease of large cardamom. Ann. Appl. Biol. 2012; 160(3): p. 225-\n236.https://doi.org/10.1111/j.1744-7348.2012.00537.x \n\n7. Saju K A, Deka T N, Sudharshan M R, Gupta U, Biswas A K. \nIncidence of Phoma leaf  spot  disease of large cardamom \n(Amomum subulatum Roxb.) and in vitro evaluation of fungicides \nagainst the pathogen. J  Spice  Arom. Crops. 2011; 20(2): p. 86-88 \n\n8. Shrestha KP. SWOT Analysis of Large Cardamom in Ilam District, \nNepal. Asian J. Res Agri. Forest. 2018; p. 1-7. \nhttp://dx.doi.org/10.9734/ AJRAF/2018/46009 \n\nhttp://dx.doi.org/10.2478/mjhr-2019-0004\nhttps://doi.org/10.1111/j.1744-7348.2012.00537.x\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 - 6      Balzer et al.  \n\n\u00a9NJB, BSN   6 \n\n9. Aryal M R, Paudel N, Ranjit M. Elimination of Chhirkey and \nFoorkey viruses from meristem culture of large Cardamom \n(Amomum subulatum Roxb.). European Online Int. J. Nat. Soc. 2018; \n7(2): p. pp. 424-443 \n\n10.Pradhan S, Pradhan S, Basistha BC, Subba KB. In vitro \nmicropropagation of Amomum subulatum (Zingiberaceae), a major \ntraditional cash crop of Sikkim Himalaya.", "start_char_idx": 20976, "end_char_idx": 23232, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b738f19f-36b8-4c8e-803b-ccc09ae1fc2f": {"__data__": {"id_": "b738f19f-36b8-4c8e-803b-ccc09ae1fc2f", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0027f0a7-4a99-44c6-8b27-c9bb404f28bd", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "d3cb2f1e52478bd0128f0c293e1fb3fc259eb21ac8fbadd312d6863b09b2ae70", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a050fbdb-9e4a-40bd-b2d7-66e9566a4ff4", "node_type": "1", "metadata": {}, "hash": "d5c88defa793325780c0ae91cce9838b873f826daa8513f075f7e0633443c9cd", "class_name": "RelatedNodeInfo"}}, "text": "International Journal of Life \nSciences Biotechnology and Pharma Research, 2014. 3(2): p. 169 \n\n11. Shah S, Shrestha R, Maharjan S, Selosse MA, Pant B. Isolation and \nCharacterization of Plant Growth-Promoting Endophytic Fungi \nfrom the Roots of Dendrobium moniliforme. Plants (Basel). \n2018;8(1):5. doi:10.3390/plants8010005 \n\n12. Poudel K, Prasai HK, Shrestha J. Micropropagation and \nAcclimatization of Large Cardamom (Amomum subulatum Roxb.) \nTurk. J  Bot. 2018; 5(3): p. 231-235. http://dx.doi.org/10.30910/ \nturkjans.448318 \n\n13. Purohit S, Nandi SK, Paul S, Tariq M, Palni LMS. \nMicropropagation and genetic fidelity analysis in Amomum \nsubulatum Roxb.: A commercially important Himalayan plant. J \nAppl. Res.  Med.  Arom. Plants. 2017; 4(1): p. 21-\n26.https://doi.org/10.1016/j.jarmap.2016.07.003 \n\n14. Jing H, Strader LC. Interplay of auxin and cytokinin in lateral root \ndevelopment. Int. J Mol. Sci. 2019. 20(3); p. 486. \nhttps://doi.org/10.3390/ ijms20030486 \n\n15.  Kieber JJ, Schaller GE. Cytokinin signaling in plant development. \nDevelopment. 2018; 145(4): p. dev149344. https://doi.org/ \n10.1242/ dev.149344 \n\n16. Laplaze L, Benkova E, Casimiro I, Maes L, Vanneste S, Swarup \nR,et al. Cytokinins act directly on lateral root founder cells to \n\ninhibit root initiation. The Plant Cell. 2007; 19(12): p. 3889-\n3900.10.1105/tpc.107.055863 \n\n17. Khairudin NA, Haida Z,   Hakiman M. In Vitro Shoot and Root \nInduction of Kaempferia parviflora (Zingiberaceae) Rhizome Using \n6-Benzylaminopurine. J. Trop. Plant Physiol. 2020; 12(2): p. 23-32 \n\n18. Nwaoguala CNC, Shittu HO. Effects of growth regulators and \ntype-variety of oil palm (Elaeis guineensis Jacq.) on direct \norganogenesis. Not. Sci. Biol. 2018; 10(2): p. 251-\n258.http://dx.doi.org/10.15835/nsb10210234 \n\n19. Srivastava A, Diengdoh LC, Rai R, Bag TK, Singh BP. In vitro \nmicropropagation and micro-tuberization potential of selected \npotato varieties. Ind. J  Hill Farm. 2012; 25(2): p. 14-17 \n\n20. Son N V, Mokashi AN, Hiegde RV, Patil VS, Lingaraju S. \nResponse of gerbera (Gerbera jamesonii Bolus) varieties to \nmicropropagation. Karnataka J Agri. Sci. 2011; 24(3) \n\n21. Eftekhari M, Alizadesh M, Mashayekhi K, Asghari HR. In vitro \npropagation of four Iranian grape varieties: influence of genotype \nand pretreatment with arbuscular mycorrhiza. Vitis.", "start_char_idx": 23233, "end_char_idx": 25560, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a050fbdb-9e4a-40bd-b2d7-66e9566a4ff4": {"__data__": {"id_": "a050fbdb-9e4a-40bd-b2d7-66e9566a4ff4", "embedding": null, "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-224", "node_type": "4", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "4073e926f72b448243ecb7eb9e4e2a643245fb2a951d447f38ad07b63839e94e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b738f19f-36b8-4c8e-803b-ccc09ae1fc2f", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "f62046787cde0571af1df24a11313db38696e5f5489c2c3950c551a1ce41f13f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2b65b3f5-a5c6-455f-ad64-603a03e48b70", "node_type": "1", "metadata": {}, "hash": "6e6de3b0b986fe87b73a68860224c73455ff0f3d367c837e8fbfb4d2ef9caa46", "class_name": "RelatedNodeInfo"}}, "text": "Vitis. 2012; 51(4): p. \n175-182 \n\n22. Lincy A,  Sasikumar B. Enhanced adventitious shoot regeneration \nfrom aerial stem explants of ginger using TDZ and its histological \nstudies. Turk. J  Bot. 2010; 34(1): p. 21-\n29.https://doi.org/10.3906/bot-0805-6 \n\n23. Abbas MS,  Taha HS, Aly UI, El-Shabrawi HM,  Gaber ES. In vitro \npropagation of ginger (Zingiber officinale Rosco). J Genet Eng \nBiotechnol. 2011; 9(2): p. 165-172.https://doi.org/10.1016/ \nj.jgeb.2011.11.002 \n\n \n\nhttp://dx.doi.org/10.15835/nsb10210234\nhttps://doi.org/10.3906/bot-0805-6", "start_char_idx": 25554, "end_char_idx": 26099, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2b65b3f5-a5c6-455f-ad64-603a03e48b70": {"__data__": {"id_": "2b65b3f5-a5c6-455f-ad64-603a03e48b70", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a050fbdb-9e4a-40bd-b2d7-66e9566a4ff4", "node_type": "1", "metadata": {"identifier": "njb-224", "author": "Balzer, Anna; Shah, Sujit; Kumar Ale, Yam; Adhikari, Dipti; Niroula, Sanjit; Mandal, Jawaharlal; Bahadur Pun, Amar; Poudel, Krishna; Bhantana, Parashuram; Timsina, Govinda; Karn, Ravindra; Karki, Sujwal", "title": "Development of Effective Protocol for Four Varieties of Large Cardamom", "date": "2022-07-30", "file": "njb-224.pdf"}, "hash": "6c5655e218d3275ceb6ea7484f7f7ccd114f207294aeaa7e4568641e08eb4c8f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bc319560-4973-4fe8-9978-c0e52de056a4", "node_type": "1", "metadata": {}, "hash": "dd480762cd96df5eab4f173831022cc22815d71f7486bf1cdddd1ebb793e2883", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 7-12  Research article  DOI: https://doi.org/10.54796/njb.v10i1.225   \n\n\u00a9NJB, BSN   7 \n\nMicrobial Quality Assessment of Raw Freshwater Fish Sold in Local \nMarkets of Kathmandu Valley \nShristi Prasai, Puja Shrestha, Sriniwas Pandey, Ishika Adhikari, Srijana Gurung, Kamil Prajapati   \n\nSt. Xavier\u2019s College, Maitighar, Kathmandu, Nepal \n\nReceived: 14 May 2022; Revised: 8 Jul 2022; Accepted: 15 Jul 2022; Published online: 30 Jul 2022 \n\nAbstract \nMicrobial quality of Labeo rohita, Cyprinus carpio and Clarias batrachus collected from the markets of Kathmandu \nvalley was evaluated. 9 freshwater fish (skin, gills, intestine) were sampled and were analyzed for Total Plate \nCount (TPC), Total Coliform Count (TCC) and Total Fecal Coliform Count (TFCC). The average TPC ranged from \n4.1 x 107 to 1.02 x 108 cfu/gm, with the highest count in C. batrachus and the lowest in C. carpio, whereas the organ \nwise load was the highest in intestine with 1.3 x 108 cfu/gm and the lowest in skin with 1.02 x 107 cfu/gm. The \nhighest TCC and TFCC was found in C. carpio and C. batrachus respectively, whereas organ wise distribution \nshowed the highest count in intestine for both TCC and TFCC. The pathogens isolated from the samples were \nEscherichia coli, Staphylococcus aureus, Coagulase negative Staphylococcus (CoNS), Vibrio cholerae, Salmonella Typhi \nand S. Paratyphi. E. coli was isolated from 67% of L. rohita, 44.44% of C. carpio and 66.67% of C. batrachus. S. aureus \nwas isolated from 44.44% of both L. rohita and C. batrachus whereas 55.55% of C. carpio. CoNS were isolated from \n33.33% of L. rohita, 22.22% of C. carpio and 33.33% of C. batrachus. S. Typhi was isolated from 11.11% of C. carpio \nand 22.22% of C. batrachus. S. Paratyphi was isolated from 11.11% of both L. rohita and C. batrachus, V. cholerae \nwas isolated from 11.11% of L. rohita, 33.33% of C. carpio and 22.22% of C. batrachus. The observation of this study \nshowed higher bacterial load in all of the fishes above the acceptance level and presence of Total Coliform, Fecal \nColiform and potential human pathogens suggests that the microbial quality of the fish available in the market \nis not satisfactory. Hence, the fishes possess a threat to public health safety and there is an urgent need to improve \nthe Quality Control and Quality Assurance Systems for fish markets of Kathmandu valley. \n\nKeywords: Raw fish, microbial quality, E. coli, S. aureus, Vibrio cholerae. \n\n Corresponding author, email:  kalkam2013@gmail.com \n\nIntroduction \nFish is one of the chief sources of protein and has \n\nremained an important part of consumption for many \n\ncenturies[1].", "start_char_idx": 48, "end_char_idx": 2729, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bc319560-4973-4fe8-9978-c0e52de056a4": {"__data__": {"id_": "bc319560-4973-4fe8-9978-c0e52de056a4", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2b65b3f5-a5c6-455f-ad64-603a03e48b70", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "1e5b0536502d067607d3fc573c54be8f85034f81c051f0d3583e66c1d3f68342", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3537470a-251b-424c-abfc-e91564527a37", "node_type": "1", "metadata": {}, "hash": "fabafbfbc60c4b9c63b236ea9005c460d04c5d31592d957635993cd6397fc613", "class_name": "RelatedNodeInfo"}}, "text": "The poikilothermic nature of fresh fish \n\nallows a wide variety of bacteria such as Pseudomonas, \n\nMoraxella, Acinetobacter, Shewanella, Flavobacterium, and \n\nVibrio among Gram negative and Gram-positive bacteria \n\nsuch as Bacillus, Micrococcus, Clostridium, Lactobacillus, \n\nand Corynebacterium [2].  The non-indigenous ones that \n\ncontaminate the fish or the habitat include Escherichia coli, \n\nClostridium botulinum, Aeromonas, Shigella dysenteriae, \n\nStaphylococcus aureus, Listeria monocytogens and Salmonella \n\nspp. The indigenous bacterial pathogens that are found \n\nnaturally in the fish habitat are Vibrio spp. and Aeromonas \n\nspp. [3].  \n\nMicrobiological quality of raw fish results from \n\nmicrobiological load of aquatic habitat, methods of \n\ncapture, transportation, chilling and storage conditions \n\nimposing a threat of food-borne infections as the \n\npathogen can be conveyed to consumers at retail level \n\nthrough raw fish. Coliform, especially Escherichia coli are \n\noften used as criteria to assess the quality and safety of \n\nfoods [1]. Extraneous bacteria, Escherichia coli is the fecal \n\nindicator capable of surviving in fish and found to be \n\nsurviving and even multiplying in the digestive tract of \n\nrainbow trout (Oncorhynchus mykiss) [4]. \n\nWater being the habitat, fish is continually bathed in \n\naqueous suspension of various microorganisms and their \n\nexterior surface, hence is in constant contact with these \n\norganisms. Some of the microorganisms may colonize the \n\nexternal parts of fish becoming the resident microflora. \n\nThe presence of the microflora adds to the defense system \n\nof fish, thereby inhibiting the accession and consequent \n\ncolonization by other potential pathogens. Obviously, the \n\nbacterial flora of fish depends on the fish\u2019s recent intake \n\ndiet and the extent of contamination in the food [5].  \n\nA study performed on fish skin sampled from the lake \n\nHawassa of Southern Ethiopia by [6], resulted in finding \n\nof the pathogenic strains of E. coli to be contaminating the \n\nfish with statistically significant results as defense to the \n\nfact that fish are contaminated enough to cause food-\n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-9739-4848\nmailto:kalkam2013@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1): 7-12     Prasai et al.  \n\n\u00a9NJB, BSN   8 \n\nborne illness. According to Sichewo et al. (2014)  [7], \n\ninspection of various organs of fish such as skin, intestine, \n\ngills and mouth collected from different fish ponds of \n\nZimbabwe showed presence of Salmonella Typhi from \n\nNhengo, Imbayago and Nyamakwe. According to \n\nKumari et al. (2001) [8], gill and intestine samples of Labeo \n\nrohita when processed revealed to be contaminated with \n\ncoagulase negative Staphylococcus spp. and Staphylococcus \n\naureus. According to Xu et al. (2019) [9], V. cholerae was \n\ndetected from the intestines of ten freshwater fish species \n\ncollected, including Astatotilapia flaviijosephi, Barbus \n\nlongiceps,  C. idella, Cyprinus carpio, Mugil cephalus, \n\nMyripristis murdjan, Oreochromis aureus, Sarotherodon \n\ngalilaeus, and Tilapia spp. and Tilapia zilli. \n\nImproper handling or consumption of undercooked or \n\nraw fish may contribute to the intake of pathogens which \n\ncan potentially cause diseases.", "start_char_idx": 2730, "end_char_idx": 6167, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3537470a-251b-424c-abfc-e91564527a37": {"__data__": {"id_": "3537470a-251b-424c-abfc-e91564527a37", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bc319560-4973-4fe8-9978-c0e52de056a4", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "cef1ad751f98ad295012dfb456a4e2e4c3f26fbbe6998780c1cfdabefcdb6334", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d53b3d6a-9f55-40e3-8c23-5a555b17e050", "node_type": "1", "metadata": {}, "hash": "433a7bcfe339550fd153f860da9841d41f6f1bb36e91aa9456cf964c49833be1", "class_name": "RelatedNodeInfo"}}, "text": "Diseases in human that \n\ncan be caused by bacteria present in fish includes food \n\npoisoning and gastroenteritis, diarrhoea, superficial \n\nwound infections and ulcers, bacillary dysentery \n\n(shigellosis), clonorchiasis, dracunculiasis and \n\nparagonimiasis due to larvae and metacercariae ingested \n\nin fish and crustaceans, cholera, typhoid and \n\nparatyphoid, etc. [4]. Salmonella, Staphylococcus spp., \n\nEscherichia spp., Vibrio parahaemolyticus, Clostridium \n\nperfringens, Clostridium botulinum E and Enteroviruses are \n\nheld responsible for majority of fish concerned with food \n\nborne diseases [2]. \n\nThe safety concerns instigated the need for the study i.e. \n\nto investigate the presence of any human pathogenic \n\nbacteria from live freshwater fishes that are popularly \n\nconsumed in Kathmandu valley. \n\nMaterials and methods  \nCollection of fish sample \nSimple random sampling method was used for selecting \n\nsamples. The freshly slaughtered 9 fish samples were \n\ncollected on different day from different retail markets of \n\nKathmandu valley (Sundhara, Baneshwor, Thapagaun, \n\nGairigaun, Ghatthaghar) and brought to the laboratory in \n\nan icebox. Fishes were killed by the retailers without \n\ncausing any physical injury just before the sample \n\ncollection. The three species of fish, L. rohita (rohu), C. \n\ncarpio (common carp) and C. batrachus (mungri) were \n\nchosen based on popularity among the consumers and \n\ncommercial availability.  \n\nSample preparation and processing \nSample preparation was done according to Sichewo et al. \n\n(2014) [7]. Fish was cut ventrally to collect intestine and \n\ngills using sterile surgical blades and forceps in aseptic \n\ncondition. One g of intestine and gills were taken and \n\ncrushed into fine solution by adding 10ml of normal \n\nsaline in sterile mortar, from where one ml of aliquot \n\nvolume was taken and serially diluted up to 10-5. Skin \n\nsamples were taken by rolling sterile cotton swabs all \n\nover the skin surfaces of all 9 fish and then inoculated into \n\n10ml of normal saline. It was then serially diluted up to a \n\ndilution of 10-5.  \n\nBacteriological analysis of fish samples \nFor total plate count (TPC), 0.1ml sample from 10-3 and \n\n10-5 dilutions were taken and spread plating was done on \n\nPCA agar. The plates were incubated for 24 hours at 37\u00b0C. \n\nFor total coliform count (TCC) and total fecal coliform \n\ncount (TFCC), spread-plating was done from 0.1 ml of \n\nevery sample of 10-3 dilutions in VRBA agar plates and \n\nincubated at 37\u00b0C and 44.5\u00b0C, respectively for 24 hours. \n\nFor isolation of Salmonella spp. one ml of sample was \n\ninoculated in 9 ml of enrichment media, Selenite F Broth, \n\nfrom where a loopful of sample was taken and cultured \n\non SS Agar. For Vibrio cholerae, one ml of sample was \n\ninoculated in 9ml of enrichment media, alkaline peptone \n\nwater, incubated at 37\u00b0C for 24 hours. A loopful of \n\nsample was taken from enrichment broth and cultured on \n\nTCBS Agar. A loopful of original sample was streaked on \n\nMacConkey Agar (for E. coli) and on Mannitol Salt Agar \n\nplate (for Staphylococcus aureus) and the plates were \n\nincubated at 37\u00b0C for 24 hours. The colonies obtained \n\nfrom TCBS, SS, MA and MSA were further sub-cultured \n\non NA [10] and identified by gram staining and \n\nbiochemical tests (IMViC, TSIA, Urease, Catalase, \n\nOxidase, Oxidative/fermentative test).", "start_char_idx": 6168, "end_char_idx": 9529, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d53b3d6a-9f55-40e3-8c23-5a555b17e050": {"__data__": {"id_": "d53b3d6a-9f55-40e3-8c23-5a555b17e050", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3537470a-251b-424c-abfc-e91564527a37", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "cb2fc4956a57d96232f1d5ee3a25e850670c96afc701d37c46845054b688a104", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5fd477f6-eb9d-41b7-8109-b6699bf9a066", "node_type": "1", "metadata": {}, "hash": "1287151e93daab7f47761d0ca77199b5ad6f096eaf5bf2736e72f753c1f43fb4", "class_name": "RelatedNodeInfo"}}, "text": "For Vibrio cholerae \n\nstring test was also performed while Staphylococcus aureus \n\nwas confirmed by coagulase test. \n\nResults  \nTable 1. Average TPC of fish samples \n\nSample \nOrgans (cfu/gm in average)  \n\nSkin Gills Intestine Average \n\nLR 6.5 x 106 8.2 x 107 8.05 x 107 5.6 x 107 \n\nCC 1.23 x 107 2.96 x 107 8.03 x 107 4.1 x 107 \n\nCB 1.2 x 107 6.6 x 107 2.3 x 108 1.02 x 108 \n\nAverage 1.02 x 107 5.9 x 107 1.3 x 108  \n\nNote: LR = Labeo rohita, CC = Cyprinus carpio and CB = Clarias batrachus \n\n \n\nIn this study, 9 fish sample of 3 different varieties, L. rohita \n\n(LR), C. carpio (CC) and C. batrachus (CB) were analyzed \n\nfor its microbial quality and antimicrobial susceptibility \n\ntesting was done for the isolated strain. The average TPC \n\nof LR, CC and CB ranged from 4.1 x 107 to 1.02 x 108 \n\ncfu/gm (Table 1). The highest bacterial load was found \n\nin C. batrachus whereas the lowest in C. carpio. Among the  \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1): 7-12     Prasai et al.  \n\n\u00a9NJB, BSN   9 \n\n body parts, intestine was found to be highly loaded with \n\nthe bacteria in all the fishes. \n\n Total coliforms were present in all parts of the three \n\nspecies of fishes. TCC ranged from 2.47 x 105 to 8.7 x 105 \n\ncfu/gm while TFCC was found to be 1.6 x 105 to 3.07 x 105 \n\ncfu/gm (Table 2). Fecal coliforms were present in all \n\nparts of all the analyzed fishes except for skin of L. rohita, \n\nand skin and gills of C. carpio. All parts of C. batrachus \n\nshowed presence of fecal coliforms. \n\n Six different organisms were isolated from gills, skin and \n\nintestine of the three fish samples (Table 3). In case of L. \n\nrohita, gills were found to harbor the diverse species of \n\nbacterial pathogens compared to skin and intestines. Gills \n\nwere found to harbor S. aureus (66.67%), V. cholerae \n\n(33.33%), E. coli (100%) and CoNS (33.33%). E. coli, S. \n\naureus and CoNS each were present in 66.67% of skin \n\nsample. E. coli was found in 100% and S. Paratyphi in \n\n33.33% of intestine. S. Typhi was not isolated from any of \n\nthe three body parts. As for C. carpio, S. aureus was \n\npresent in 100% of gills, 66.67% of skin. V. cholerae was \n\nfound in 66.67% of gills and 33.33% of intestine. E. coli \n\nwas found in 66.67% of gills and 33.33% of both skin and \n\nintestine. CoNS were present in 33.33% of both skin and \n\ngills. S. Typhi was present in only 33.33% of intestine \n\nwhereas S. Paratyphi was not isolated from any of the \n\nthree body parts. In case of C. batrachus, gills showed the \n\nprevalence of most of the type of bacteria. E. coli was \n\npresent in 100% of gills, 66.67% of intestines and 33.33% \n\nof skin.", "start_char_idx": 9530, "end_char_idx": 12156, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5fd477f6-eb9d-41b7-8109-b6699bf9a066": {"__data__": {"id_": "5fd477f6-eb9d-41b7-8109-b6699bf9a066", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d53b3d6a-9f55-40e3-8c23-5a555b17e050", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "7b9cf2a0cccefc1a034c83d5644407fea6f6f407b4908f67ca58481c81efd204", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e13b9180-e5a7-4cd3-bcc5-cb2d8a293f41", "node_type": "1", "metadata": {}, "hash": "5995ed0f629ee57cdb424ead36ce634f03fab7a13e426852f999be5e36b99583", "class_name": "RelatedNodeInfo"}}, "text": "S. aurues was in 100% of skin and 33.33% of gills. \n\nCoNS was present in 66.67% of skin and 33.33% of gills. \n\nS. Typhi was present in 33.33% of gills and intestines \n\neach. \n\nDiscussions \nThis study was carried out in the quest to determine the \n\nmicrobial quality of raw and freshly killed freshwater \n\nfishes available in retail markets of Kathmandu valley. \n\nThe skin, gills, and intestine of the samples were \n\nanalyzed for TPC, TCC and TFCC.  \n\nThe average total plate count of L. rohita, C. carpio and C. \n\nbatrachus was found to be 5.6 x 106, 4.1 x 107, 1.02 x 108, \n\nrespectively indicating that latter had higher microbial \n\nload than the other two (Table 1). As for organ-wise \n\ndistribution, skin of L. rohita contained least microbial \n\nload compared to gills and intestine where as in C. \n\nbatrachus intestine had the highest microbial load.  \n\nHowever, in C. carpio, all three organs had similar \n\nTable 2. Average TCC and TFCC of fish samples \n\n \n\nSample \n\nTotal Coliform Count (cfu/gm) Total Fecal Coliform Count (cfu/gm) \n\nSkin Gills Intestine Average Skin Gills Intestine Average \n\nLR 1.5 x 105 3.5 x 105 2.4 x 105 2.47 x 105 No growth 9.4 x 106 2.6 x 105 1.77 x 105 \n\nCC 1.2 x 106 2.1 x 105 1.2 x 106 8.7 x 105 No growth No growth 1.6 x 105 1.6 x 105 \n\nCB 7.6 x 105 2 x 105 TMTC 4.8 x 105 1.7 x 105 2.9 x 105 4.6 x 105 3.07 x 105 \n\nAverage 7.03 x 105 2.53 x 105 7.2 x 105  1.7 x 105 1.92 x 105 2.93 x 105  \n\nNote: LR = Labeo rohita, CC = Cyprinus carpio, CB = Clarias batrachus, TMTC = Too many to count \n\n \n\n \nTable 3. Distribution of bacteria in skin, gills and intestine of fish samples. \n\nSample \nOrgan \n(N=3) \n\nIsolates \n\nS. aureus S. Paratyphi S. Typhi V. cholerae E. coli CoNS \n\nLR \n\nSkin  2(66.67%) ND ND ND ND 2 (66.67%) \n\nGills 2 (66.67%) ND ND 1 (33.33%) 3 (100%) 1 (33.33%) \n\nIntestine  ND 1 (33.33%) ND ND 3 (100%) ND \n\nCC \n\nSkin  2(66.67%) ND ND ND 1 (33.33%) 1 (33.33%) \n\nGills  3 (100%) ND ND 2 (66.67%) 2 (66.67%) 1 (33.33%) \n\nIntestine  ND 1 (33.33%) ND 1 (33.33%) 1 (33.33%) ND \n\nCB \n\nSkin  3 (100%) ND ND ND 1 (33.33%) 2 (66.67%) \n\nGills  1 (33.33%) 1 (33.33%) ND 2 (66.67%) 3 (100%) 1 (33.33%) \n\nIntestine  ND 1 (33.33%) 1 (33.33%) ND 2 (66.67%) ND \n\n% = calculated according to total number of fish samples (3 each) \nND= Not detected \nNote: LR = Labeo rohita, CC = Cyprinus carpio, CB = Clarias batrachus, TMTC = Too many to count", "start_char_idx": 12157, "end_char_idx": 14529, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e13b9180-e5a7-4cd3-bcc5-cb2d8a293f41": {"__data__": {"id_": "e13b9180-e5a7-4cd3-bcc5-cb2d8a293f41", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5fd477f6-eb9d-41b7-8109-b6699bf9a066", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "78f15185c6db5f35f453136f7ced2c64398b0411d08682d59fb7ba1f9213c3c1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f000ccba-089c-40d7-8a08-261180ee4afb", "node_type": "1", "metadata": {}, "hash": "f0f19c2c500933f41807d620bb712bebe8b0c63cf0266bab54ed464da9cc70bc", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1): 7-12     Prasai et al.  \n\n\u00a9NJB, BSN   10 \n\nmicrobial load. High load of bacteria possibly resulted \n\nfrom keeping fishes in wells with contaminated water. \n\nTotal bacterial count of more than 105 cfu per gram \n\nelevates the concern of hygiene. According to ICMSF \n\n(2011), for a newly caught fish, aerobic count may range \n\nfrom 104 to 107 cfu per cm2. Much lower count is \n\nassociated with properly skinned ones [11]. Detectable \n\nspoilage is usually associated with spoilage bacteria \n\nexceeding 107 cfu per gram. Goja (2013) did similar study \n\nin three freshwater fish and found that viable bacterial \n\ncounts in intestine and skin ranged from 1.5 x 103 to 8.4 x \n\n104 cfu per gram and 2.8 x 103 to 9.8 x 103 cfu per gram \n\nrespectively which is much lower than in the three fish \n\nsamples in this study[12]. Comparable research \n\nconducted on 150 fish samples from Nile bream by \n\nSichewo et al. (2014) revealed the TPC of intestine, skin, \n\ngills ranging from 4.6 x 103 to 8.03 x 103 cfu per gram [7].  \n\nEvery organ of the fish samples was found to contain \n\ntotal coliform (Table 2). Similarly, presence of fecal \n\ncoliforms was also true for all samples analyzed except \n\nskin of L. rohita and skin and gills of C. carpio. All samples \n\ncontained fecal coliform in at least one of the body part \n\nwhereas the coliform count was the highest in the \n\nintestines. According to Liu et al. (2016), gut microbiota \n\nand their diversity is influenced by many independent \n\nfactors as different niches have variation in diet \n\navailability [13]. In addition, gut microbiota is also \n\ninfluenced by metabolic capacity and gut content enzyme \n\nactivity. Coliform or fecal coliforms are not considered to \n\nbe normal flora of fish which reflects contamination of \n\nfish during transportation, handling or during rearing in \n\nwater contaminated with human or animal waste [3]. \n\nFour of the fish samples had a very high count of coliform \n\nin their intestines (TMTC). The feed and trophic level \n\n(carnivores, omnivores and herbivores) might have some \n\nassociation with the intestinal microbiota. The salinities \n\nof water in fish habitat also have some influence on the \n\nmicrobiota of fish intestine [14]. \n\nPrevalence of microorganisms varied among the three \n\nfish samples analyzed with E. coli being most common \n\nisolate followed by S. aureus and CoNS (Figure 1). \n\nPrevalence of V. cholerae was higher among C. carpio, \n\nwhile S. Typhi is absent in L. rohita and S. Paratyphi was \n\nabsent in C. carpio. E. coli was present in the gills of L. \n\nrohita (100%), C. carpio (66.67%) and C. batrachus (100%) \n\nrespectively (Table 3). Similar study conducted by \n\nYogoub (2009) revealed that E. coli was the most \n\ndominant isolate from fishes [2]. Enterobacteriaceae \n\ngenera were isolated from gills, skin, intestine and \n\nmuscles of 83 out of 150 randomly collected fishes which \n\nalso included pathogenic Salmonella and Shigella spp. \n\nDuring rainy season due to rain surface runoff of organic \n\nmatters into water bodies are increased which favors \n\nmultiplication of bacteria. Previous study performed by \n\nShabeeb et al. (2016) revealed that most isolates were \n\nGram negative rods [15].", "start_char_idx": 14537, "end_char_idx": 17776, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f000ccba-089c-40d7-8a08-261180ee4afb": {"__data__": {"id_": "f000ccba-089c-40d7-8a08-261180ee4afb", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e13b9180-e5a7-4cd3-bcc5-cb2d8a293f41", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "6137ac91bf5bebd112c8103a8ff5b1d0ae9425d6268d6ce6f5498a84a5e24a05", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6faefa82-705d-43cc-a80b-e2e440b5dffc", "node_type": "1", "metadata": {}, "hash": "f6d030bf64628d528487a69d9af5508b6a6b5f7ee56cb395df8baefa7971007a", "class_name": "RelatedNodeInfo"}}, "text": "(2016) revealed that most isolates were \n\nGram negative rods [15]. \n\n \nFigure 1: Distribution of pathogenic bacteria in L. rohita, C. \ncarpio and C. batrachus \n%= Calculated according to the total number of samples (9 each) \n\nSalmonella being enteric bacteria, their presence in \n\nfreshwater fish undoubtedly attributes to fecal \n\ncontamination of such source from where it is harvested. \n\nPonds with higher temperature or from hot regions \n\nharbors more Salmonella with high prevalence rates. \n\nAccording to Bibi et al. (2015) there has been occurrence \n\nof Salmonella Typhi in freshwater fish like Labeo rohita \n\n(skin, gills, intestine) and Cyprinus carpio (intestine)[16]. \n\nIn our study, S. Typhi was isolated from one sample each \n\nof gills and intestine of C. batrachus and a sample of \n\nintestine of C. carpio while none of the sample from L. \n\nrohita showed the presence of the bacterium (Table 3). As \n\nfor S. Paratyphi, it was not isolated from any of the tested \n\nsamples of C. carpio but isolated from one sample of \n\nintestine of both L. rohita and C. batrachus. It was noted \n\nthat none of the skin samples of all the three fish tested \n\nshowed the presence of Salmonella spp. However, these \n\nisolates were not confirmed through serological tests.   \n\nAll 3 gills samples of the C. carpio contained \n\nStaphylococcus aureus (Table 3). Intestines of L. rohita and \n\nC. batrachus were not found to contain the pathogen. \n\nStaphylococcus is related with unhygienic handling as \n\nthese are inhabitant of human skin [17]. Presence of such \n\nopportunistic human pathogens advocates the possibility \n\nof cross contamination between handlers and fish. A \n\nstudy conducted in Andhra Pradesh, India by Bujjamma \n\nand Padmavathi (2015) in 192 fish samples disclosed that \n\nover 24.47% of fish were contaminated with S. aureus \n\nwhich also included similar samples [18]. Similarly, \n\nsampled skins were mostly found to harbor these \n\n0\n\n10\n\n20\n\n30\n\n40\n\n50\n\n60\n\n70\n\nS. aureus S.\nParatyphi\n\nS. Typhi V.\ncholerae\n\nE. coli CoNS\n\nP\ner\n\nce\nn\n\nta\ng\n\ne \n(%\n\n)\n\nLabeo rohita\n\nCyprinus carpio\n\nClarias batrachus\n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1): 7-12     Prasai et al.  \n\n\u00a9NJB, BSN   11 \n\npathogens. However, all intestine samples were negative \n\nfor CoNS. Skin was observed as the most contaminated \n\narea with CoNS and Staphylococcus aureus. Composition \n\nand count of microflora in fish is a function of water \n\nquality, age, type of species and fish [19] along with \n\nrearing density and diet impact [20]. In relation to this, \n\nhazard escalates when livestock manure is fed to fish \n\nwhich is also the case in Nepal. As an established fact, \n\nchicken has been remarkably associated with a high load \n\nof Salmonella.  \n\nIn this study, V. cholerae was isolated from gills of all three \n\ntypes of fish and from intestines of C. carpio (Table 3). \n\nSamples were expected to yield positive results for V. \n\ncholerae as this bacterium is the indigenous member of \n\nfish associated aquatic habitats. However, their \n\ndistribution was outnumbered by that of E coli and S. \n\naureus. Gills bear a high load of bacteria as a consequence \n\nof water and organic substance precipitation[19, 21]. A \n\nstudy conducted in Bangladesh on Hilsha, a freshwater \n\nfish by Hossain et al.", "start_char_idx": 17710, "end_char_idx": 20988, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6faefa82-705d-43cc-a80b-e2e440b5dffc": {"__data__": {"id_": "6faefa82-705d-43cc-a80b-e2e440b5dffc", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f000ccba-089c-40d7-8a08-261180ee4afb", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "29b1c139cefbf8481f6a1f81b6b846970217914df50a1fbf198fa7a71f7c0e58", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63bab80c-7b11-4955-9769-3f3d3a3913ab", "node_type": "1", "metadata": {}, "hash": "8f969e72bad6c386b18e640b7d87b16b0d73ca3d27c101a9ef345ed4d9a0f5b8", "class_name": "RelatedNodeInfo"}}, "text": "(2018) revealed that out of 48 fishes, \n\n39 fishes tested positive for V. cholerae in specific OmpW \n\ngene assay [22]. Gills tested positive in highest number \n\n(79%) on market fish whereas the same happened for \n\nscale swab in case of fresh fishes which is similar to our \n\nfindings as most gills tested positive for the pathogen. \n\nFurthermore, PCR results in this study, revealed (66.7%) \n\nhigher prevalence of Vibrio cholerae in fish purchased \n\nfrom local markets than those collected from river banks.  \n\nE. coli being highly prevalent in all intestinal samples, \n\nlower yield of V. cholerae can probably be attributed to \n\nglucose metabolism of E. coli yielding acid products \n\nwhich can potentially reduce survival chances of V. \n\ncholerae as evidenced by in-vitro studies [23]. Halpern \n\nand Izhaki (2017) emphasized the mutualistic relation \n\nbetween V. cholerae and fish with the evidence of the \n\nformer helping fish to properly digest chitinous prey like \n\nzooplanktons [21]. However, fish serve as an important \n\nvehicle for V. cholerae and hence is the key to the \n\ndissemination process for epidemics. Vibrio \n\nparahemolyticus has been sporadically associated with \n\nfreshwater fish and is seasonal. None of the freshwater \n\nfish contained V. parahemolyticus [22]. \n\nThese findings and observations seem alarming and \n\ndemand assessing of the freshwater fish and other \n\naquatic products that are kept for sale in the markets. \n\nHigh load of bacteria in the samples including the \n\npresence of total coliforms, fecal coliforms and \n\npathogenic bacteria can be attributed to the type of water \n\nused in the shops for keeping the fishes. The bacterial \n\nload in the fish can probably be the function of frequency \n\nof change of water. Besides, indigenous bacteria on \n\nsurface as well as normal flora of other sites, rapidly \n\nmultiply and invade the previously sterile part after \n\ndeath of fish and hence, spoilage is a consequence if \n\nproper keeping conditions are not maintained after \n\nharvesting or killing (after being bought). In addition to \n\nthat, fish handlers at different stages of the supply chain \n\nare at greater risk apart from consumers eating the fish. \n\nBacteria can pave a way into wounds, cracks or lacerated \n\nregions of skin of fish handlers and might potentially \n\ncause various infections. Moreover, the bad hygiene \n\npractice of the handler can potentially cause fish borne \n\ninfection. Conventional cooking systems might reduce \n\nthe bacterial load to acceptable or possibly kill pathogens. \n\nHowever, the risks remain high when consumers prefer \n\nit raw or smoked. In addition, undercooking of such \n\ncontaminated fish can pose dangers to public health. \n\nConclusion  \nNine (3 L. rohita, 3 C. batrachus, 3 C. carpio) fish samples \n\nwere taken from retailers of Kathmandu which were \n\nprocessed for assessing their microbial quality and were \n\nassayed for presence of potential pathogenic bacteria. \n\nThe samples were found to contain a high load of bacteria \n\nalong with coliforms. The samples were also found to \n\ncontain potential pathogens like S. aureus, S. Typhi, S. \n\nParatyphi, V. cholerae, E. coli and CoNS with the most \n\nprevalent being E. coli. These findings suggested the poor \n\nmicrobial quality of freshwater raw fishes that are being \n\nsold in the market. C. batrachus was found to be the most \n\ncontaminated fish. The research indicated that there is an \n\nimmediate need for quality assessment of fish and such \n\naquatic products on a large scale.  To conclude, the fish \n\nwhether alive, dead, dried or frozen should be \n\nconsistently monitored for bacterial load and presence of \n\npathogenic forms.", "start_char_idx": 20989, "end_char_idx": 24649, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63bab80c-7b11-4955-9769-3f3d3a3913ab": {"__data__": {"id_": "63bab80c-7b11-4955-9769-3f3d3a3913ab", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6faefa82-705d-43cc-a80b-e2e440b5dffc", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "7b07cbf7990b34da490316e7eb6429d7ccee1c3e9fd2c29ddc0460b76b4596e4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "218d58a8-a98f-4697-b6a3-27b57289f07c", "node_type": "1", "metadata": {}, "hash": "18c8fe5fd066b99ba010095a1f5fae7b1c189c671959d19f1b076b8199440d6f", "class_name": "RelatedNodeInfo"}}, "text": "Author\u2019s Contribution  \nProject coordinator: KP \n\nConceptualization: SP, PS, SP, IA, SG and KP  \n\nWriting \u2013 Original Draft Preparation: SP, PS \n\nWriting \u2013 Review & Editing: KP \n\nAll authors read and approved the final manuscript: Yes \n\nCompeting Interests  \nNo competing interests were disclosed \n\nFunding  \nNo grants were involved in supporting this work \n\nAcknowledgements  \nWe would like to express our deepest gratitude to the \n\nDepartment of Microbiology, St Xavier\u2019s College, \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1): 7-12     Prasai et al.  \n\n\u00a9NJB, BSN   12 \n\nMaitighar. We are also thankful to staff members for their \n\nhelp, support and their kind cooperation during the \n\nresearch period. \n\nEthical Approval and Consent  \nNot Applicable \n\nReferences \n1. Eizenberga I, Terentjeva M, Valci\u0146a O, et al. \n\nMicrobiological quality of raw fish at retail market in \nLatvia. In: Food Quality and Safety. NJF Latvia, 2015, pp. \n16\u201318. \n\n2. Yagoub SO. Isolation of Enterobacteriaceae and \nPseudomonas spp. from raw fish sold in fish market in \nKhartoum state. J Bacteriol Res 2009; 1: 85\u201388. \n\n3. Atwa EI. Bacteriological Study of Fish Samples \nCollected from Different Markets in Some Egyptian \nGovernorates and Antimicrobial Sensitivity of \nIsolates. Int J Curr Microbiol Appl Sci 2017; 6: 2765\u2013\n2776. \n\n4. Megha P U and Harikumar P S. Isolation and \nIdentification of Pathogenic Bacteria in Edible Fish: A \nCase Study of Mogral River, Kasargod, Kerala, India. \nBio Sci 2016; 100: 43672\u201343677. \n\n5. Jalal, K.C.A., Akbar John, B., Nurul Lyana, M.S., \nFaizul, H.N., Noor Isma, Yanti, M., Irwandi, J. and \nMahbuba Bulbul. Comparative study on spoilage and \npathogenic bacteria in selected commercial marine \nand freshwater fishes. Int Food Res J 2017; 24: S298\u2013\nS304. \n\n6. Tilahun A, Engdawork A. Isolation, Identification and \nAntimicrobial Susceptibility Profile of E. coli \n(O157:H7) From Fish in Lake Hawassa, Southern \nEthiopia. 2019; 3: 14\u201319. \n\n7. Sichewo PR, Gono RK, Muzondiwa J, et al. Isolation \nand identification of pathogenic bacteria in edible fish: \nA case study of rural aquaculture projects feeding \nlivestock manure to fish in Zimbabwe. \nIntJCurrMicrobiolAppSci 2014; 3: 8897\u20138904. \n\n8. Kumari SP, Prasad BN, Kumari G, et al. \nMicrobiological quality of fish, rohu marketed in \nPatna and its public health significance. J Food Sci \nTechnol -Mysore- 2001; 38: 607\u2013608. \n\n9. Xu M, Wu J, Chen L. Virulence, antimicrobial and \nheavy metal tolerance, and genetic diversity of Vibrio \ncholerae recovered from commonly consumed \nfreshwater fish. Environ Sci Pollut Res 2019; 26: 27338\u2013\n27352. \n\n10. Cheesebrough M. District Laboratory Practice in \nTropical Countries Part 2. Cambridge      University    \nPress, 2006. \n\n11. International Comission on Microbiological \nSpecifications for Food (ICMSF). Microorganisms in \nFoods. 2. Sampling for Microbiological Analysis: Principles \nand Specific Applications. 2nd ed. Toronto, Canada: \nUniversity of Toronto Press. \n\n12. Goja AM.", "start_char_idx": 24652, "end_char_idx": 27649, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "218d58a8-a98f-4697-b6a3-27b57289f07c": {"__data__": {"id_": "218d58a8-a98f-4697-b6a3-27b57289f07c", "embedding": null, "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-225", "node_type": "4", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "54e3a16b44595d25de6684dd7b668b471516073bf5efd06b388de9de8ae6b028", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63bab80c-7b11-4955-9769-3f3d3a3913ab", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "c07af15381c022e9da5cf58724ac67104e4d628e209fc98f88d9d29ab620bfd1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "001b91ea-7566-4207-89c9-7c15fcfb4084", "node_type": "1", "metadata": {}, "hash": "6d925d3132c84288eab367400d1595a8d2a6e180b27f39123a8911a95bc2eda9", "class_name": "RelatedNodeInfo"}}, "text": "Toronto, Canada: \nUniversity of Toronto Press. \n\n12. Goja AM. Microbiological assessment of three types of \nfresh fish (Tilapia niloticus, Labeo niloticus and \n\nHydrocynus spp.) sold in Ed Dueim, Sudan. N Y Sci J \n2013; 6: 49\u201354. \n\n13. Liu H, Guo X, Gooneratne R, et al. The gut microbiome \nand degradation enzyme activity of wild freshwater \nfishes influenced by their trophic levels. Sci Rep 2016; \n6: 0\u201312. \n\n14. Wong S, Rawls JF. Intestinal microbiota composition \nin fishes is influenced by host ecology and \nenvironment: News and Views: Perspective. Mol Ecol \n2012; 21: 3100\u20133102. \n\n15. Al Shabeeb SS, Ibrahim MAM, Ramadhan GHA. A \nComparative Microbial Quality Assessment among \nFishes, Prawns and Cuttlefishes collected from \nDammam Fish Market. Int J Curr Microbiol Appl Sci \n2016; 5: 405\u2013418. \n\n16. Bibi F, Qaisrani SN, Ahmad AN, et al. Occurence of \nSalmonella in freshwater fishes: A review. J Anim Plant \nSci 2015; 25: 330\u2013310. \n\n17. Mhango M, Mpuchane SF, Mpuchane BA. Incidence \nof indicator organisms, opportunistic and pathogenic \nbacteria in fish. Afr J Food Agric Nutr Dev 2010; 10: \n4203\u20134218. \n\n18. Bujjamma P, Padmavathi P. Prevalence of \nStaphylococcus aureus in Fish Samples of Local \nDomestic Fish Market. 2015; 4: 427\u2013433. \n\n19. Kluga A, Kacaniova M, K\u00e1ntor A, et al. Identification \nof microflora of freshwater fish caught in the Driksna \nriver and pond in Latvia. Foodbalt, 2017, pp. 164\u2013168. \n\n20. Wong S, Waldrop T, Summerfelt S, et al. \nAquacultured Rainbow Trout (Oncorhynchus mykiss) \nPossess a Large Core Intestinal Microbiota That Is \nResistant to Variation in Diet and Rearing Density. \nAppl Environ Microbiol 2013; 79: 4974\u20134984. \n\n21. Halpern M, Izhaki I. Fish as Hosts of Vibrio cholerae. \nFront Microbiol 2017; 8: 282. \n\n22. Hossain ZZ, Farhana I, Tulsiani SM, et al. \nTransmission and Toxigenic Potential of Vibrio cholerae \nin Hilsha Fish (Tenualosa ilisha) for Human \nConsumption in Bangladesh. Front Microbiol 2018; 9: \n222. \n\n23. Nag D, Breen P, Raychaudhuri S, et al. Glucose \nMetabolism by Escherichia coli Inhibits Vibrio cholerae \nIntestinal Colonization of Zebrafish. Infect Immun \n2018; 86: e00486-18.", "start_char_idx": 27588, "end_char_idx": 29740, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "001b91ea-7566-4207-89c9-7c15fcfb4084": {"__data__": {"id_": "001b91ea-7566-4207-89c9-7c15fcfb4084", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "218d58a8-a98f-4697-b6a3-27b57289f07c", "node_type": "1", "metadata": {"identifier": "njb-225", "author": "Prasai, Shristi; Shrestha, Puja; Pandey, Sriniwas; Adhikari, Ishika; Gurung, Srijana; Prajapati, Kamil", "title": "Microbial Quality Assessment of Raw Freshwater Fish Sold in Local Markets of Kathmandu Valley", "date": "2022-07-30", "file": "njb-225.pdf"}, "hash": "6716c63c520f84aa47412d35d333abe0742172f42dc179cc7d72e204b72f4a2c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4a2f0d2a-8046-4bbe-92b3-b6b6a1533764", "node_type": "1", "metadata": {}, "hash": "7ee4289515ac1a00864ed44ca57a182323abb10b48fb2aebe014ec5fa13d05c0", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 1 3 - 2 4   Research article  DOI: https://doi.org/10.54796/njb.v10i1.226 \n  \n \n\n\u00a9NJB, BSN    13 \n\nOccurrence of Purple Blotch Disease Associated with Selected Garlic \nVarieties and its Management Through Bio-Agent, Botanicals and \nFungicides \nUmme Habiba Akter  , Fatema Begum, M. R. Islam, Jannatun Nahar Prinky, Mst. Rehena Khatun \n\nDepartment of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh \n\nReceived: 5 Jul 2021; Revised: 21 Jul 2022; Accepted: 26 Jul 2022; Published online: 30 Jul 2022 \n\nAbstract  \nPurple blotch of garlic caused by Alternaria porri is recognized as a prominent diseases posing threat to garlic cultivation \nthroughout the world including Bangladesh. The experiments were conducted to determine the prevalence of purple blotch \ndisease on garlic varieties in field condition, to test the pathogenicity of isolated causal organism and to find out the suitable \nmanagement options of the disease. Eight garlic varieties viz. BAU Rashun-1, BAU Rashun-2, BARI Rashun-1, BARI \nRashun-2, BARI Rashun-3, BARI Rashun-4, Local Deshi and Local Indian were explored in prevalence study and nine \nmanagement option comprising a bio-agent Trichoderma harzianum (T1), five botanicals viz.  Lantana camara (T2), Spilanthes \npaniculata (T3), Ocimum sanctum (T4), Raphanas raphanistrum (T5) and Azadirachta indica (T6), two fungicides Mancozeb 80% \nWP (T7) and Sulcox 50% WP (T8) and, an untreated control (T9) were explored in the experiments. BARI Rashun-3 showed \nthe highest disease incidence (40.00%) and severity (92.00%) of purple blotch disease. Isolation, identification of pathogen \nand pathogenicity test was carried out as well. In case of management, all botanicals and bio-agent were tested significantly \nbeneficial in lessening the disease incidence and severity of purple blotch disease. The results revealed that Lantana camara \n(T2) was found most effective for minimizing the disease incidence (26.67, 26.67 and 33.33%) at 30, 45 and 60 DAS, \nrespectively while maximum disease incidence was recorded in control (T9) (86.67, 96.67 and 100.00%). T2 also reduced \ndisease severity at 30 DAS (11.00%) whereas, at 45 DAS (18.67%) and 60 DAS (19.33%) T1 performed well against the \ndisease.  \n\nKeywords: Purple blotch; Alternaria porri; in-vivo; bio-agent; plant extracts and fungicides.  \n\n Corresponding author, email:  sumonahabiba31@gmail.com \n\nIntroduction \nGarlic (Allium sativum) belongs to Alliaceae family is \n\nconsidered as the most demanded and universal spices \n\nwithin the world especially in Bangladesh. The position \n\nof garlic is second among all the important spices in the \n\nworld [1]. Garlic helps in controlling hypertension, \n\ndiabetes, cancer, ulcer, rheumatism, germs, fungal and \n\nbacterial diseases etc. [2]. According to Asfand et al. \n\n(2019)[3] the pungent taste of garlic is reduced by the \n\nfield fresh exfoliated garlic which consist just about 0.8% \n\nfiber, 63% water, 7% protein, 28% carbohydrate, 0.2% fat \n\nand an excellent amount of sulphur compounds.", "start_char_idx": 48, "end_char_idx": 3138, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4a2f0d2a-8046-4bbe-92b3-b6b6a1533764": {"__data__": {"id_": "4a2f0d2a-8046-4bbe-92b3-b6b6a1533764", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "001b91ea-7566-4207-89c9-7c15fcfb4084", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "bf8d00f764c3b94052c71d1e6beba1a7fef0926d860ce68fd96951a4e7a84f11", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "99ff7b37-b0c0-44f3-b2ca-ddd93905e6a1", "node_type": "1", "metadata": {}, "hash": "783cd6870b80bd5fb364f345284826ba704eee6058c904760c1cdadd71b70411", "class_name": "RelatedNodeInfo"}}, "text": "From the report of FAOSTAT, (2021)[4] the United \n\nNations Food and Agriculture Organization reported \n\nthat from 1,634,634 hectares production with 30,708,243 \n\ntonnes of garlic globally each year. Total production of \n\nGarlic in the year of 2019 in Bangladesh was 466, 389 \n\ntonnes from 71734 ha land. Though garlic production is \n\nenhancing gradually, but due to expanding population \n\nrate in the low income country Bangladesh, the domestic \n\nneed cannot be fulfilled. To meet up the demand \n\nBangladesh imports enormous amounts of garlic from \n\nabroad every year [5]. \n\nThough garlic has many importance, the yield is below \n\naverage in many parts around the world. Various \n\nfactors viz. diseases, insects, soil, climatic condition and \n\nlack of technical knowledge etc. affect the quality of \n\ngarlic bulb and the yield greatly. [6]. Soil borne diseases \n\nare major in garlic. Among the fungal diseases, purple \n\nblotch caused by Alternaria porri (Ellis) Cif., is a major \n\nconstraint that leads to considerable loss in yield and \n\nquality of garlic [7]. The disease is considered as a \n\ncrucial disease all over the world including Bangladesh \n\n[8]. Epidemic may cause total failure of the crop in \n\nfavorable conditions. Gupta and Srivastava (1993) [9] \n\ncarried out a study in Maharashtra during Kharif where \n\nextreme loss was noted owing to purple blotch disease. \n\nIn Punjab, Haryana and Maharashtra purple blotch \n\nspotted as serious disease and caused 20-60% loss [10, \n\n11, 12]. The purple blotch disease acting as more \n\ndreadful for seed crops in contrast to bulb crops that \n\ncaused sometimes 100% loss on productivity of onion \n\nseed [13, 14, 15]. Hence, in existing situation convenient \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0003-3361-0473\nmailto:mailtosumonahabiba31@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    14 \n\nmanagement strategies of purple blotch of garlic has \n\nbecome a turning topic. To control the plant pathogens, \n\nfarmers around the world need the chemical pesticides \n\nwith a view to sustain the standard and dismissal of \n\nagricultural products [16]. Sharma et al. (2012)[17] \n\napproximated in her study that because of pests cause \n\n37% of crop loss and 12% crop loss is due to pathogens.  \n\nOn the contrast, issues of environmental pollution and \n\nvarious health complications arose because of the \n\nimmoderate and the inappropriate use of pesticides over \n\nthe past decades around the world. Patent resistant \n\norganisms can be developed by the extreme use of \n\nchemical pesticides [18]. However, now a days strict \n\nregulation are applied on the implementation of \n\nchemical fungicides because of their carcinogenic effects, \n\nproblems of residual toxicity, environmental pollution \n\nand development of fungicide-resistant strains [19, 20]. \n\nKumar & Palakshappa, (2008) [21] stated that, biological \n\ncontrol of plant pathogens through antagonistic \n\nmicroorganisms is proved as an effective, not harmful to \n\nthe environment and a suitable strategy other than an \n\noptimistic alternative of chemical uses. Trichoderma sp. is \n\na biological control agent and botanicals have been \n\nfound to be very effective for several soil borne plant \n\npathogenic fungi. Plant extract possess an anti-fungal \n\nactivity in opposition to a wide range of plant \n\npathogenic fungi. These are less phytotoxic, \n\nbiodegradable and host metabolism stimulatory.", "start_char_idx": 3143, "end_char_idx": 6755, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "99ff7b37-b0c0-44f3-b2ca-ddd93905e6a1": {"__data__": {"id_": "99ff7b37-b0c0-44f3-b2ca-ddd93905e6a1", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4a2f0d2a-8046-4bbe-92b3-b6b6a1533764", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "478ebb30a82bed8d9fc298060e9a8c1898803c49471b49ea58bb1b621a9d5e02", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "dc3fec88-631e-45e4-8626-11c1ebf8e35e", "node_type": "1", "metadata": {}, "hash": "8148f4b1752b43d6e11d49b9db0021a4f78dd8756c2f6d41c51cbc93e806a309", "class_name": "RelatedNodeInfo"}}, "text": "These are less phytotoxic, \n\nbiodegradable and host metabolism stimulatory. \n\nVarious experiments were undertaken over the past \n\nmany years to control purple blotch disease through \n\nbio-agents, botanicals and fungicides [12, 15, 21, 22, 23, \n\n24, 25]. The current research work was aimed to assess \n\nthe occurrence of purple blotch disease incidence and \n\nseverity of selected garlic varieties, to isolate and \n\nidentify purple blotch disease and pathogenicity test of \n\nAlternaria porri, and its management using bio-agent, \n\nplant extracts and fungicides.  \n\nMaterials and methods \nThe experiments were conducted at the central farm of \n\nSher-e-Bangla Agricultural University and in the central \n\nLaboratory, Department of Plant Pathology, Sher-e-\n\nBangla Agricultural University, Dhaka, Bangladesh \n\n(23\u00b041\u2032N latitude and 90\u00b022\u2032E longitudes at the elevation \n\nof 8.6 m above the sea level, AEZ-28) during the Rabi \n\nseason of 2018-2019 and 2019-2020 with three \n\nreplications consists of 24 units plots in Randomized \n\ncomplete block design (RCBD). Recommended doses of \n\nfertilizer (Cowdung @10 tons/ha, Triple Super \n\nPhosphate (TSP) @417kg/ha, Muriate of Potash (MP) \n\n@165kg/ha, Urea @320kg/ha, Gypsum 100kg/ha, Zinc \n\noxide @5kg/ha and Boric acid @5kg/ha) were applied \n\nduring field preparation and after sowing of garlic clove \n\nand, 2 packets of Sevin were applied to control from the \n\nattack of ant [26].  \n\nSource of garlic seeds \nEight different fresh and disease-free garlic variety seed \n\nwere collected from three different places. BAU Rashun-\n\n1 (V1) and BAU Rashun-2 (V2) varieties were collected \n\nfrom Bangladesh Agricultural University, Horticulture \n\nDepartment, Mymensingh. BARI Rashun-1 (V3), BARI \n\nRashun-2(V4), BARI Rashun-3 (V5) and BARI Rashun-4 \n\n(V6) varieties were collected from Bangladesh \n\nAgricultural Research Institute, Joydebpur, Gazipur and \n\nlast two local varieties naming Local Deshi (V7) and \n\nIndian Local (V8) were collected from Siddik bazar, \n\nDhaka, Bangledesh. \n\nDisease incidence \n\nGarlic varieties were assessed on the basis of symptoms \n\nappeared on the above ground plants and recorded. For \n\ncalculation of disease incidence each plant was counted \n\nincluding infected one in the field and then expressed in \n\npercentage. For the determination of disease incidence \n\nof garlic the following formula was used: [27] \n\n \n\n% DI = \nNumber of diseased plants \n\n  Number of total plants observed\n\u00d7 100 \n\nDisease severity \nDisease severity of purple blotch was assessed using 0-5 \n\nscale [28], as follows by randomly selected 10 plants \n\nfrom each plot and final data were calculated for PDI \n\n(percent disease index) estimation. \n\n \n\nGrade Symptoms description \n\n0 Free from infection \n\n1 Small sized lesion towards the tip, covering \nless than 10% leaf area \n\n2 Several dark purplish brown patches covering \nless than 20% leaf area \n\n3 Several patches with paler outer zone, \ncovering up to 40% leaf area \n\n4 Long streaks covering up to 75% leaf area or \nbreaking of leaves/stems   from the center  \n\n5 Complete drying of the leaves/stems or \nbreaking of the leaves/stems from the base \n\nThe percent disease index (PDI) was computed \n\naccording to the formula given by (Wheeler, 1969 and \n\nIslam et al., 2003) [29, 30].  \n\n \n\nPDI=\nTotal sum of numerical ratings\n\nNumber of observations x Maximum disease rating\n \ud835\udc65 100", "start_char_idx": 6680, "end_char_idx": 10050, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "dc3fec88-631e-45e4-8626-11c1ebf8e35e": {"__data__": {"id_": "dc3fec88-631e-45e4-8626-11c1ebf8e35e", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "99ff7b37-b0c0-44f3-b2ca-ddd93905e6a1", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "4f9f72cbe3ea51d8328f0d473bb9f6e21d7430cbf6fb0b673a9fe8399e23a89b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "346080a2-cef0-4e74-a681-b5cd69757c75", "node_type": "1", "metadata": {}, "hash": "ce00285007b6652a75ea0d9ce4e3371500d7b021b220b3de16b7fc3e6ac7ef26", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    15 \n\nYield per hectare \n\nYields of harvested garlic bulbs were computed using \n\nelectric balance after solar drying of bulbs for 10 days. \n\nThe yields was expressed as Kg/hectare. \n\nIsolation and identification of Alternaria \nporri  \nDiseased leaf samples were collected from field, put into \n\nbrown paper envelope and, taken to the central \n\nlaboratory, Department of Plant Pathology, Sher-e-\n\nBangla Agricultural University, Dhaka for isolation. \n\nCollected diseased leaves were cut into pieces regarding \n\n1 to 1.5 mm with the help of sterilized scalpel, cleaned \n\nwith sterilized distilled water and disinfected using 0.1 \n\npercent MgCl2 solution (30 to 60 seconds). Then \n\nsterilized cut pieces were washed three times right away \n\nwith double sterilized distilled water frequently to \n\nremove the traces of mercuric chloride and dried with a \n\ntowel on sterilized filter paper, then placed to petri \n\nplates containing 20 ml of autoclaved water agar (Agar \n\n20 g with 1000 ml distilled water) in a laminar flow and \n\nincubated at 25\u00b11\u00b0C for 10 days. After 10 days the \n\ngrowing mycelia on water agar petri plates were \n\ntransferred to potato dextrose agar media (200g of \n\npeeled potatoes, 20 g of dextrose, and 20 g of agar and \n\n1000 ml of distilled water). At 14 days the fungus grew \n\nwell and sporulated then freshly prepare slide was \n\nobserved under compound microscope and digital \n\nmicroscope for the identification of the pathogen using \n\nrelevant literature. After identification of Alternaria porri \n\nthe pure culture was maintained by sub culturing at an \n\ninterval every 15 days and preserved at low temperature \n\n(4\u2070C) in refrigerator for future purpose. The \n\nobservations were equated with the standard \n\nmeasurements following by Ellis (1971) [31] for the \n\nidentification of the pathogens (Plate 1).  \n\nDesignation of cultured isolates \nThe cultured isolates were designated based on variety \n\nand location [32]. For example BAU1I1 represents that \n\nthis isolate was cultured from BAU Rashun-1 variety. \n\nCultural variability of Alternaria porri \nColony diameter was recorded on the 2nd, 7th and 14th \n\ndays after incubation. The data on radial growth was \n\nanalyzed statistically [33]. Growth per day was \n\ncalculated by the followed formula: \n\n mm/ day = (growth observed on a day - growth on \n\nprevious observation) \u2215 2. \n\nMorphological variability of Alternaria porri \nFourteen days old cultures of A. porri isolates were \n\nstudied for morphological variations viz. conidia color, \n\nshape, size, colony character and surface structure. \n\nPathogenicity test of Alternaria porri \nTo prove the association of isolated organism with the \n\ndisease pathogenicity test was done using Koch\u2019s \n\nPostulates conducted on the Agri found onion red \n\nvarieties [34, 35]. For testing the virulence level of \n\nAlternaria porri isolates, BARI Rashun-3 variety was \n\nselected. The plants were raised in sterilized plastic pot \n\nunder greenhouse condition. Soil was sterilized for three \n\nconsecutive days in an autoclave at 20 lbs per sq. inch \n\npressure for one and half an hour. The plastic pots were \n\ncleaned entirely with water, rinsed with two per cent \n\nformalin and before using it was dried in the sunlight. \n\nThe fumigated pots were filled with this sterilized soil \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    16 \n\nand covered with disinfected polythene sheet to block \n\naerial contamination.", "start_char_idx": 10059, "end_char_idx": 13612, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "346080a2-cef0-4e74-a681-b5cd69757c75": {"__data__": {"id_": "346080a2-cef0-4e74-a681-b5cd69757c75", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "dc3fec88-631e-45e4-8626-11c1ebf8e35e", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "0c9cdb220058c1db03c96ca3cabb3ab2482ede950345fb31894774a2e812b79e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2cb82c21-ce98-4c85-806f-17544ed26e76", "node_type": "1", "metadata": {}, "hash": "819f24afb6cbbbe6b9eec3ac5dd1edc10830896898d6a481137a671f7c622c8e", "class_name": "RelatedNodeInfo"}}, "text": "After that air dried sandy loam \n\nsoil and cow dung were mixed thoroughly at the ratio of \n\n4:1 and then filled in earthen pots (20 cm diameter). No \n\nchemical fertilizers were used in the pot soil. The \n\nconidial suspension (5x105 spores mL-1) was mixed in \n\nprepared distilled water from 10 days old culture of A. \n\nporri isolates. The garlic plants of 30 days old were \n\ninoculated with these spore suspensions after garlic \n\nleaves were injured by sterile toothpick. Water was \n\nsprayed consequently to the plants both before 24 hour \n\nand after inoculation the plants were covered with moist \n\npolythene bag to keep up high relative humidity (%RH) \n\nand also to inhibit natural contamination with other \n\nfungal conidia or spores. The inoculation was done on \n\ncool evening hours. The inoculated plants were \n\nmaintained in greenhouse condition. On the 17 days \n\nafter inoculation the severe symptoms were observed \n\nand compared with original symptoms. \n\nCollection of data on leaf infection  \nAfter 5 days of inoculation on garlic leaves the size of \n\nlesions was recorded on 5th, 7th, 9th, 11th, 13th, 15th, and \n\n17th days after inoculation. Size of lesions increased per \n\nday was calculated by the formula:  \n\nLeaf infection per day = \n\nLeaf infection observed on a day\u2212Leaf infection on previous observation\n\n2\n  \n\nManagement of Purple blotch of garlic caused \nby Alternaria porri through Trichoderma, \nbotanicals and fungicides   \nExperimental design \nThe experiment was conducted in a Randomized \n\nComplete Block Design (RCBD) with three replication \n\nand nine treatments with the objective to attain \n\nmanagement of Purple blotch of garlic caused by \n\nAlternaria porri. It was conducted in Rabi season 2019-\n\n2020 at Central Farm, Sher-e-Bangla Agricultural \n\nUniversity, Dhaka-1207, Bangladesh. \n\nVariety and Treatments \nFrom previous evaluation, most susceptible variety \n\nBARI Rashun-3 was used for the management strategies \n\nof purple blotch disease. One bio-agent, five botanicals, \n\ntwo fungicides and a control used as treatments (Table \n\n1). \n\nPreparation of plant extract \n\nFresh and healthy leaves of all five test plants were \n\ncollected from the surrounding field of university for \n\nthe preparation of plant extract. For the removal of dust \n\nmaterial adhering to surfaces the collected leaves were \n\nfirst washed under running tap water and then in \n\ndistilled water. One hundred grams (100 g) leaves from \n\neach sample were then mixed with sterile water (100 ml) \n\nat 1:1 (w/w) with the help of mortar and pestle. After \n\nthrough grinding the extract was filtered through \n\nmuslin cloth and then through Whatman filters paper \n\nno1. Then the extract was passed through sieve filter to \n\neliminate contamination. After that the extract is used as \n\nstandard plant extract solution of 100% concentration of \n\n1:1 ratio. Prepared plant extract was treated at 60\u00b0C for \n\n15 minutes for demolition of other microorganism \n\ncontamination. Then after the initial appearance of \n\ndisease at 30 days after sowing, the foliar spray of \n\nbotanicals was applied given for 3 times at 15 days \n\ninterval. The foliar spray was given using hand sprayer \n\nat afternoon for better result [36]. \n\n \n\nTable 1.", "start_char_idx": 13613, "end_char_idx": 16833, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2cb82c21-ce98-4c85-806f-17544ed26e76": {"__data__": {"id_": "2cb82c21-ce98-4c85-806f-17544ed26e76", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "346080a2-cef0-4e74-a681-b5cd69757c75", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "543b750e6b54c3cec14757640f848274501af1158a0bdc9c91b686aead54f20b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6c1d04c1-d828-41b7-bebc-e37765b93603", "node_type": "1", "metadata": {}, "hash": "2417c8adbb26762f24395f42395c9fe600d9e1e00cd08e8b94043522c273ffc8", "class_name": "RelatedNodeInfo"}}, "text": "Table 1. Treatments used for management of Purple blotch of \ngarlic \n\nTreatment \nName \n\nCommon \nName \n\nScientific Name / \nChemical name \n\nPlant \npart/media \n\nused \n\nT1 Trichoderma Trichoderma \n\nharzianum \n\nLiquid \n\nsolution \n\nT2 Lantana Lantana camara Leaf \n\nT3 Shormoni Spilanthes \n\npaniculata \n\nLeaf \n\nT4 Tulsi Ocimum sanctum Leaf \n\nT5 Bon mula Raphanas \n\nraphanistrum \n\nLeaf \n\nT6 Neem Azadirachta indica Leaf \n\nT7 Mancozeb \n\n80% WP \n\nEthylene (bis) di \n\nthio carbamate \n\nPowder \n\nT8 Sulcox 50% \n\nWP \n\nCopper \n\noxychloride \n\nPowder \n\nT9 Control - - \n\n \nPreparation of bio-agent and fungicides \nThe bio-agent Trichoderma harzianum liquid solution was \n\nsprayed 3 times at 15 days interval after the initial \n\nappearance of disease at 30 DAS [36].  \n\nFungicidal solutions were prepared following the \n\nrecommended doses of selected fungicides. The \n\nfungicides were mixed thoroughly using required \n\nquantity with sterilized water. It was required 2 \n\ngm/liter of Mancozeb 80% WP and 3 gm/liter of Sulcox \n\n50% WP for preparation of solution for recommended \n\nconcentration. The solutions of the fungicides were \n\nsprayed 3 times at 15 days interval at afternoon by hand \n\nsprayer [37]. A control treatment was maintained in \n\neach block where spraying was done with normal water \n\nonly. \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    17 \n\n  \nParameters observed \nData were noted on plant height (cm), number of leaves, \n\ndisease incidence, and disease severity. Observations on \n\nAlternaria porri disease intensity were recorded on \n\nrandomly selected six plants from the diseased infected \n\nleaves. Screening was assessed using 0-5 rating scale \n\n[28] based on leaf area covered by the pustules. \n\nMeasurement of PDI described before.  \n\nStatistical Analysis \nThe data of different characters were statistically \n\nanalyzed which were obtained from the experiment to \n\nobserve the significant difference among the treatment \n\nby using the MSTAT-C program. Conversions of the \n\ndata were required when necessary. The mean values of \n\ntreatments were calculated and analyzed using \n\nDuncan\u2019s Multiple Range test. DMRT test were executed \n\nto determine the level of significant differences and to \n\nseparate the means within the parameters at 5% level of \n\nprobability [38]. \n\nResults \nEvaluation of selected garlic varieties against \npurple blotch diseases at field condition \nSmall, whitish and sunken like lesions were marked on \n\nleaves and stalks initially as the symptoms of purple \n\nblotch disease. Subsequently watersoaked lesions \n\ndeveloped and transferred to brown. While the disease \n\nadvanced, these lesions expanded and became zonate \n\nand turned into purplish color. The border of the lesions \n\nturned to purplish red encircling by yellowish brown or \n\npale color margin. Upwards and downwards extension \n\nwere founded in these lesions. Infected leaves turned \n\nyellow and wilted at advanced stages (Plate 2). \n\nPrevalence of % disease incidence and \nseverity of purple blotch disease among \nselected garlic varieties  \nSignificant variation was found at different days after \n\nplanting in % disease incidence and severity. The results \n\nare presented in Table 2. Disease incidence and severity \n\nvaried depending on cultivars and climatic condition. \n\nThe disease incidence varied from 1.47 to 34.44% and \n\n1.47 to 40.00% at 60 DAS and 90 DAS, respectively. \n\nWhereas, the disease severity varied from 36.00 to \n\n70.67% and 64.00 to 92.00% at 60 DAS and 90 DAS, \n\nrespectively.", "start_char_idx": 16825, "end_char_idx": 20367, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6c1d04c1-d828-41b7-bebc-e37765b93603": {"__data__": {"id_": "6c1d04c1-d828-41b7-bebc-e37765b93603", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2cb82c21-ce98-4c85-806f-17544ed26e76", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "107b506b2291e00d7b595c00fb5bce1b342815477d5f3c31fd4cefdd602a17ff", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70b38eaa-2a71-4f17-a8ca-ab5212132a37", "node_type": "1", "metadata": {}, "hash": "4d0cd3d029469413e30c697a01aaa2741125cf5211d090e5645991269163e3d0", "class_name": "RelatedNodeInfo"}}, "text": "At 60 DAS, the highest disease incidence (34.44%) and \n\ndisease severity (70.67%) was noted on BARI Rashun-3, \n\nrespectively. Statistically similar disease severity \n\n(62.67%) was reported on BARI Rashun-4. On the \n\ncontrary, the lowest disease incidence (1.47%) was \n\nobserved on BAU Rashun-2 which was statistically alike \n\nwith BAU Rashun-1 (1.80%). The lowest disease severity \n\n(36.00%) was found in BAU Rashun-1 and BAU Rashun-\n\n2 variety that was statistically similar to BARI Rashun-1 \n\n(49.33%) and on Local Indian variety (44.00%), \n\nrespectively.  \n\n  \n\nTable 2. Prevalence of % disease incidence and disease severity \nof purple blotch disease among selected garlic varieties \n\nVariety (%) Disease \nIncidence \n\n(%) Disease Severity \n\n60 DAS 90 DAS 60 DAS 90 DAS \n\nBAU Rashun-1 1.80    c 1.80     d 36.00   d 68.00   c  \n\nBAU Rashun-2 1.47    c 1.47     d 36.00   d 68.00   c \n\nBARI Rashun-1 8.33    b 8.33     c 49.33   b-d 76.00   bc \n\nBARI Rashun-2 8.88    b 15.00   b 56.00   bc 89.33   ab \n\nBARI Rashun-3 34.44  a 40.00   a 70.67   a 92.00   a \n\nBARI Rashun-4 6.67    b 13.89   b 62.67   ab 82.67   ab \n\nLocal Deshi 7.78    b 7.78     c 51.33   bc 76.00   bc \n\nLocal Indian 6.66    b 7.22     c 44.00   cd 64.00   c \n\nCV 14.80 18.89 15.39 9.46 \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    18 \n\nAt 90 DAS, the highest disease incidence (40.00%) and \n\ndisease severity (92.00%) was recorded on BARI \n\nRashun-3 variety. BARI Rashun-2 showed statistically \n\nsimilar disease severity (89.33%) with BARI Rashun-3 \n\nalong with BARI Rashun-4 (82.67%). Conversely, the \n\nlowest disease incidence (1.47%) was observed BAU \n\nRashun-2 followed by BAU Rashun-1(1.80%), Local \n\nIndian (7.22%), Local Deshi (7.78%) and BARI Rashun-2 \n\n(8.88%). Local Indian was found as less infected variety \n\nwith lowest disease severity of 64.00% which was \n\nstatistically similar to BAU Rashun-1 and BAU Rashun-\n\n2(68.00%) respectively.  \n\nCV = Coefficient of variance; in a column mean values \n\nhaving similar letter(s) are statistically similar and those \n\nhaving dissimilar letter(s) differ significantly as per \n\n0.01% level of significance. \n\nCorrelation of purple blotch percent disease severity \n\nwith yield (t/ha) \n\nThe yield of the garlic plant affected due to disease \n\nseverity. Correlation analysis was done to find out the \n\nassessment of yield loss owing to disease severity. To \n\ndetermine the effect of disease severity on yield of \n\nselected garlic varieties correlation of coefficient was \n\nconsidered at 0.01% level of probability. From this \n\ncorrelation, it was estimated that the fresh and dry \n\nweight of yield showed negative correlation with \n\ndisease severity of purple blotch of garlic and became \n\nsignificant at 0.01% level of probability.", "start_char_idx": 20371, "end_char_idx": 23175, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70b38eaa-2a71-4f17-a8ca-ab5212132a37": {"__data__": {"id_": "70b38eaa-2a71-4f17-a8ca-ab5212132a37", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6c1d04c1-d828-41b7-bebc-e37765b93603", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "f4e967d3227f292426a14c30e7ac65f582550114f19a4360873efa782ddcd994", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63e08b1a-dc4f-4e5b-bdda-dd67fd77f239", "node_type": "1", "metadata": {}, "hash": "96925f61acff6e38ff23e9412782c49fb4ec844296787953e3776cd0d984afcf", "class_name": "RelatedNodeInfo"}}, "text": "These values \n\nclearly expressed that less disease severity provide \n\nhigher yield and vice versa (Figure 1). \n\n \nFigure 1. Correlation between % disease severity of purple \n\nblotch with yield (t/ha) \n\nIsolation, identification and pathogenicity of \nAlternaria porri \nCultural studies of Alternaria porri    \n\nIsolated pathogens (Alternaria porri) from infected leaves \n\nof garlic transferred to water agar thereafter cultured in \n\npetri plates on potato dextrose agar (PDA). Cultured \n\npathogen incubated at 25\u00b11\u00baC, afterwards sub cultured \n\nfor future use. The colony appearance and growth of the \n\npathogens were monitored and noted for 14 successive \n\ndays (Table 3). \n\nTable 3. Radial mycelial growth of Alternaria porri on \nPDA media \n\nIsolates Radial mycelial growth (mm) \n\n2 DAI 7 DAI 14 DAI \n\nBAU1 I1 2.80 7.65 9.00 \nBAU2 I2 4.00 6.50 9.00 \nBARI1 I3 3.05 7.00 9.00 \nBARI2 I4 2.90 7.50 9.00 \nBARI3 I5 4.10 7.65 9.00 \nBARI3 I6 2.60 6.45 9.00 \nBARI4 I7 4.10 6.50 9.00 \nBARI4 I8 2.65 7.65 9.00 \nLD I9 3.10 7.45 9.00 \nLIND I10 4.00 7.65 9.00 \nIn the column BAU1 I1= BAU1 Isolate 1; BAU2 I2= BAU2 Isolate 2; \nBARI1 I3= BARI1 Isolate 3; BARI2 I4= BARI2 Isolate 4; BARI3 I5= \nBARI3 Isolate 5; BARI3 I6= BARI3 Isolate 6; BARI4 I7= BARI4 \nIsolate 7; BARI4 I8= BARI4 Isolates 8; LD I9= Local Deshi Isolate 9 \nand LIND I10= Local Indian Isolate 10 \n\nRadial mycelial growth of 10 isolate of A. porri from \n\neight different varieties of garlic varied significantly on \n\nPDA media. Alternaria porri is a fast growing pathogen. \n\nColony growth of the pathogen appeared after 2 days of \n\nincubation; maximum increase (4.10 mm) of colony \n\ndiameter was recorded in BARI3 I5 and BARI4 I7 isolates \n\nalong with isolates BAU2 I2 and LIND I10 (4.00 mm), \n\nBARI1 I3 (3.05 mm), LD I9 (3.10 mm). The minimum \n\nincrement (2.60 mm) of colony diameter was found in \n\nBARI3 I6 followed by isolates BARI4 I8 (2.65 mm), BAU1 I1 \n\n(2.80 mm) and BARI2 I4 (2.90 mm). \n\nAfter 7 days of incubation, the maximum increment \n\n(7.65 mm) of colony diameter was recorded in BAU1 I1, \n\nBARI3 I5, BARI4 I8 and LIND I10 respectively followed by \n\nBARI2 I4 (7.50mm), LD I9 (7.45 mm) and BARI1 I3 \n\n(7.00mm). On the other hand, the minimum increase \n\n(6.45 mm) of colony diameter was recorded in BARI3 I6.  \n\nAfter 14 days of incubation all the colonies covered \n\nwhole petridish which was 9.00mm.  \n\nMorphological studies of Alternaria porri \nRegular inspection was done to the pure culture of \n\npathogen Alternaria porri under the microscope to \n\ndetermine morphological characteristics of the pathogen \n\nviz. color, shape and surface texture.", "start_char_idx": 23176, "end_char_idx": 25783, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63e08b1a-dc4f-4e5b-bdda-dd67fd77f239": {"__data__": {"id_": "63e08b1a-dc4f-4e5b-bdda-dd67fd77f239", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70b38eaa-2a71-4f17-a8ca-ab5212132a37", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "4fb9dcd38448386d40a981b44fee896565c4f572aa82a6426fb3d4e8422b3e44", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "865f2ce7-2997-4ea1-9637-9bf3a2211e96", "node_type": "1", "metadata": {}, "hash": "35388e73b61e36b5f9db9c2ca02ed0ce73a5d7bf9f4bf74d6cb4ae101c5d7ec0", "class_name": "RelatedNodeInfo"}}, "text": "color, shape and surface texture. The results of \n\nmorphological studies of A. porri are shown in Table 4, \n\nPlate 3 and Plate 4. \n\nAlmost all the isolates showed fluffy growth appearance \n\non potato dextrose agar. The colony color varied from \n\nolivaceous green in BAU2 I2 to greyish white in BARI1 I3, \n\nashy black in BAU1 I1, BARI2 I4 and LIND I10, off-white in \n\nBARI4 I8 and black in rest of the isolates. Most of the \n\nsurface texture of the isolates was smooth, cottony black \n\ncenter with whitish to greyish periphery.  \n\nFresh Weight Dry Weight\n\n60das -0.257 -0.073\n\n90das -0.323 -0.089\n\n-0.35\n\n-0.3\n\n-0.25\n\n-0.2\n\n-0.15\n\n-0.1\n\n-0.05\n\n0\n\nY\nie\n\nld", "start_char_idx": 25750, "end_char_idx": 26404, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "865f2ce7-2997-4ea1-9637-9bf3a2211e96": {"__data__": {"id_": "865f2ce7-2997-4ea1-9637-9bf3a2211e96", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63e08b1a-dc4f-4e5b-bdda-dd67fd77f239", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "381cf3072df1f305386f216b865d83c5363c5a1daf9c3bab9a7660a089eb11f0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a4ab008b-6ffa-4258-92b7-00e828ad7baf", "node_type": "1", "metadata": {}, "hash": "ae76d79c29fdd6609884e20a5aaff3b33b2afbf36632744e3a75da248ce41c34", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    19 \n\nTable 4 Colony characteristics of Alternaria porri on PDA \nmedia \n\nIsolates Colony characteristics \n\nColor Surface texture Shape \n\nBAU1 I1 Ashy black Velvety smooth Regular \nBAU2 I2 Olivaceous \n\ngreen \nFluffy Regular \n\nBARI1 I3 Greyish \nwhite \n\nFluffy Regular \n\nBARI2 I4 Ashy black Velvety \nsmooth \n\nIrregular \n\nBARI3 I5 Olivaceous \ngreen \n\nCottony Regular \n\nBARI3 I6 Smoky ash Fluffy Regular \nBARI4 I7 Black Fluffy Irregular \nBARI4 I8 Off white Fluffy Regular \nLD I9 Dark Velvety \n\nsmooth \nRegular \n\nLIND I10 Ashy Black Velvety \nsmooth \n\nIrregular \n\nIn the column BAU1 I1= BAU1 Isolate 1; BAU2 I2= BAU2 \nIsolate 2; BARI1 I3= BARI1 Isolate 3; BARI2 I4= BARI2 \nIsolate 4; BARI3 I5= BARI3 Isolate 5; BARI3 I6= BARI3 \nIsolate 6; BARI4 I7= BARI4 Isolate 7; BARI4 I8= BARI4 \nIsolates 8; LD I9= Local Deshi Isolate 9 and LIND I10= \nLocal Indian Isolate 10  \n\nAfter 14 days of inoculation all the isolates had \n\nsuppressed growth on PDA media. From the \n\nmicroscopic study of Alternaria porri it was found that at \n\nfirst the mycelium of the fungus was hyaline then \n\neventually turned to pale brown through olivaceous \n\nbrown, smoky ash to black blended black tinge. The \n\nhypha of the conidia was septate. Colony shape of the \n\nisolates was regular with concentric ring sometimes \n\nirregular. \n\n \n\nPathogenicity test of Alternaria porri \n \n\nSymptoms developed on inoculated plants was \n\nrecorded from time to time. After 5 days of inoculation, \n\ntiny, water soaked, sunken, and whitish lesions on the \n\ninoculated leaves were visible. While the disease \n\nprogressed, the lesions expanded, changed to elliptical \n\nto oblong, zonate and transformed reddish brown to \n\npurple encircled by pale yellow halo enlarging upwards \n\nand downwards. After 15 days of inoculation, chlorosis \n\nof the leaves was observed on the plants. The inoculated \n\nplants were dried completely after 21 days of \n\ninoculation. The symptoms were almost identical to \n\nthose under field experiment. Conidia of A. porri were \n\nfound under compound microscope from the sectioned \n\ndiseased leaves (Plate 5). \n\n Efficacy of selected treatment on percent disease \n\nincidence and severity of purple blotch of garlic in \n\ntreated condition \n\nIn case of % disease incidence, all botanicals and \n\nTrichoderma were found significantly effective in \n\nreducing disease incidence as compared to control and \n\nfungicides (Table 5).  \n\n \n  \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    20 \n\nThe results revealed that Lantana camera (Lantana leaf) \n\nextracts designated as T2 was  \n\nfound most effective in minimizing the disease \n\nincidence (26.67%, 26.67% and 33.33%) at all 30, 45 and \n\n60 DAS, respectively.", "start_char_idx": 26408, "end_char_idx": 29213, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a4ab008b-6ffa-4258-92b7-00e828ad7baf": {"__data__": {"id_": "a4ab008b-6ffa-4258-92b7-00e828ad7baf", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "865f2ce7-2997-4ea1-9637-9bf3a2211e96", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "cefb3448e9ec806822cc411323f0af9ffd211c1bdeef350b03cd3360c57e20a6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "68be28db-6e61-48d0-b5d9-a26326e11912", "node_type": "1", "metadata": {}, "hash": "1d3e682d5f43dbc6dff465525bad894efd9e3a5cd5800aa16f4dc74394bfbf09", "class_name": "RelatedNodeInfo"}}, "text": "The second effective treatment \n\nwas T1 (33.33% and 43.33%) which was statistically alike \n\nwith T8 (43.33%, 50% and 56.67%) followed by T5 (50%, \n\n53.33% and 56.67%) respectively, whereas the maximum \n\ndisease incidence was recorded in control T9 (86.67%, \n\n96.67% and 100.00%) at 30, 45 and 60 DAS.  \n\nIn case of % disease severity, Lantana camara (T2) was \n\nfound most effective in reducing disease severity at 30 \n\nDAS (11.00%) whereas, at 45 DAS and 60 DAS (18.67% \n\nand 19.33%) T1 (Trichoderma harzianum) was found most \n\neffective. At 30 DAS the second effective treatment was \n\n11.33% in T3 and T8 in reducing the disease severity. On \n\nthe other hand, at 45 and 60 DAS T2 gave the second \n\nlowest severity of (19.33% and 20.67%). All the \n\ntreatments showed the statistical similar result in \n\nreducing disease incidence and severity over control. \n\nMean performance of different treatment on growth \n\nand yield parameters against purple blotch disease of \n\ngarlic \n\nThe results of mean performance of yield parameter due \n\nto treatment are presented in the Table 6. The data \n\nrevealed that all the botanical plant extracts, bio-agent \n\nand fungicides given significantly better results in \n\ncomparison to control. The best plant height was noted \n\non T4 treatment (21.55 cm, 29.07 cm and 32.67cm) at 30, \n\n45 and 60 DAS, respectively in contrast to control. \n\nMaximum no. of leaf per plant was found on T2, T3, T4, \n\nT5, T6 and T9 (4.00) and minimum no. of leaf per plant \n\nwas found on other remaining treatment (3.67). \n\nMaximum fresh weight and dry weight was found on T5 \n\n(99.33 and 75.00gm/m2) statistically similar with T6 \n\n(91.00 and 65.67gm/m2) in contrary, T9 gave poor fresh \n\nweight and dry weight of 41.67 and 24.33gm/m2.  \n\nDiscussions \n\nPreliminary symptoms of purple blotch disease were \n\nvisible on the leaves in the shape of tiny, whitish, \n\nTable 5.", "start_char_idx": 29214, "end_char_idx": 31094, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "68be28db-6e61-48d0-b5d9-a26326e11912": {"__data__": {"id_": "68be28db-6e61-48d0-b5d9-a26326e11912", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a4ab008b-6ffa-4258-92b7-00e828ad7baf", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "85fed9c4ccaf7e2813a4d3e335862220ab097fa6ba96dfed98217db208c98355", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "639ba91f-9e76-45b6-8ec8-c33a897fb128", "node_type": "1", "metadata": {}, "hash": "756798344dd5b25716722ebcca5e106468adb1a23857130a1d847f69dd5d34f3", "class_name": "RelatedNodeInfo"}}, "text": "Efficacy of selected treatment on percent disease incidence and severity of purple blotch of garlic     \n\nTreatment (%) Disease incidence (%) Disease severity  \n\n30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS  \n\nT1 33.33 cd 43.33 cd 43.33   cd 13.33 c-e 18.67 e 19.33   e  \nT2 26.67 d 33.33 d 33.33   d 11.00 e 19.33 de 20.67   e  \n\nT3 53.33 b 60.00 b 60.00   bc 11.33 de 22.00 c-e 26.00   de  \n\nT4 46.67 bc 46.67 b-d 53.33  bc 14.67 b-e 23.33 c-e 26.00   de  \nT5 50.00 b 53.33 bc 56.67  bc 15.33 b-d 26.00 cd 46.67   c  \n\nT6 56.67 b 56.67 bc 60.00  bc 16.00 bc 26.67 c 30.67   d  \n\nT7 53.33 b 56.67 bc 70.00  b 18.00 b 46.00 b 55.33   b  \nT8 43.33 bc 50.00 bc 56.67  bc 11.33 de 44.00 b 44.00   c  \n\nT9 86.67 a 96.67 a 100.00 a 28.67 a 81.00 a 100.00 a  \n\nCV 16.67 16.50 17.22 15.50 11.91 11.17  \nCV= Coefficient of variance; In a column mean values having similar letter(s) are statistically similar and those having dissimilar \nletter(s) differ significantly as per 0.01 level of significance. Here, T1=Trichoderma harzianum; T2=Lantana camara; T3=Spilanthes \npaniculata; T4=Ocimum sanctum; T5=Raphanas raphanistrum; T6=Azadirachta indica; T7=Mancozeb 80% WP; T8=Sulcox 50% WP and \nT9=Control \n\n \n\nTable 6. Mean performance of different treatments on growth and yield parameters against purple blotch disease of \ngarlic \n\n \n\nTreatments Plant height (cm) No.", "start_char_idx": 31095, "end_char_idx": 32450, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "639ba91f-9e76-45b6-8ec8-c33a897fb128": {"__data__": {"id_": "639ba91f-9e76-45b6-8ec8-c33a897fb128", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "68be28db-6e61-48d0-b5d9-a26326e11912", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "1cabfaa9a5a85e03345c8924260c4f8cfa204197f3a6330d7082e4c534330acf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "faf57922-a3a2-4cab-ad00-c080612ca975", "node_type": "1", "metadata": {}, "hash": "3b6196a944120d485abb7f2ea41d17115b1ec18a2bae91a04dfa625535dff36e", "class_name": "RelatedNodeInfo"}}, "text": "of leaf per plant Fresh \nweight \n(gm/m2) \n\nDry weight \n(gm/m2) \n\n \n\n30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS  \n\nT1 17.58 c 24.77 bc 28.80 b-d 3.67 a 4.33 cd 5.33 a-c 69.33 c 48.33  c  \n\nT2 21.55 ab 25.60 bc 29.08 b-d 4.00 a 5.00 a-c 5.67 a-c 64.33 cd 42.00  cd  \n\nT3 18.48 bc 23.53 c 28.12 cd 4.00 a 4.00 d 4.33 c 62.00 d 39.00  de  \n\nT4 21.55 ab 29.07 a 32.67 a 4.00 a 5.00 a-c 6.33 a 87.00 b 66.33  b  \n\nT5 18.70 bc 26.07 b 29.16 a-d 4.00 a 5.33 ab 6.33 a 99.33 a 75.00  a  \n\nT6 21.55 a 27.20 ab 32.02 ab 4.00 a 5.67 a 6.67 a 91.00 b 65.67  b  \n\nT7 16.98 c 27.17 ab 30.58 a-c 3.67 a 4.67 b-d 6.00 ab 52.67 e 33.67  e  \n\nT8 16.72 c 25.24 bc 28.12 cd 3.67 a 5.00 a-c 6.33 a 52.00 e 33.67  e  \n\nT9 17.97 c 23.20 c 25.77 d 4.00 a 4.00 d 4.67 bc 41.67 f 24.33  f  \n\nCV 7.46 5.66 6.91 7.42 8.18 15.59 5.96 9.09  \n\nCV= Coefficient of variance; In a column mean values having similar letter(s) are statistically similar and those having dissimilar \nletter(s) differ significantly as per 0.01 level of significance. Here, T1=Trichoderma harzianum; T2=Lantana camara; T3=Spilanthes \npaniculata; T4=Ocimum sanctum; T5=Raphanas raphanistrum; T6=Azadirachta indica; T7=Mancozeb 80% WP; T8=Sulcox 50% WP \nand T9=Control \n\n \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    21 \n\nsunken like lesions. At the final stage, water soaked \n\nlarge zonate lesions of purplish red color enclosed by \n\nyellowish pale brown border was found. The symptoms \n\nand advancement of the disease were similar and have \n\nbeen stated by completely different researchers who \n\nrecorded that white flecks like symptoms appeared at \n\ninitial stage on older leaves, that enlarged and \n\ndeveloped into elliptical to rectangular sunken zonate \n\npurple lesions with a yellow to pale brown margin \n\nunder favorable environmental conditions [39, 40, 41, \n\n42]. \n\nThe highest disease incidence (34.44% and 40.00%) and \n\nseverity (70.67% and 92.00%) was found on BARI \n\nRashun-3 variety at 60 DAS and 90 DAS, respectively. \n\nOn the contrary, the lowest disease incidence (1.47% and \n\n36.00%) was recorded on BAU Rashun-2 variety.", "start_char_idx": 32451, "end_char_idx": 34580, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "faf57922-a3a2-4cab-ad00-c080612ca975": {"__data__": {"id_": "faf57922-a3a2-4cab-ad00-c080612ca975", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "639ba91f-9e76-45b6-8ec8-c33a897fb128", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "bccba26974eb9a020cebd61051691eeead4fcdc1be6a937d2bf66a0ade72375e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "859a7ca0-3efa-46da-bf81-3ee5f501faa8", "node_type": "1", "metadata": {}, "hash": "b2e46e1851c28d69c6c93e6ed342aca4d0c81291e81e7cf024279ab380a6d96d", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nstudy was almost similar to Patil (1999) [43] throughout \n\nsurvey of purple blotch incidence of garlic he reported, \n\nmaximum incidence of disease in Dharwad and Gokak \n\ntaluks during kharif 1998 and rabi 1998-99. An Indian \n\nStudy carried out in the horticulture garden of Raichur, \n\nwhere the highest disease severity (49.63%) was noted \n\nand least (10.00%) was in Neermanvi [44]. Yadav (2013) \n\n[45] studied on the onion purple leaf blotch (PLB) \n\ndisease in Navsari district of Gujrat over two Rabi \n\nseasons where the severity of the disease ranging from \n\n11.29 to 63.73%. Relative results were mentioned in [46] \n\nwherever, the highest per cent disease index (46.00) \n\nwere found at the fields of Sangreshkoppa village in \n\nBelgaum district and at Hulkund village in Belgaum \n\ndistrict the least per cent disease index (3.00) of purple \n\nblotch disease was recorded. Angadi et al. (2018)[47] also \n\nattained purple blotch disease from his survey.  \n\nNegative correlation with yield at 0.01% level was \n\nfound. These values clearly expressed that less infection \n\nof disease severity provide higher yield and the more \n\nthe disease is severe the more the degradation of yield \n\noccurred. Similar correlation was done by Jannatun et al. \n\n(2020) [48] wherever leaf height (cm) showed negative \n\ncorrelation with the entire yield parameters considered \n\nand at 0.01% levels of probability it become significant. \n\nNumber of leaves showed positive correlation with total \n\nyield defining characters except clove diameter (-.859).  \n\nAlmost all of the A. porri isolates showed fluffy growth \n\non potato dextrose agar. The colony color varied from \n\nolivaceous green in BAU2 I2 to greyish white in BARI1 I3, \n\nAshy black in BAU1 I1, BARI2 I4 and LIND I10, off-white \n\nin BARI4 I8 and black in rest of the isolates. After 14 days \n\nof incubation all the isolates had suppressed growth on \n\nPDA media. The causal agent, A. porri was isolated and \n\npure culture was cultured on PDA media. The isolated \n\npathogen was identified as A. porri following \n\nmorphological features given by Neergaard (1938) [49]. \n\nIn accordance with Chethana (2010), Chowdhury (2013) \n\nand Yadav et al. (2017) [25, 50, 51] potato dextrose agar \n\nwas the most acceptable culture media for mycelial \n\ngrowth and sporulation of Alternaria porri. The cultural \n\ncharacteristics of different isolates of A. porri were \n\nexamined by Shahnaz et al. ( 2013) [52] where she \n\nrecorded that almost all of the isolates had fluffy growth \n\non PDA with colony color varied from pinkish white \n\nthrough dull orange to olivaceous and black with \n\ndistinct to diffuse patterns of zonation.  Mohsin et al. \n\n(2016) [53] used 27 isolates of Alternaria porri which were \n\nisolated from diseased leaf samples collected from \n\ncompletely different onion growing regions of \n\nBangladesh and later characterized for cultural, \n\nmorphological and pathogenic variabilities where \n\ncolony color ranged between light to dark olivaceous \n\nand grayish white with irregular, regular with \n\nconcentric ring and regular without concentric ring \n\nshape. Isolates impregnated media with color ranged \n\nbetween grey to brown on the reverse of the plates. \n\nAll botanicals and Trichoderma harzianum were found \n\nsignificantly effective in reducing disease incidence and \n\nseverity compare to untreated control and fungicides. \n\nThe result revealed that Lantana camara designated as T2 \n\nwas found most effective in minimizing the disease \n\nincidence (26.67%, 26.67% and 33.33%) at 30 DAS, 45 \n\nDAS and 60 DAS.", "start_char_idx": 34581, "end_char_idx": 38136, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "859a7ca0-3efa-46da-bf81-3ee5f501faa8": {"__data__": {"id_": "859a7ca0-3efa-46da-bf81-3ee5f501faa8", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "faf57922-a3a2-4cab-ad00-c080612ca975", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "5c78107ace8f7038b88d6a7d2d3cf6844fa63f1d5122103efe1e4d44bfeda130", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "36094e5a-5a5e-44a9-ac1d-f39cef123606", "node_type": "1", "metadata": {}, "hash": "5ea1c6d23322247c9806623d7b92d0cadc55a92e3b5853dc698c815bdfc4d409", "class_name": "RelatedNodeInfo"}}, "text": "However, the maximum disease \n\nincidence was recorded in control T9 (86.67%, 96.67% \n\nand 100.00%). Again, Lantana camera (T2) was found \n\nmost effective in reducing disease severity at 30 DAS \n\n(11.00%) contrarily, at 45 DAS and 60 DAS (18.67% and \n\n19.33%) T1 (Trichoderma harzianum) was found most \n\neffective. At 45 DAS and 60 DAS T2 gave the second \n\nlowest severity of (19.33% and 20.67%). Datar (1994) [54] \n\nevaluated six plant extracts under field condition and \n\nnoticed that maximum depletion of purple blotch was \n\nattained with leaf extract of. Polyalthia longifolia. A field \n\ntrial were assessed by Prasad and Barnwal (2004) [55] on \n\nStemphylium blight of onion (cv. N-53) during rabi, \n\n1998-1999 and 1999-2000 crop season in Bihar where, \n\ndisease intensity was lowest (38.1% and 38.2%) with \n\n20% leaf extracts of Azadirachta indica. The obtained \n\nresults correspond with [36, 56]. Consistent with Uddin \n\net al. (2006) [57] after 10 days of sowing  disease \n\nincidence (19.95 %, 13.63 %) and severity (38.87 %, 34.59 \n\n%) were reduced due to the bulb treatment with either \n\nDithane M-45 (0.45 %) or Rovral 50 WP (0.2 %) followed \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    22 \n\nby foliar spraying with the same, and increased seed \n\nyield by 64.82 % and 42.18 % respectively. Similar \n\nresults were obtained by [23, 37, 58, 59, 60, 61, 62, 63] \n\nwhere different treatments had inhibitory effect on \n\nfungus based on phytochemical present in plants. \n\nConclusions \nThe present investigations showed purple blotch highest \n\ndisease incidence (40.00%) and severity (92.00%) was \n\nfound on BARI Rashun-3 variety at 90 DAS, \n\nrespectively. On the contrary, the lowest disease \n\nincidence (36.00%) was recorded on BAU Rashun-2 \n\nvariety. Negative correlation was found between disease \n\nseverity and yield (t/ha) against all identified diseases. \n\nIn laboratory Alternaria porri was isolated from infected \n\nleaves. After 14 days of incubation mycelial growth \n\ncovered the whole petridish by Alternaria porri. Cultural \n\nand morphological variability exits in purple blotch \n\n(Alternaria porri). All botanicals and Trichoderma were \n\nfound significantly effective in reducing disease \n\nincidence and severity compare to control. Lantana \n\ncamara (T2) was found most effective in minimizing the \n\ndisease incidence (26.67%, 26.67% and 33.33%) at 30 \n\nDAS, 45 DAS and 60 DAS. Again, Lantana camara (T2) \n\nwas found most effective in reducing disease severity \n\n(11.00%) at 30 DAS and Trichoderma harzianum (T1) \n\n(18.67% and 19.33%) at 45 DAS and 60 DAS against \n\npurple blotch disease in compare to control and \n\nfungicides. \n\nAcknowledgements  \nWe would like to express cordial gratitude to Prof. Dr. \n\nAbdur Rahim, Horticulture Department, Bangladesh \n\nAgricultural University, Mymensingh and Late Arpon \n\nHaider, Scientific officer, Bangladesh Agricultural \n\nResearch Institute, Joydebpur, Gazipur for providing \n\nthe different garlic varieties and good cooperation.", "start_char_idx": 38137, "end_char_idx": 41172, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "36094e5a-5a5e-44a9-ac1d-f39cef123606": {"__data__": {"id_": "36094e5a-5a5e-44a9-ac1d-f39cef123606", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "859a7ca0-3efa-46da-bf81-3ee5f501faa8", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "6e231ef369dcd951b6cf3b8de3de71109feb882c16b0d5a42593906c923e924d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ea29ee1e-1e7b-4959-b190-76bc676879ef", "node_type": "1", "metadata": {}, "hash": "30fc27da848c0764693ba61321e85ea7af061aabb6f39b4cf8b27e3f0b3e7116", "class_name": "RelatedNodeInfo"}}, "text": "Special thanks to all the teacher and staffs of Plant \n\nPathology department, Sher-e-Bangla Agricultural \n\nUniversity, Dhaka, Bangladesh for helping throughout \n\nthe research work. \n\nConflict of interest  \nAll authors declare that they have no conflict of interest. \n\nCompliance with ethical standards  \nThe present manuscript does not contain any studies \n\nwith human participants or animals performed by the \n\nauthors. \n\nFunding  \nThe research work did not receive any funding from any \n\ninstitution or organization. \n\nAuthor contributions  \nUmme Habiba Akter conducted the whole research and \n\nwrote the manuscript; Jannatun Nahar Prinky and Mst. \n\nRehena Khatun collected the purple blotch samples \n\nfrom field and helped in analyzing the data; Fatema \n\nBegum designed and supervised the research work and \n\nhelped to correct the manuscript; M. R. Islam co-\n\nsupervised the research work, read the manuscript \n\ncontributed to the conceptualization, and methodology \n\nof the study. \n\nReferences \n1. Voigt, C. Glorious garlic herb of the year 2004. J Int Herb Pp. 1-6. \n\nAssociation Horticulture Committee, Virginia State University. \n2004. \n\n2. Kilgori, M, MagaJi, M and Yakubu, A. Effect of plant spacing and \ndate of planting on yield of two garlic (Allium Sativum L.) cultivars \nin Sokoto, Nigeria. Am-Eurasian J Agric Environ Sci. 2007;2(2): 153-\n157. \n\n3. Asfand, R, Hidayatullah, Raheel, B, Arshad, MU, Zaffar, A and \nMaouz, I. Adaptability studies of garlic (Allium sativum) advanced \nlines. J Sci Agric. 2019;3: 19-21. \n\n4. FAOSTAT. Crops. FAOSTAT. 2021. http:/ww  \nw.fao.org/faostat/en/#data/QC/ \n\n5. Hossain, MM and Abdullah, F. Forecasting the Garlic Production \nin Bangladesh by ARIMA Model. Asian J Crop Sci. 2015;7(2): 147-\n153. \n\n6. Nonnecke, I. Vegetable Production, New York. 1989; Pp. 657. \n7. Mishra, RK, Verma, A, Singh, S and Gupta, RP. Screening of garlic \n\nlines against purple blotch and Stemphylium blight. Pest Manage \nHort Ecosys. 2009;15: 138-40. \n\n8. Islam, MR, Akter, N, Chowdhury, SM, Ali, M and Ahamed, KU. \nEvaluation of fungicides against Alternaria porri causing purple \nblotch of onion. J Agric Sci Technol. 2001;2(1):27-30. \n\n9. Gupta, RP and Srivastava, PK. Studies on the intervals and \nquantity of solution of mancozeb for mixed pesticides on the \ncontrol of diseases and thrips of kharif onion. AADF Newsletter. \n1993;13(1): 1214. \n\n10. Sandhu, KS, Gill, SPS and Sindhu, JS. Field evaluation of onion \nvarieties against Purple blotch. Indian Phytopathol. 1981;35: 540. \n\n11. Thind, TS and Jhooty, JS. Association of trips with purple blotch \ninfection on onion plants caused by Alternaria porri. Indian \nPhytopathol. 1982;35: 696-698. \n\n12. Prakasam, V and Sharma, P. Trichoderma harzianum (Th-3) a \npotential strain to manage the purple blotch of onion (Allium cepa \nL.) caused by Alternaria porri under North Indian Plains. J Agric Sci. \n2012;4(10): 266-272. \n\n13.", "start_char_idx": 41175, "end_char_idx": 44085, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ea29ee1e-1e7b-4959-b190-76bc676879ef": {"__data__": {"id_": "ea29ee1e-1e7b-4959-b190-76bc676879ef", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "36094e5a-5a5e-44a9-ac1d-f39cef123606", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "4f65b10407eeb771add3e974b26553a2c56caa5b30c4597decf8a39f384b168f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "194b4776-c3cf-4f0f-a55e-1aeeb3aea060", "node_type": "1", "metadata": {}, "hash": "5d924b87f24cf71e895c23e5e7256aa05855c7d693cd01d4c2514a4c47ce8933", "class_name": "RelatedNodeInfo"}}, "text": "2012;4(10): 266-272. \n\n13. Singh, D, Dhiman, J, Sidhu, A and Singh, H. Current status of \nonions in India: strategies for disease resistance breeding for \nsustained production. Onion Newsletter for the Tropics. 1992;43\u201344. \n\n14. Schwartz, H. Botrytis, downy mildew and purple blotch of onion. \nExtension No. 2.941. Colorado State University Cooperative. 2004. \n\n15. Rahman, SMM, Maniruzzaman, SM, Nusrat, S and Khair, A. In \nvitro evaluation of botanical extract, bio-agents and fungicides \nagainst purple blotch diseases of bunch onion in Bangladesh. \nAdvan Zoolo Bot. 2015;3(4): 179\u2013183 \n\n16. Junaid, JM, Dar, NA, Bhat, TA, Bhat, AH and Bhat, MA. \nCommercial bio-control agents and their mechanism of action in \nthe management of plant pathogens. Int J Modern Plant Animal Sci. \n2013;1(2): 39-57. \n\n17. Sharma, R, Joshi, A and Dhaker, RC. A brief review on mechanism \nof Trichoderma fungus use as biological control agents. Int J \nInnovations in Bio-Sc.i 2012;2: 200-210. \n\n18. Naher, L, Yusuf, U, Ismail, A and Hossain, K. Trichoderma spp: A \nbio control agent for sustainable management of plant diseases. \nPakistan J Botany. 2014; 46(4): 1489-1493. \nwww.researchgate.net/publication/281736621 \n\nhttp://www.fao.org/faostat/en/#data/QC/\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    23 \n\n19. Mar\u00edn, A, Oliva, J, Garcia, C, Navarro, S and Barba, A. Dissipation \nrates of cyprodinil and fludioxonil in lettuce and table grape in the \nfield and under cold storage conditions. J Agric Food Chem. 2003;51: \n4708\u20134711. \n\n20. Rial-Otero, R, Arias-Est\u00e9vez, M, L\u00f3pez-Periago, E, CanchoGrande, \nB and Simal-G\u00e1ndara, J. Variation in concentrations of the \nfungicides tebuconazole and dichlofluanid following successive \napplications to greenhouse-grown lettuces. J Agric Food Chem. \n2005;53: 4471\u20134475. \n\n21. Kumar, TP and Palakshappa, MG. Management of purple blotch \nof onion through bioagents. Karnataka J Agric Sci. 2008;21(2): 306-\n308. \n\n22. Chethana, BS, Ganeshan, G, Rao, AS and Bellishree, K. In vitro \nevaluation of plant extracts, bio-agents and fungicides against \nAlternaria porri (Ellis) Cif., causing purple blotch disease of onion. \nPest Manage Hort Ecosys. 2012;18(2): 194-198. \n\n23. Ali, H, Nisha, HAC, Hossain, MB and Islam, MR. Evaluation of \ncombined effect of micronutrients (ZnSO4 + Borax) and fungicides \nto control the purple blotch complex of onion (Allium cepa). Am J \nPlant Sci. 2016;7: 715-723. \n\n24. [24] Jhala, P and Bali, ML. Effective management of purple blotch \nof onion caused by Alternaria porri (Ellis) through host resistance, \nfungicides and botanicals. Int J Current Microbiol Appl Sci.", "start_char_idx": 44059, "end_char_idx": 46724, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "194b4776-c3cf-4f0f-a55e-1aeeb3aea060": {"__data__": {"id_": "194b4776-c3cf-4f0f-a55e-1aeeb3aea060", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ea29ee1e-1e7b-4959-b190-76bc676879ef", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "f14ad84296656e1842817ef26ca744a6887fbff730677d1d1cea20ec3ce40b60", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d2a06c6f-9dac-4dcf-8670-26a678a90c93", "node_type": "1", "metadata": {}, "hash": "01824a1eabb9faa2045e751adcca6ca92e134369ad44dac63c9ac70215a132c9", "class_name": "RelatedNodeInfo"}}, "text": "Int J Current Microbiol Appl Sci. \n2017;6(5): 1737-1745. https://doi.org/10.20546/ijcmas.2017.605.188 \n\n25. [25] Yadav, RK, Singh, A, Jain, S and Dhatt, AS. Management of \npurple blotch complex of onion in Indian Punjab. Int J Appl Sci \nBiotechnol. 2017;5(4): 454-464. doi:10.326/ijasbt.v5i4.18632 \n\n26. [26] Hossain, M, Chowdhury, MN, Khan, AL. Effect of fungicides \non the production of healthy onion seeds. Abstract of Fifth \nBiennial Conference, Bangladesh Phytopathological Society. 1993. \n\n27. [27] Manandhar, HK, Timila, RD, Sharma, S, Joshi, S, Manandhar, \nS, Gurung, SB, Sthapit, S, Palikhey, E, Pandey, A, Joshi, BK, \nManandhar, G, Gauchan, D, Jarvis, DI and Sthapit, BR. A field \nguide for identification and scoring methods of diseases in the \nmountain crops of Nepal. NARC, LI-BIRD and Bioversity Int. \n2016;P: 12. \n\n28. [28] Islam, MR, Ashrafuzzaman, MH, Adhikari, SK, Rahman, MH \nand Rashid, MH. Effect of fungicidal treatments in controlling \nAlternaria porri causing purple blotch of onion. Progress Agric. \n1999;10(1&2): 43-46. \n\n29. [29] Wheeler, BE. An introduction to plant diseases. John Willey \nand Sons, Ltd., U. K. 1969; Pp. 301. \n\n30. [30] Islam, MR, Akhter, N, Chowdhury, SM, Ali, M and Ahmed, \nKU. Evaluation of Fungicides against Alternaria porri Causing \nPurple Blotch of Onion. J Agric Sci Technol. 2003;2: 27-30. \n\n31. [31] Ellis, MB. Dematiaceous Hyphomycetes. Commonwealth \nMycological Institute, Kew, England. 1971;Pp 608. \n\n32. [32] Aminuzzaman, FM, Hossain, I, Ahmed, F. Cultural variation \nand pathogenicity of Bipolaris sorokiniana on wheat in Bangladesh. \nInt J Agric Environ Biotechnol. 2010;3(1):76-81. \n\n33. [33] Ainsworth, GC. Dictionary of fungi by Ainsworth and Bisby\u2019s. \nCommon Wealth Mycological Institute, Ferrylane, Kew Surrey, \nUK. 1971;p. 663. \n\n34. [34] Jain, S, Verma, KS and Shah, MD.  Pathological studies \non Alternaria alternata (Fr.) Keiss. Causing leaf blight of pear. Plant \nPathol J. 2005;4: 51-53. \n\n35. [35] Elwakil, MA, El-Refai, IM, Awadallah, OA, El-Metwally, MA \nand Mohammed, MS.  Seed-borne pathogens of faba bean in \nEgypt: Detection and pathogencity. Plant Pathol J. 2009;8: 90-97. \n\n36. [36] Kumar, U, Naresh, S and Biswas, SK. Ecofriendly \nmanagement of Stemphylium blight (Stemphylium botryosum) of \ngarlic by plant extract and bioagents. HortFlora Res Spect. 2012;1(1): \n42-45.  \n\n37. [37] Abd El- Samad, EH, Khalifa, RKHM, Lashire, ZA and Shafeek, \nMR.", "start_char_idx": 46691, "end_char_idx": 49120, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d2a06c6f-9dac-4dcf-8670-26a678a90c93": {"__data__": {"id_": "d2a06c6f-9dac-4dcf-8670-26a678a90c93", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "194b4776-c3cf-4f0f-a55e-1aeeb3aea060", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "064ceccd1f84341651f63ed393b12ef8c1f3b8b315d9f17efe9c0d00eb8233c0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d46b1bf4-6641-425c-89e0-3cecfad9bc4d", "node_type": "1", "metadata": {}, "hash": "13103090f8675b7d0350b4079fc18a82e88d5d4123c37120052a4cce42de2837", "class_name": "RelatedNodeInfo"}}, "text": "Influenced of urea fertilization and foliar application of some \nmicronutrients on growth, yield and bulb quality of onion. Aust J \nBasic Appl Sci. 2011;5 (5): 96-103. \n\n38. [38] Gomez, KA and Gomez, AA. Statistical Procedure for \nAgricultural Res. (2nd Ed.) Manila, Philippines. 1984;139-207. \n\n39. [39] Aveling. TAS. Purple blotch (Alternaria porri) of onion. Recent \nRes Div Plant Pathol. 1998;2: 63-76. \n\n40. [40] Suheri, H and Price, TV. Purple Leaf Blotch Disease of Allium \nspp. in Australia. Acta Hort. 2001;Pp: 555. \n\n41. Vijayalakhmi, M, Madhavi, M and Kavita, A. Studies of Alternaria \nporri (Ellis) Cifferi pathogenic to onion (Allium cepa L.). Arch of Appl \nSci Res. 2012;4(1): 1-9.  \n\n42. Agale, RC, Kadam, J, Joshi, MS and Borkar, PG. Symptomatology \nof purple blotch disease of onion and exploration of fungicides, \nphytoextract and bio-agents against causal fungus Alternaria porri. \nSpecies. 2014;11: 63-69. \n\n43. Patil, SB. Leaf blight of Garlic (Allium sativum) caused by Alternaria \nspp., M.Sc. (Agri.) Thesis, University of Agric Sci Dharwad (India). \n1999. \n\n44. Prakash, AJ. Studies on purple blotch of onion caused by Alternaria \nporri (Ellis). Cif. M. Sc. Thesis, University of Agricultural Science, \nDharwad (India). 2007. \n\n45. Yadav, PM. Management of purple blotch of onion caused by \nAlternaria porri (Ellis) Cif. Ph.D. Dissertation, Navsari Agricultural \nUniversity, Navsari, Gujrat. 2013. \n\n46. Pradnyarani, P, Nidagundi and Kulkarni, MS. Roving survey and \nin vitro identification of the fungus Alternaria porri causing purple \nblotch of onion in different growing areas of Northern Karnataka. \nTrends Biosci. 2014;7(10): 885-888. \n\n47. [47] Angadi, P, Aswathanarayana, DS, Amaresh, YS, Mallesh, SB, \nRamesh, G, Savitha, AS and Ajithkumar, K. Survey for severity of \npurple blotch and screening of onion varieties against purple \nblotch disease caused by Alternaria porri in North Eastern \nKarnataka. J Farm Sci. 2018;31(1): 79-83.  \n\n48. [48] Jannatun, NP, Fatema, B, Md, RI, Habiba, UA and Morshed, \nM. Screening of selected garlic varieties against Fusarium rot \ncaused by Fusarium proliferatum. SSRG Int J Agric Environ Sci. \n2020;7(4): 23-32. http://doi.org/10.14445/23942568/IJAES -\nV7I4P103 \n\n49. [49] Neergaard, P. Annual Report of the Phytopathological \nLaboratory of J.E. Ohlen\u2019s Window from 1st April, 1937 to 31st \nMarch 1938. Copenhagen. 1938;Pp. 12. \n\n50. [50] Chethana, BS, Kachapur, MR, Manjunath, B, Kumawat, GL. \nEffect of culture media and various carbon and nitrogen sources \non growth of Alternaria porri (Ellis) Cif. causing purple blotch of \nonion.", "start_char_idx": 49121, "end_char_idx": 51726, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d46b1bf4-6641-425c-89e0-3cecfad9bc4d": {"__data__": {"id_": "d46b1bf4-6641-425c-89e0-3cecfad9bc4d", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d2a06c6f-9dac-4dcf-8670-26a678a90c93", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "4b805dea6fe7bb48358cd07b3a7e23f9deb972b071b35797d6776b3576a1cce6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "adfc889f-601f-4177-8666-30047481bd5b", "node_type": "1", "metadata": {}, "hash": "88b5e00eb6a63892eb08d6ce361c76a93bf049e37690a233842fe740c23b95cc", "class_name": "RelatedNodeInfo"}}, "text": "causing purple blotch of \nonion. Environ Ecol. 2010;28: 2393-2395. \n\n51. [51] Chowdhury, NHA. Cultural, morphological and molecular \ncharacterization of Stemphylium vesicarium causing white blotch of \nonion. M.Sc. Thesis, Sher-e-Bangla Agricultural University, Dhaka, \nBangladesh. 2013. \n\n52. [52] Shahnaz, E, Razdan, VK, Andrabi, M and Rather, TR. \nVariability among Alternaria porri isolates. Indian Phytopathol. \n2013;66: 164-67. \n\n53. [53] Mohsin, SM, Rafiqul, MI, Noman, AFA, Nisha, HAC and \nHasanuzzaman, M. Cultural, Morphological and Pathogenic \nCharacterization of Alternaria porri Causing Purple Blotch of \nOnion. Notulae Botanicae Hort Agrobotanici Cluj-Napaco. 2016;44(1): \n222-227. doi:10.15835/nbha44110110 \n\n54. [54] Datar, VV. Investigation of purple blotch of onion in India. In \nInternational Symposium of Allium for the tropics, Bankok and \nChiang Mai, Thailand, 15-19 Feb., 1993. Acta Hort. 1994;358: 259-\n263. doi: 10.17660/ActaHortic.1994.358.42 \n\n55. [55] Prasad, SM and Barnwal, MK. Evaluation of plant extracts in \nmanagement of Stemphylium blight of onion. Indian Phytopathol. \n2004;57(1): 834-835. \nhttps://www.cabdirect.org/cabdirect/abstract/20043139584 \n\n56. [56] Islam, MM, Begum, F, Nahar, N,  Habiba, UA and \nFakruzzaman, KM. In vivo and In vitro Management of Purple \nBlotch of Onion by Using Fungicides and Plant Extracts. Int J Sci \nRes. 2020;9(10): 930-938. http://doi.org/10.21275/SR201008003034 \n\n57. [57] Uddin, MN, Islam, MR, Akhtar, N and Faruq, AN. Evaluation \nof fungicides against purple blotch complex of onion (Alternaira \nporri & Stemphylium boytryosum) for seed production. J Agric Educ \nTechnol. 2006;9(1&2): 83-86. \nhttps://www.researchgate.net/publication/275021495 \n\n58. [58] El- Sallami, IH and Gad, MM. Growth and flowering response \nof New York aster to a slow release fertilizer and foliar applied \nZinc. Assuit J Agric Sci. 2005;36(2): 121\u2013136. www. aun.edu.eg/ \nfaculty_agriculture/journal/papers.php?page=4&P_ID=234 \n\n59. [59] El- Tohamy, WA, Khalid, AKH, El-Abagy, HM and Abou- \nHussein, SD. Essential oil, growth and yield of onion (Allium cepa \nL.) in response to foliar application of some micronutrients. Aust J \n\nhttps://doi.org/10.20546/ijcmas.2017.605.188\nhttps://doi.org/10.17660/ActaHortic.1994.358.42\nhttps://www.ijsr.net/archive/v9i10/SR201008003034.pdf\nhttps://www.ijsr.net/archive/v9i10/SR201008003034.pdf\nhttps://www.ijsr.net/archive/v9i10/SR201008003034.pdf\nhttp://doi.org/10.21275/SR201008003034", "start_char_idx": 51694, "end_char_idx": 54166, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "adfc889f-601f-4177-8666-30047481bd5b": {"__data__": {"id_": "adfc889f-601f-4177-8666-30047481bd5b", "embedding": null, "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-226", "node_type": "4", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "2de8a57efae5a5191b930f77d56a6f3f7083ff4673337c8a2b21fd87b61dbfd3", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d46b1bf4-6641-425c-89e0-3cecfad9bc4d", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "566d189bcf9146e0d098e301e988b683c843a76d41df10141d257cda543eba65", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a2ddbcae-0ca2-4a12-b9e8-19655bee1e25", "node_type": "1", "metadata": {}, "hash": "a793bd9312515e6bc5fcfbadece9c9185b67229c278aedcfb64b0b24808e59fc", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):1 3 - 2 4      Akter et al.  \n\n\u00a9NJB, BSN    24 \n\nBasic Appl Sci. 2009;3(1): 201\u2013205. \nwww.researchgate.net/publication/289436650  \n\n60. [60] Alam, MN, Abedin, MD and Azad, MAK. Effect of \nmicronutrients on growth and yield of onion under calcareous soil \nenvironment. Int Res J Plant Sci. 2010;1(3): 56\u201361. \nwww.researchgate.net/publication/284880915 \n\n61. [61] Brahma, S, Yousef, MN and  Ara, R. Effect of micro- nutrients \non growth, yield and quality of summer onion. Research Report \n2009, Spices Research Center, BARI, Shibganj, Bogra. 2012. \n\n62. [62] Chanchan, M, Hore, JK and Ghanti, S. Response of garlic to \nfoliar application of some micronutrients. J Crop Weed. 2013;9(2): \n138\u2013141. \nwww.cropandweed.com/archives/2013/vol9issue2/25.pdf \n\n63. [63] Rizk, FA, Shaheen, AM, Abd El- Samad, EH and El- Labban, \nTT. Response of onion plants to organic fertilizer and foliar \nspraying of some micronutrients under sandy soil condition. J Appl \nSci Res. 2014;10(5): 383 \u2013 392. https://ssrn.com/abstract=2812463", "start_char_idx": 54169, "end_char_idx": 55228, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a2ddbcae-0ca2-4a12-b9e8-19655bee1e25": {"__data__": {"id_": "a2ddbcae-0ca2-4a12-b9e8-19655bee1e25", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "adfc889f-601f-4177-8666-30047481bd5b", "node_type": "1", "metadata": {"identifier": "njb-226", "author": "Habiba Akter, Umme; Begum, Fatema; Islam, M. R.; Nahar Prinky, Jannatun; Khatun, Mst. Rehena", "title": "Occurrence of Purple Blotch Disease Associated with Selected Garlic Varieties and its Management Through Bio-Agent, Botanicals and Fungicides", "date": "2022-07-30", "file": "njb-226.pdf"}, "hash": "f5d84a37b37a2c2efc193c211169834aefd9e29fcebc05793511120854fcd9be", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9f2faed1-fdd5-48e0-b26b-adfccc41d808", "node_type": "1", "metadata": {}, "hash": "4a5561447829dee26709179ec052177c71c55d138369f0b7c3a868ce4f82d5c4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 25-31  Research article  DOI: https://doi.org/10.54796/njb.v10i1.227 \n \n\n\u00a9NJB, BSN   25 \n\nPhytochemical and Antimicrobial Screening of Bark Extract of Shorea \nrobusta (Sal) \nBijay Kumar Shrestha , Bidhya Dhungana , Jenish Shakya , Romika Shrestha , Sujata Chauhan  \n\nDepartment of Microbiology, Central Campus of Technology, Tribhuvan University, Hattisar, Dharan, Nepal \n\nReceived: 10 Oct 2020; Revised: 29 Dec 2021; Accepted: 21 Jul 2022; Published online: 30 Jul 2022 \n\nAbstract \nDifferent parts of Shorea robusta (Sal) are being used in ancestral and ayurvedic medicines and are known to cure health \n\nailments. The different phytochemicals present in S. robusta is known to possess antimicrobial property. The different \n\nbotanical parts of this plant have been used in ayurvedic medicines to cure certain infectious diseases. The main aim of this \n\nstudy was to screen phytochemicals and antimicrobial activity of bark extract of S. robusta. Literatures were collected through \n\nbooks, journals and further additional information were collected from residents and traditional ayurvedic practitioners. The \n\nethanolic bark extract of S. robusta was obtained through 70% ethanol in rotatory shaker for 72 hours at 37 \u2103 and then the \n\ncrude extract was dried, preserved and analyzed for phytochemical analysis and antimicrobial activity. The phytochemical \n\nscreening of ethanolic extract of bark of S. robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, \n\nsteroids, anthraquinone and absence of phlobatannins, terpenoids, starch and proteins. The extract of S. robusta on \n\nStaphylococcus aureus exhibited clear zone of inhibition of 21mm at minimum inhibitory concentration (MIC) of 2 mg/mL \n\nwhile on Escherichia coli exhibited clear zone of inhibition of 9 mm at MIC of 4 mg/mL. The antimicrobial activity may be \n\nconferred due to the presence of plant phytochemicals. S. robusta bark extract exhibiting significant minimum inhibitory \n\nconcentration and antimicrobial activity indicates the efficacy of this plant to be considered for discovering and extracting \n\nnew antimicrobial products against the pathogens. These findings need further support for appropriate formulation of the \n\ndrug and its therapeutic use in clinical settings. \n\nKeywords: Antimicrobial screening, Bark extract, Minimum Inhibitory concentration, pathogens, phytochemical profile, \n\nShorea robusta \n\nCorresponding author, e-mail: interfacebj@gmail.com \n\nIntroduction \nShorea robusta is a deciduous large tree, exceptionally \n\nreaching a height of 50 m. [1]. S. robusta has always been \n\na tree of medical, cultural and economic importance and \n\nis known by common name \u2018Sal\u2019. S. robusta belongs to \n\nfamily Dipterocarpaceae and is reported to possess \n\nantimicrobial properties [2]. The entire tree of S. robusta is \n\nused for different purposes such as timber in house \n\nconstruction, firewood, leaves for making leaf-plates and \n\ncups [3]. The different chemical composition of S. robusta \n\nplant such as Asiatic acid, triterpenic acid, tannic acid and \n\nphenol is known to possess antimicrobial property [4]. \n\nThe different parts of S. robusta like bark, flower, resins \n\nare known to possess pharmacological action to treat \n\ndiarrheal diseases, Diabetes mellitus and bacterial \n\ninfections etc. [5]. \n\nStaphylococcus aureus is a pathogenic superbug that \n\ncauses bloodstream infection, tissue infections and often \n\nassociated with infective endocarditis [6]. Whereas, \n\nEscherichia coli is prominent cause of gastroenteritis, urine \n\ninfection and bloodstream infections [7].", "start_char_idx": 48, "end_char_idx": 3692, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9f2faed1-fdd5-48e0-b26b-adfccc41d808": {"__data__": {"id_": "9f2faed1-fdd5-48e0-b26b-adfccc41d808", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a2ddbcae-0ca2-4a12-b9e8-19655bee1e25", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "b437604603913ef77f48ea1d52e60d035107efa9d9f75ff82333a5f7d0a706a5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9a5462d5-e282-4bb1-b9f9-bca15fc1a409", "node_type": "1", "metadata": {}, "hash": "2d1f2f4ffbda09ecc17935d3e9d7ac26460e09cc1d8786e15220da85806f562c", "class_name": "RelatedNodeInfo"}}, "text": "These two are \n\nthe most prevalent and common cause of human \n\ninfections that induce clinical mastitis and need to be \n\ncorrectly cured by appropriate antimicrobials for \n\npreventing and controlling emerging drug resistance and \n\nnosocomial infections [8]. \n\nSince the pathogens are emerging with drug resistance, \n\nthe need for research in antimicrobial products from \n\nmedicinal plants can address the issue brought by drug \n\nresistant strains in clinical settings [9, 10]. The number of \n\ninfections caused by emerging drug resistant pathogens \n\ngrow daily and the hospitalized patients with \n\nimmunocompromised condition are more prone to \n\nsevere infections [11]. There is a line of antibiotics on \n\nmarket, but bacterial species shows resistance to most \n\nantimicrobials used in the clinical treatment [12]. \n\nIt has been documented that the bark of S. robusta is \n\ntraditionally used as astringent, acrid, cooling, \n\nanthelminthic, anodyne, constipating, and urinary \n\nastringent, union promoter depurative and tonic [13]. \n\nThey are useful in vitiated conditions of cough, ulcers, \n\nwounds, bacterial infections diarrhea, dysentery, \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nmailto:interfacebj@gmail.com\nhttps://orcid.org/0000-0002-6542-829X\nmailto:interfacebj@gmail.com\nhttps://orcid.org/0000-0002-5390-9889\nhttps://orcid.org/0000-0002-9397-4511\nhttps://orcid.org/0000-0002-4925-9297\nhttps://orcid.org/0000-0002-7528-3792\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.  \n\n\u00a9NJB, BSN   26 \n\ngonorrhea, leucorrhea, pruritus, leprosy, cough, and \n\nanemia [14]. Therefore, present study was aimed to \n\nscreen and evaluate phytochemicals and antibacterial \n\nactivity of crude ethanolic extracts of S. robusta bark \n\nextract against S. aureus and E. coli. \n\nMaterial and Methodology \nResearch Design \nThis study was conducted from February 2017 to April \n\n2017 at Microbiology Laboratory of Central Campus of \n\nTechnology, Dharan. Bark of S. robusta was collected \n\nfrom Bijaypur hill forest of Dharan-14 which extends at \n\nan altitude of 390 meters (Latitude 26\u00b0 49\u2032 12\u2033 N and \n\nLongitude: 87\u00b0 18\u2032 0\u2033 E). The selected plants were firstly \n\nidentified from herbarium collection of Postgraduate \n\nCampus Biratnagar, Nepal. The different botanical \n\ninformation was collected by field study, research articles \n\nand books. Medicinal, cultural and economic information \n\nabout the plants was obtained from experienced \n\ntraditional ayurvedic practitioners and local individuals. \n\nAll the information about plants, medicinal values and \n\nuses were documented. About 500 grams bark samples \n\nwere collected from the forest and were brought in \n\nMicrobiology lab of Central Campus of Technology, \n\nDharan. \n\nMicroorganisms used \n The bacterial strains used in this research were S. aureus \n\n(25923) and E. coli (CFT073) strain. These microbial \n\ncultures were requested and received from Microbiology \n\nDepartment of Central Campus of Technology, Dharan. \n\nThe growth media used were Mannitol Salt agar (MSA) \n\n(HiMedia, India) for S. aureus and MacConkey agar \n\n(MAC) (HiMedia, India) for E. coli. These selective \n\ngrowth medias were used for recovering the bacterial \n\nstrains from preserved culture for further study. The \n\nisolated bacterial strains from selective media were \n\nfurther subcultured in brainheart infusion broth media \n\n(HiMedia, India). \n\nExtract Preparation \nThe extraction methodology was carried out according to \n\nAgrawal and Paridhavi (2012) [15].", "start_char_idx": 3693, "end_char_idx": 7348, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9a5462d5-e282-4bb1-b9f9-bca15fc1a409": {"__data__": {"id_": "9a5462d5-e282-4bb1-b9f9-bca15fc1a409", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9f2faed1-fdd5-48e0-b26b-adfccc41d808", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "5b87bd5ec6a4e185d8b0c6760a94dade4fe41aa04bcd3b3df369d583f54adba5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "845a5248-3a77-485d-b2a7-604fdd2ff57a", "node_type": "1", "metadata": {}, "hash": "e93783bf7d2063b661bead3fbc10bc71eb25e99e817846cd8d5218023943a274", "class_name": "RelatedNodeInfo"}}, "text": "The bark of S. robusta \n\nwas chopped and dried under shade at room \n\ntemperature of 25 \u2103 for two weeks. The dried bark of S. \n\nrobusta was powdered using mortar and pestle at room \n\ntemperature. About 20 grams of the powdered plants was \n\nextracted with 400 mL of 70% ethanol in rotatory shaker \n\nfor 72 hours at 37 \u2103. The obtained extract was \n\nconcentrated and dried by evaporation in hot air oven at \n\n60 \u2103. Stock solutions of 32 mg/ mL was prepared in 10% \n\nsterile dimethyl sulfoxide (DMSO). The stock solution \n\nwas stored at 4 \u00b0C until use. Crude powder extract or \n\naqueous suspension of ethanolic dried extract was used \n\nin phytochemical screening. \n\nPhytochemical Assays \nScreening of the phytochemical constituents was carried \n\nout for detection of documented chemical constituents of \n\nbark extract as described by Thilagavathi et al., (2015) [17] \n\nand Harborne (1998) [18]. \n\nTest for Alkaloids \nIn a test tube, 2 mL ethanolic bark extract was inoculated \n\nwith 2-3 drops of HCl (dilute hydrochloric acid). To this \n\nsuspension about 1 mL of Dragendorff\u2019s reagent was \n\nadded. The presence of alkaloids is indicated by the \n\nappearance of orange to red precipitate. \n\nTest for flavonoids \nIn a test tube, 4 mL ethanolic bark extract was suspended \n\nwith 1.5 mL methanol. The solution was gently heated to \n\nwarm with addition of magnesium with 4 drops of Conc. \n\nHCL (Concentrated Hydrochloric acid). Development of \n\ncolor change is indicative for presence of flavonoids. \n\nTest for Phlobatannins \nBark extract sample was boiled with 1% aqueous \n\nhydrochloric acid. Suspension of red precipitate is \n\nevidence for the Phlobatannins.  \n\nTest for tannins \nAbout 2 mL of ethanolic bark extract was treated with 2-\n\n3 drops of 10% lead acetate. Tannins are indicated by the \n\ndevelopment of white precipitate. \n\nTest for steroids  \nAbout 10 mL of chloroform was taken in a test tube and \n\n2 mL of ethanolic bark extract was added to it. This \n\nsuspension was treated with 1 mL of acetic anhydride \n\nand then 2 mL of concentrated sulphuric acid. The \n\npresence of steroids is indicated by the development of \n\nblue green color at the junction. \n\nTest for Anthraquinone \nIn a test tube, dilute Sulphuric acid and 1 mL of diluted \n\nammonia were added to 5 mL of the ethanolic bark \n\nextract. Development of pink color indicates the existence \n\nof anthraquinone. \n\nTest for terpenoids \nAbout 10 mL of chloroform was added to 2 mL of \n\nethanolic bark extract. The resulting suspension was \n\ninoculated with 1 mL of acetic anhydride and 2 mL of \n\nconcentrated sulphuric acid. The existence of terpenoids \n\nis indicated by development of red, pink or violet color at \n\nthe junction. \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.  \n\n\u00a9NJB, BSN   27 \n\nTest for Starch \nBenedict\u2019s test: One litre of Benedict\u2019s solution was be \n\nprepared from 100 gm of anhydrous sodium carbonate, \n\n173 gm of sodium citrate and 17.3 gm of copper (II) \n\nsulfate pentahydrate. In a test tube, 0.5 mL of Benedict\u2019s \n\nreagent was added to 0.5 mL of the ethanolic bark extract. \n\nThe suspension was heated on water bath at 100 \u00baC for 2 \n\nminutes. The existence of starch is indicated by the \n\ndevelopment of red color precipitate.", "start_char_idx": 7349, "end_char_idx": 10604, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "845a5248-3a77-485d-b2a7-604fdd2ff57a": {"__data__": {"id_": "845a5248-3a77-485d-b2a7-604fdd2ff57a", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9a5462d5-e282-4bb1-b9f9-bca15fc1a409", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "27d10a82c54f32b181dcd1bee41a3a1767ec3ec56f4f8c01003addf24df4bf49", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4244fbbc-0eca-48c0-8ccf-14c8961f68bf", "node_type": "1", "metadata": {}, "hash": "1363f51dcc230da04799a5c2be6cc5266afce6089302284c7d9778485d99adcf", "class_name": "RelatedNodeInfo"}}, "text": "The existence of starch is indicated by the \n\ndevelopment of red color precipitate. \n\nTest for proteins \nNinhydrin Test: About 1 mL of the ethanolic bark extract \n\nwas treated with 2-3 drops of Ninhydrin agent and \n\nheated in a boiling water bath. The presence of proteins \n\nis indicated by the appearance of purple blue color. \n\nAntimicrobial Assay \nAntibacterial tests were carried out by well diffusion \n\nmethod as described by Aneja (2009) [16]. For \n\nantibacterial bioassay, the fresh bacterial inoculum with \n\nstandard turbidity of 0.5 McFarland standard for \n\nmicroorganism was arranged in Mueller Hinton broth \n\n(HiMedia, India) and about 100 \u00b5L culture was seeded \n\nover the Mueller Hinton agar (HiMedia, India). Mueller \n\nHinton agar (MHA) with 2% NaCl was seeded by S. \n\naureus culture and MHA without 2% NaCl was seeded by \n\nE. coli culture. With the cork borer no. 6, the wells of about \n\n6mm were created in the media plates. The different test \n\nconcentrations ranging from, 0.0625-16 mg/mL of bark \n\nextract was developed in 10% DMSO solution. About 50 \n\n\u00b5L aliquot of extracts with different concentrations were \n\ninoculated into the wells of MHA plates seeded by the S. \n\naureus strains. Similarly, about 50 \u00b5L aliquot of extract \n\nwith different concentrations were inoculated into the \n\nwells of MHA plates seeded by the E. coli strains. The \n\ninoculated culture medias were allowed to incubate at 37 \n\n\u2103 for 24 hours. About 50 \u00b5L of 10% sterile DMSO \n\nsolution was used for the negative control and penicillin \n\n(10 \u00b5g) and Gentamicin (10 \u00b5g) was used as the positive \n\ncontrol. After the incubation, the plates were observed for \n\nthe halo zone around the well. The halo zone around the \n\nwell represented zone of inhibition which was measured \n\nand documented. The experiments were performed for \n\nthree times and the mean zone of inhibition was \n\ncomputed. \n\nMinimum Inhibitory Concentration \nThe minimum inhibitory concentration (MIC) of plant \n\nextracts was studied using 96-well microtitre plates as \n\nexplained by CLSI (2012) [19]. The 96-well plates were \n\nprepared from 95 \u00b5L aliquot of Mueller Hinton Broth \n\n(MHB) (HiMedia, India) suspended in wells. In each well, \n\n5 \u00b5L bacterial culture of 0.5 McFarland standard, \n\nprepared in MHB medium was inoculated. About 100 \u00b5L \n\nof stock extract was suspended in first well and so was \n\nthe same volume of suspension serially diluted to achieve \n\ntwo-fold dilution ranging from 16-0.0625 mg/mL. For \n\nnegative control DMSO solution was used. The microtitre \n\nplates were covered with sterile lid and incubated at 37 \n\n\u00b0C exactly for 24 hours. The minimum concentration of \n\nthe extract sample, that inhibited growth of tested \n\norganism after overnight incubation, was determined as \n\nMIC. \n\nQuality Control \nComplete aseptic condition was maintained during \n\nmedia preparation, sample collection, sample processing. \n\nReagents and culture media were regularly monitored \n\nfor their manufacture and expiry date and proper \n\nstorage. Laboratory equipment like incubator, \n\nrefrigerator, autoclave and hot air oven were regularly \n\nmonitored for their efficiency. The temperature of the \n\nincubator and refrigerator was monitored every day. \n\nContamination of biological samples was prevented by \n\nperforming the work in laminar flow cabinet. \n\nData analysis \nThe information collected was documented and the data \n\nwould be analyzed by Microsoft Excel 2010. \n\nResults \nPhytochemical Screening \nThe phytochemical screening of ethanolic extract of bark \n\nof Shorea robusta indicated presence of phytochemicals \n\nlike, alkaloids, flavonoids, tannins, steroids, \n\nanthraquinone and absence of phlobatannins terpenoids, \n\nstarch and proteins (Table 1).", "start_char_idx": 10521, "end_char_idx": 14243, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4244fbbc-0eca-48c0-8ccf-14c8961f68bf": {"__data__": {"id_": "4244fbbc-0eca-48c0-8ccf-14c8961f68bf", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "845a5248-3a77-485d-b2a7-604fdd2ff57a", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "6575ffb9a0662284c653bb49addb933235524630491bd069c65aa5f68fbae3e1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91f6134e-7da0-4529-ae72-360bde3d81ad", "node_type": "1", "metadata": {}, "hash": "63739423f9834de2e29f720b7fbb888d8a9c7f76d7dc597ded2d9e257fae8108", "class_name": "RelatedNodeInfo"}}, "text": "Antimicrobial assay and MIC \nThe ethanolic bark extract was tested for antimicrobial \n\nactivity against both bacteria S. aureus (25923) and E. coli \n\n(CFT073). The antimicrobial assay with zone of inhibition \n\nwith positive controls are shown in Table 2.  \n\n \n\nTable 1. Phytochemicals screening of bark of Shorea robusta \n\nPhytochemicals Results Intensity of color \n\nAlkaloids Present +++ \n\nFlavonoids Present ++ \n\nPhlobatannins  Absent - \n\nTannins Present ++ \n\nSteroids Present + \n\nAnthraquinone  Present ++ \n\nTerpenoids Absent - \n\nStarch Absent - \n\nProteins Absent -", "start_char_idx": 14246, "end_char_idx": 14814, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91f6134e-7da0-4529-ae72-360bde3d81ad": {"__data__": {"id_": "91f6134e-7da0-4529-ae72-360bde3d81ad", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4244fbbc-0eca-48c0-8ccf-14c8961f68bf", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "e9bf8018bdb003ba5edfcdfc668643d63fe4d9fefc8e8dc2f8b785a1544ba827", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a3d998d0-0a9a-415c-870d-ccffcf05323c", "node_type": "1", "metadata": {}, "hash": "4dbf271414ad4412cc6e725dd875abfd15bb1bf76330daa5ab8a8d57283ee7a4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.  \n\n\u00a9NJB, BSN   28 \n\n \n\nThe ethanolic bark extract of S. robusta on S. aureus \n\nexhibited clear zone of inhibition of 21 mm at MIC of 2 \n\nmg/mL. The ethanolic bark extract of S. robusta on E. coli \n\nexhibited clear zone of inhibition of 9 mm at MIC of 4 \n\nmg/mL (Figure 1 and 2).  \n\n \n\n \n\nThe ethanolic bark extract of S. robusta expressed \n\nsuppressive activity on both S. aureus (25923) and E. coli \n\n(CFT073). The findings of this study provide significant \n\nin vitro antimicrobial activity of ethanolic extract of bark \n\nof S. robusta against S. aureus and E. coli. \n\n \n\nFigure 1. Antimicrobial Screening of bark extract of S. robusta \n\nagainst S. aureus \n\n \n\nFigure 2. Antimicrobial Screening of bark extract of S. robusta \nagainst E. coli \n \n\n \n\n \nFigure 3: Antimicrobial susceptibility test by S. robusta extract. \n\n \n\nMedicinal information \nThe medicinal, cultural, and economic information was \n\ncollected from Ayurvedic practitioners and locals are \n\nincluded in Table 3. \n\nTable 3. The medicinal, cultural, and economic information \n\nof S. robusta \n\nPlant Parts Medicinal use Other cultural \nand economic \nuse \n\nS. \nrobusta \n\nBark Enhance \nimmunity power, \ntreat typhoid, \ndiarrhea, ulcer. \n\nUsed in incense \nstick \n\nLeaves Reduce obesity, \ninhibit pain. \n\nBiodegradable \nleaf plates and \ncups \n\nResins Lower Fever, skin \ndisorder, \ndiarrhea. \n\nUsed in incense \nstick \n\nDiscussion \nNepal has always stood a nation of natural biodiversity \n\nrich in natural vegetation that includes herbs, shrubs at \n\ndifferent climate and altitude [20]. Many of such herbs \n\nhave been used as traditional medicines by local people \n\nincluding some even been market as ayurvedic medicine \n\n[21]. Nowadays, pharmacology industries are also \n\nseemed interested in incorporating natural drugs since \n\nbecause of modern drugs imposing many health side \n\neffects with growing incidence of antibiotic resistance. \n\nThe S. robusta which is one of the species of plant found \n\nin Nepal has been known to be best timber producing tree \n\n[22]. Its many parts have been used by local people for \n\nmedicinal and cultural purposes [23]. \n\nIn this study the phytochemical screening of ethanolic \n\nextract of S. robusta bark showed the presence of \n\nalkaloids, flavonoids, anthraquinone tannins and \n\nsteroids whereas phytochemicals like terpenoids, starch, \n\nphlobatannins and proteins were absent. This result \n\nTable 2. Antimicrobial assay of positive controls \n\nMicroorganisms \nType of \n\nBacteria \n\nAntibiotic Zone of \n\ninhibition in mm \n\nS. aureus Gram + Penicillin G (10\u00b5g)- 30 mm \n\nE. coli Gram - Gentamicin (10 \u00b5g) \u2013 17 mm \n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\n1 2 3 4 5 6 7 8 9\n\n16\n\n8\n\n4\n\n2\n1\n\n0.5 0.25 0.125 0.0625\n\n14\n\n11\n\n9\n\n0 0 0 0 0 0\n\nMIC (mg/mL )\n\nZone of Inhibition\n(mm)\n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\n30\n\n1 2 3 4 5 6 7 8 9\n\n16\n\n8\n\n4\n2 1 0.5 0.25 0.125 0.0625\n\n26\n24\n\n23\n21\n\n0\n\nMIC (mg/mL ) Zone of Inhibition (mm)\n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.", "start_char_idx": 14819, "end_char_idx": 17889, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a3d998d0-0a9a-415c-870d-ccffcf05323c": {"__data__": {"id_": "a3d998d0-0a9a-415c-870d-ccffcf05323c", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91f6134e-7da0-4529-ae72-360bde3d81ad", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "6ec4f8a8ed968ca2ac57d825d7a0ada4e2c4eec1c4acdf39342706977d221cbf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "602d6b5c-f1f1-4477-b9d1-ef306cd319a5", "node_type": "1", "metadata": {}, "hash": "2ceecc6e0a2811a5263ab5b251e2971da5b6273e9608680e0bdcd15dc147fd38", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN   29 \n\ncoincides with the study performed by [2, 24] which \n\nshowed the presence of common phytochemicals. \n\nSimilarly, the study performed by [3] showed the absence \n\nof anthraquinone which has shown similarity with this \n\npresent study. There are other many factors that affect \n\nphytochemical composition such as geographical \n\ncondition, climatic condition, collection procedure, \n\nstorage condition, etc. [25, 26, 27]. The effectiveness of the \n\nplant extract against selected bacterial strains may be due \n\nto the collective antimicrobial action of different \n\nphytochemical constituents [28]. The botanical \n\nbiomolecules like flavonoids, alkaloids and variety of \n\nother phenolic constituents have been identified with \n\nantimicrobial properties [29]. \n\nThe phytochemical screening of ethanolic extract of bark \n\nof S. robusta showed the presence of flavonoids, which is \n\nan antioxidant compound having antimicrobial property \n\nto suppress both certain species of Gram-positive and \n\nGram-negative bacteria [30]. Studies have forwarded \n\ndifferent antimicrobial mechanism of phytochemicals. \n\nSome phytochemicals are supposed to suppress growth \n\nand development of microorganism, bacterial cell \n\nmembrane disarrangement, halting microbial \n\nmetabolism and modifying microbial genetic expression \n\n[31]. Investigators have proved antibacterial activity of S. \n\nrobusta extracts against bacterial pathogens [32]. In one \n\nstudy, S. robusta extract exhibited antibacterial activity \n\nagainst different clinical pathogens [2]. In agreement \n\nwith this study even in the present study the ethanolic \n\nextract of bark of S. robusta exhibited significant \n\nantimicrobial activity with significant level of Minimum \n\ninhibitory concentration against both bacterial \n\npathogens. These results clearly suggest its antimicrobial \n\nefficacy in treating the infection caused by those \n\npathogens. \n\nPlant extract for natural antibiotic could be a strong \n\npotent drug against many pathogenic species and could \n\nbe drug of choice against emerging drug resistant strains \n\n[33]. The extended study for purification, activation and \n\ntherapeutic uses of plant extracts should be conducted to \n\nexamine its effective antimicrobial role against \n\npathogenic microorganisms. If being effective the \n\nnature\u2019s best metabolite could be effective in treating \n\ninfections caused by antibiotic drug resistance pathogens \n\nthat have become one of the major therapeutic challenges \n\nin clinical settings. \n\nThe indigenous community of developing nations are \n\nstill using the plant based traditional drugs for treating \n\nmany diseases. Resins, leaves, flower and bark extracts of \n\nS. robusta has been identified with rich medicinal \n\nimportance [34]. Potent antimicrobial drugs extracted \n\nfrom S. robusta may be safe for treating many infections \n\nand inflammations. In some Asian nations the research \n\non antimicrobial and immunomodulatory effect of the \n\nplant has been carried out and even they have come up \n\nwith excellent results on its antimicrobial and \n\nimmunological property [35]. These findings will \n\nsupport for scientific research on this plant for \n\npharmacological importance against nosocomial and \n\ncommunity acquired pathogens. With new emerging \n\ninfectious diseases people have been depending upon \n\nallopathic, homeopathic and ayurvedic medicines. If \n\nproper research could be carried out, then this botanical \n\nspecies could be used for excellent antimicrobial and \n\nimmunomodulatory medicine. \n\nIn Nepal, the study has been limited to Taxonomic study \n\nand conservation. The bark of S. robusta is not available \n\nlike that of other plant products. It is the tree which \n\nrequires long tenure for its proper growth and \n\ndevelopment [36]. Therefore, scientific tissue culture, \n\nconservation and promotion can be achieved through \n\ncommunity forest and commercial forestry. Where in \n\ncontext of Nepal the scientific screening and study of S. \n\nrobusta based drugs are not adequately performed and \n\nanalyzed, thus this study will help to provide lead to \n\nexplore more about the pharmacological importance of S. \n\nrobusta. \n\nConclusion \nEthanolic extract of S. robusta bark extract displayed \n\nacceptable inhibitory activity against both S. aureus and \n\nE. coli. The antimicrobial activity may be conferred due to \n\nthe presence of plant phytochemicals like alkaloids, \n\ntannins, anthraquinone etc.", "start_char_idx": 17893, "end_char_idx": 22317, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "602d6b5c-f1f1-4477-b9d1-ef306cd319a5": {"__data__": {"id_": "602d6b5c-f1f1-4477-b9d1-ef306cd319a5", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a3d998d0-0a9a-415c-870d-ccffcf05323c", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "70ed515348593e4b54e94a0c0622998b4f3b2f08599f38851962abffd4f7d476", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bcea9ebd-1f6f-4cd5-b3d0-90a22f0debd5", "node_type": "1", "metadata": {}, "hash": "858a0bfac9ea13739c971b8886954e8de26451cd9f91d14260d4a9bd0e33d80a", "class_name": "RelatedNodeInfo"}}, "text": "Further development of the \n\nplant-based drugs could bring sustained drug release \n\nand would help to reduce side effects of synthetic drugs. \n\nIn-vivo studies of these plant-based extracts are required \n\nfor therapeutic application. \n\nAuthors Contribution \nBKS and BD designed the study, participated in sample \n\ncollection, extraction and processing. BKS and JS \n\nparticipated in literature review, sample extraction, \n\nsample processing quality control, data analysis and \n\nresult interpretation. RS and SC participated in drafting \n\nand proofreading the manuscript. All five authors \n\nparticipated in drafting the manuscript and approved for \n\npublication. \n\nCompeting Interests \nThere is not any competing interest. \n\nFunding \nSelf-financed \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.  \n\n\u00a9NJB, BSN   30 \n\nAcknowledgements \nAuthors want to thank the Microbiology Department of \n\nCentral Campus of Technology, Hattisar, Dharan for \n\nlaboratory support. Authors extend sincere thanks to the \n\nresidents, traditional ayurvedic medicine practitioners \n\nand herbarium collection of Postgraduate Campus \n\nBiratnagar, Nepal for immense support and response. \n\nEthical Approval and Consent \nNot applicable. \n\nAbbreviations \nMSA: Mannitol Salt Agar \n\nMAC: MacConkey agar \n\nMHA: Mueller Hinton Agar \n\nMHB: Mueller Hinton Broth \n\nCLSI: Clinical & Laboratory Standards Institute  \n\nDMSO: Dimethyl sulfoxide \n\nMIC: Minimum Inhibitory Concentration \n\nCFU/mL: Colony Forming Unit \n\nNaCl: Sodium Chloride \n\nReferences \n1. Tah J, Mukherjee AK. Ecological Hindrances for \n\nEstablishment of Mass Population of Sal (Shorea robusta) in \nForest Gardens Overtaking. Asian Journal of Applied \nScience and Technology.2018;2(2):857-872. \n\n2. Murthy KSR, Lakshmi N, Ramulu DR. Biological Activity \nand Phytochemical Screening of The Oleoresin of Shorea \nrobusta Gaertn. F. Tropical and Subtropical \nAgroecosystems.2011;14(3):787-791. \n\n3. Marandi RR, Britto SJ, Soreng PK.Phytochemical Profiling, \nAntimicrobial Screening and Antioxidant Properties of the \nSacred Tree(Shorea Robusta Gaertn.) of Jharkhand. \nInternational Journal of Pharmaceutical Sciences and \nResearch. 2016;7(7): 2874-2888. DOI: 10.13040/IJPSR.0975-\n8232.7(7).2874-88. \n\n4. Poornima B. Comparative phytochemical analysis of Shorea \nrobusta Gaertn (oleoresin) WSR to its seasonalcollection. Anc \nSci Life. 2009;29(1):26-8. \n\n5. Satyanarayan P, Subash S, Madan SAK. Medicinal \nProperties of Shorea robusta Gaertn. F.-A Review. \nInternational Journal of Advanced Research and \nPublications.2019; 3(6):219-222. \n\n6. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG \nJr. Staphylococcus aureus infections: epidemiology, \npathophysiology, clinical manifestations, and management. \nClin Microbiol Rev. 2015;28(3):603-61. DOI: \n10.1128/CMR.00134-14. \n\n7. Allocati N, Masulli M, Alexeyev MF, Di Ilio C. Escherichia \ncoli in Europe: an overview. Int J Environ Res Public Health. \n2013;10(12):6235-54. DOI: 10.3390/ijerph10126235. \n\n8. Bannerman DD, Paape MJ, Lee JW, Zhao X, Hope JC, \nRainard P. Escherichia coli and Staphylococcus aureus elicit \ndifferential innate immune responses following \nintramammary infection.", "start_char_idx": 22318, "end_char_idx": 25526, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bcea9ebd-1f6f-4cd5-b3d0-90a22f0debd5": {"__data__": {"id_": "bcea9ebd-1f6f-4cd5-b3d0-90a22f0debd5", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "602d6b5c-f1f1-4477-b9d1-ef306cd319a5", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "1cc6e829166343006d6700fbea9c19deff2b13ce4718a20d120c213af83abd08", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e77b4b69-9658-43ae-a663-d687fdcabe16", "node_type": "1", "metadata": {}, "hash": "c34b2ce81405eb0bc4c9b6aa31b9ddd4cbf7b99d3ab819b46814ca323cd6944c", "class_name": "RelatedNodeInfo"}}, "text": "Clin Diagn Lab Immunol. \n2004;11(3):463-72. DOI: 10.1128/CDLI.11.3.463-472.2004. \n\n9. Alviano DS, Alviano, CS. Plant extracts: search for new \nalternatives to treat microbial diseases. Current \n\npharmaceutical biotechnology.2009;10(1):106-121.DOI: \nhttps://doi.org/10.2174/138920109787048607\". \n\n10. Hemaiswarya S, Kruthiventi AK, Doble M. Synergism \nbetween natural products and antibiotics against infectious \ndiseases. Phytomedicine.2009;15(8): 639-652. DOI: \nhttps://doi.org/10.1016/j.phymed.2008.06.008. \n\n11. Yapar N, Erdenizmenli M, Oguz VA, Kuruuzum Z, Senger \nSS, \u00c7ak\u0131r N, Yuce A. Infectious disease consultations and \nantibiotic usage in a Turkish university \nhospital.International journal of infectious diseases. \n2006;10(1): 61-65. DOI: \nhttps://doi.org/10.1016/j.ijid.2005.03.008. \n\n12. Ventola CL. The antibiotic resistance crisis: part 1: causes \nand threats. P T. 2015;40(4):277-83. \n\n13. Kalaiselvan A, Anand T, Gokulakrishnan K, Kamaraj MC, \nVelavan S. Modulatory role of Shorea robusta bark on \nGlucose-metabolizing enzymes in diethylnitrosamine \ninduced hepatocellular carcinoma in rats. Pharmacognosy \nmagazine. 2015;11(3):S496-S500. DOI: 10.4103/0973-\n1296.168981. \n\n14. Soni RK, Dixit V, Irchhaiya R, Singh H. A review updates on \nShorea robusta Gaertn f.(Sal). Journal of Drug Delivery & \nTherapeutics.2013;3(6):127-132. \n\n15. Agrawal SS and Paridhavi M. Herbal Drug Technology. 2nd \nedition. India. Unversity Press Pvt. Ltd; 2012; 667-669p. \n\n16. Aneja KR, Joshi R. Evaluation of antimicrobial properties of \nfruit extracts of Terminalia chebula against dental caries \npathogens. Jundishapur J Microbiol. 2009;2(3):105\u2013111. \n\n17. Thilagavathi T, Arvindganth R, Vidhya D, Dhivya R. \nPreliminary phytochemical screening of different solvent \nmediated medicinal plant extracts evaluated. Int. Res. J. \nPharm. 2015;6(4):246-248. DOI:10.7897/2230-8407.06455. \n\n18. Harborne JB. Phytochemical methods: A guide to modern \ntechniques of plant analysis.3rd edition. London: Chapman \nand Hall; 1998; 107-108p. \n\n19. Clinical and Laboratory Standards Institute: Methods for \ndilution antimicrobial susceptibility tests for bacteria that \ngrow aerobically, Approved Standard, 9th edn. In: CLSI \ndocument M07-A9. 950 West Valley Road, Suite 2500, \nWayne, Pennsylvania 19087, USA; 2012. \n\n20. Paudel PK, Bhattarai BP, Kindlmann P. An overview of the \nbiodiversity in Nepal. In Himalayan biodiversity in the \nchanging world. Springer, Dordrecht.2012;1-40. DOI: \nhttps://doi.org/10.1007/978-94-007-1802-9_1. \n\n21.", "start_char_idx": 25527, "end_char_idx": 28050, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e77b4b69-9658-43ae-a663-d687fdcabe16": {"__data__": {"id_": "e77b4b69-9658-43ae-a663-d687fdcabe16", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bcea9ebd-1f6f-4cd5-b3d0-90a22f0debd5", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "9776c05f8a017044939c71ebf0ebf0f3fec3bb21962e6768838cc870bb3c6cec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0d8836a3-9d0e-43d8-9af5-36cf2b4ec8ef", "node_type": "1", "metadata": {}, "hash": "640759ce88d39901e162d949a02c48dc04bb9581e550d43ff21035d9ab790354", "class_name": "RelatedNodeInfo"}}, "text": "21. Pan SY, Litscher G, Gao SH, Zhou, SF, Yu ZL, Chen HQ, \nZhang SF, Tang MK, Sun JN, Ko KM. Historical perspective \nof traditional indigenous medical practices: the current \nrenaissance and conservation of herbal resources. Evidence-\nBased Complementary and Alternative Medicine; 2014;20. \nDOI: https://doi.org/10.1155/2014/525340. \n\n22. Sah SP. Management Options for Sal Forests (Shorea robusta \nGaertn.) in the Nepal Terai. Selbyana.2000; 21(1/2):112-117. \n\n23. Jackson JK .Manual of Afforestation in Nepal. Nepal-UK \nForestry Research Project. Kathmandu. 1994. \n\n24. Mohod PS, Jangde CR, Narnaware SD, Raut S. \nPhytochemical analysis of bark skin of Saraca indica (ashoka) \nand Shorea robusta (shal). World Journal of Pharmaceutical \nResearch. 2014;3(7):556-64. \n\n25. Tiwari, U., and E. Cummins. \"Factors influencing levels of \nphytochemicals in selected fruit and vegetables during pre-\nand post-harvest food processing operations.\" Food \nResearch International 50.2 (2013): 497-506. \n\n26. Bazargani, Mitra Mohammadi, Mohsen Falahati-Anbaran, \nand Jens Rohloff. \"Comparative analyses of phytochemical \nvariation within and between congeneric species of willow", "start_char_idx": 28047, "end_char_idx": 29212, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0d8836a3-9d0e-43d8-9af5-36cf2b4ec8ef": {"__data__": {"id_": "0d8836a3-9d0e-43d8-9af5-36cf2b4ec8ef", "embedding": null, "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-227", "node_type": "4", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "a1ae77f592527f06bce19df1ca56540dac00aa6b686f1f37b302104b081c3a6d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e77b4b69-9658-43ae-a663-d687fdcabe16", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "6269a410d774ecb4586a6b6b3fbe65dcab2b30664ce50be3545d8e21394b7c9e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f2494cdf-997d-43f3-ac67-6289bc993e74", "node_type": "1", "metadata": {}, "hash": "91cc305dc373a5990fa65faddea380cccc88e20ea18a3ff3ab08dfccf9b981b2", "class_name": "RelatedNodeInfo"}}, "text": "\"Comparative analyses of phytochemical \nvariation within and between congeneric species of willow \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):2 5 - 3 1               Shrestha et al.  \n\n\u00a9NJB, BSN   31 \n\nherb, Epilobium hirsutum and E. parviflorum: Contribution \nof environmental factors.\" Frontiers in Plant Science 11 \n(2020). \n\n27. Liu, W., Yin, D., Li, N. et al. Influence of Environmental \nFactors on the Active Substance Production and Antioxidant \nActivity in Potentilla fruticosa L. and Its Quality \nAssessment. Sci Rep 6, 28591 (2016). \nhttps://doi.org/10.1038/srep28591 \n\n28. Bai D. Traditional Chinese material; a respect and prospect. \nPlant Medica. 1990;56(06):502. \n\n29. Rojas A, Hernandez L, Pereda-Miranda R, Mata R. \nScreening for antimicrobial activity of crude drug extracts \nand pure natural products from Mexican medicinal plants. \nJournal of ethnopharmacology. 1992;35(3):275-83. DOI: \nhttps://doi.org/10.1016/0378-8741(92)90025-M. \n\n30. Duddukuri GR, Rao DE, Kaladhar DS, Sastry N, Rao KK, \nChaitanya KK, Sireesha C. Preliminary studies on in vitro \nantibacterial activity and phytochemical analysis of aqueous \ncrude extract of Shorea robusta floral parts. International \nJournal of Current Research. 2011;3(8):21-3. \n\n31. 28. Omojate Godstime C, Enwa Felix O, Jewo Augustina O, \nEze Christopher O. Mechanisms of antimicrobial actions of \nphytochemicals against enteric pathogens\u2013a review. J \nPharm Chem Biol Sci. 2014; 2(2):77-85. \n\n32. 32. Vashisht S, Singh MP, Chawla V. In-vitro antioxidant \nand antibacterial activity of methanolic extract of Shorea \nrobusta Gaertn. F. resin. International Journal of \nPharmaceutical and Phytopharmacological Research. \n2016;6(4):68-71. DOI: 10.24896/eijppr.2016641. \n\n33. 33. Subramani R, Narayanasamy M, Feussner K. Plant-\nderived antimicrobials to fight against multi-drug-resistant \nhuman pathogens. 3 Biotech. 2017; 7(172). DOI: \nhttps://doi.org/10.1007/s13205-017-0848-9. \n\n34. 34. Luitel DR, Rokaya MB, Timsina B, M\u00fcnzbergov\u00e1 Z. \nMedicinal plants used by the Tamang community in the \nMakawanpur district of central Nepal. J Ethnobiol \nEthnomed. 2014;10(5):1-11. DOI: 10.1186/1746-4269-10-5. \n\n35. 35. Dhama K, Tiwari R, Chakraborty S, Saminathan M, \nKumar A, Karthik K, Wani MY, Amarpal SS, Rahal A. \nEvidence based antibacterial potentials of medicinal plants \nand herbs countering bacterial pathogens especially in the \nera of emerging drug resistance: An integrated update. \nInternational Journal of pharmacology. 2014;10(1):1-43. DOI: \n10.3923/ijp.2014.1.43. \n\n36. 36. Sapkota P, Meilby H. Modelling the growth of Shorea \nrobusta using growth ring measurements. BankoJanakari. \n2009;19(2):25-32. DOI: \nhttps://doi.org/10.3126/banko.v19i2.2982.", "start_char_idx": 29115, "end_char_idx": 31836, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f2494cdf-997d-43f3-ac67-6289bc993e74": {"__data__": {"id_": "f2494cdf-997d-43f3-ac67-6289bc993e74", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0d8836a3-9d0e-43d8-9af5-36cf2b4ec8ef", "node_type": "1", "metadata": {"identifier": "njb-227", "author": "Kumar Shrestha, Bijay; Dhungana, Bidhya; Shakya, Jenish; Shrestha, Romika; Chauhan, Sujata", "title": "Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)", "date": "2022-07-30", "file": "njb-227.pdf"}, "hash": "50b2cf2c9047c8ac01f7bb1a5519319711ee1df25e0c17265d031a25bfa5509c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5f4e18e6-ae89-4663-b3d0-715dce183223", "node_type": "1", "metadata": {}, "hash": "5473dcd16bf00e2ab0dceb890dba59f9a66de3d441edaffdcb6a226e6b35d2ee", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 3 2 - 3 9   Research article  DOI: https://doi.org/10.54796/njb.v10i1.228 \n \n\n32 \n\u00a9NJB, BSN   32 \n\nQuality Evaluation of Apis laboriosa and Apis mellifera Honey Collected \n\nfrom Bagmati Province, Nepal \nAbhishek Bajgain, Bashu Dev Neupane, Diwakar Sarraf, Jwalant Karmacharya, Saksham Ranjitkar,  \n\nRajan Shrestha, Rajendra Gyawali  \n\nDepartment of Pharmacy, School of Science, Kathmandu University, Dhulikhel, Kavre, Nepal \n\nReceived: 9 Jun 2022; Revised: 22 Jun 2022; Accepted: 4 Jul 2022; Published online: 30 Jul 2022 \n\nAbstract \nHoney is a natural sweet substance produced by Apis sp. from floral nectar or other plant parts which are gathered, modified \nand stored in the honeycombs by honeybees. The current research was aimed to analyze the quality parameters of locally \navailable honey. Honey samples of Apis laboriosa and Apis mellifera were collected during spring of 2019 & 2021 and autumn \n2021 from the Bagmati province, Nepal. Samples were analyzed their physicochemical and phytochemical properties. The result \nshows that, the pH was ranged between [4.467\u00b10.0306 - 5.05\u00b10.02], rheological studies showed Newtonian flow and pseudo-\nplastic type of Non-Newtonian flow, specific optical rotation was ranged between [(+) 5.75\u00b10.4684 - (-) 12.71\u00b10.234], specific \ngravity was ranged between [1.35\u00b10.00017 - 1.409\u00b10.00022], moisture content was ranged between [19.2% - 25%]. Secondary \nMetabolite screening showed the honey samples possesses flavonoids, saponins, glycosides, tannins, amino acids, protein and \nreducing sugar. Total phenolic content was ranged between [1.0427 - 6.86288] gm GAE/Kg honey while total flavonoid content \nranged between [0.016755 - 0.353132] gm QE/Kg Honey. IC50 obtained from DPPH assay ranged between [649.6465 - 9867.1617] \nppm. Properties and qualities of honey are affected by seasonal factors and various floral sources. The samples were in positive \ncorrelation between flavanoid content, phenolic content and their respective anti-oxidant potency. \n\nKeywords: Honey, Quality, Physicochemical Properties, Phytochemicals, TPC, TFC, Antioxidant. \n\n Corresponding author, email: ragyawali@gmail.com \n\nIntroduction \nHoney is a natural sweet substance produced by honey \n\nbee, which can be classified based on type of honey \n\nsource, floral and extra floral honey[1]. Apis laboriosa is \n\nworld\u2019s largest honeybee species with measurement of \n\nup to 3.0 cm, which are found at an altitude range from \n\n2,500 m to 4,000 m above the sea level, building their nests \n\ncommonly in higher altitude, 1,200 m. above the sea level. \n\nHoney is harvested twice a year in spring season and in \n\nautumn season, with Spring one showing strong \n\nmedicinal property than autumn one [2].The medicinal \n\nproperty of honey is that, the floral distribution of the \n\nregion, where Apis laboriosa lives; is the plants that belong \n\nto Ericaceae (Rhododendron) family, which have a \n\npsychoactive and hallucinogenic group of \n\nphytochemistry known as Grayanotoxins[3]. In Nepal, it \n\nis estimated that over 10,000 MT of honey is produced \n\nand every year growing the production.", "start_char_idx": 48, "end_char_idx": 3193, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5f4e18e6-ae89-4663-b3d0-715dce183223": {"__data__": {"id_": "5f4e18e6-ae89-4663-b3d0-715dce183223", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f2494cdf-997d-43f3-ac67-6289bc993e74", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "64c5d32607c05a9ad67fb07f6f814490af0a6789c191e462c76405553d4bb29d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "671ee396-7393-4c6e-84c9-711c16515cb5", "node_type": "1", "metadata": {}, "hash": "eaf6a54b0246ac27960b5ac2afb57f023781be76fb92d3dda9847e0190bf3587", "class_name": "RelatedNodeInfo"}}, "text": "Honey is \n\nreported to contain at least 181 substances, high \n\nnutritional value, high refractive index, high viscosity \n\nand Specific gravity [4][5][6]. To the best our knowledge, \n\nthis is the first comparative study on Nepalese honey to \n\ninvestigate wide range of quality parameters. The present \n\nstudy was aimed to carry out the quality assessment and \n\ncharacterization of honey of Apis laboriosa harvested from \n\nBagmati province, Nepal. \n\nMaterial and methods \nHoney Collection \nIn this study, honey samples of Apis laboriosa and Apis \n\nmellifera were harvested on spring of 2019 & 2021 and \n\nautumn 2021 from Bagmati Province, Nepal. The \n\ninformation about samples profile is as given below in \n\nthe Figure 1, Figure 2 and Table 1. \n\nPhysical Properties \nIn a view to identifying the purity of honey based on \n\nphysical properties following preliminary tests were \n\nperformed. \n\nSand Sinking Test \nIn this test, 50 g sand was taken from nearby and was \n\nsieved through Mesh Size 30. The sand was then allowed \n\nto dry completely in oven at a temperature of 95\u2103 for 15 \n\nMinutes. The particle distribution profile of the sand was \n\nexamined using electromagnetic sieve shaker (Model: \n\nEMS-8, Instrument Manufacturer: Electropharma). After \n\nthat, four petri plates were kept in a row. \n\n \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-5745-0702\nmailto:ragyawali@gmail.com", "start_char_idx": 3194, "end_char_idx": 4742, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "671ee396-7393-4c6e-84c9-711c16515cb5": {"__data__": {"id_": "671ee396-7393-4c6e-84c9-711c16515cb5", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5f4e18e6-ae89-4663-b3d0-715dce183223", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "89d7273a17a87faf87eec657c31c71770d3c7c63e8b36b589ce4d69efab0c2bd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b553a290-208e-4e75-acb7-55fec05a293b", "node_type": "1", "metadata": {}, "hash": "31babfc7aa6feb2735bbfc7b198513b0fc3d6c34c4519c41db8475bceefda68f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n33 \n\u00a9NJB, BSN   33 \n\nFigure 1. Google Earth Snapshot showing Honey Harvested Location \n\n \n\nFigure 2.f Different honey samples (From Left to Right; S1, S2, \n\nS3, and S4). \n\nWith the help of a tripod stand, the funnel was set and \n\nthe sand was allowed to flow through the funnel, which \n\ncreated a conical shape of sand over the petriplates. To \n\nthe sandhill, three drops of each honey sample were \n\nplaced in each petriplates, and the time required to sink \n\nthrough the sand surface was recorded. Delayed \n\nsinking Honey samples are considered to be pure as \n\nthose honey samples contain less amount of moisture \n\nand are not adulterated [7]. \n\nWater Sinking Test \nIn this test, a 5 g of each sample was taken and it was \n\npoured into a beaker containing deionized water. After \n\nthat, the nature by which it interacts with water was \n\nobserved. Directly sinking honey samples to the bottom \n\nof the vessel without mixing with water unless stirred \n\nare considered to be pure [7]. \n\nAir-Flow Test \nIn this test, a clean glass stirrer was taken and was \n\ndipped into the vessel containing the honey sample. \n\nThe stirrer was then rotated through the honey sample \n\nTable 1. Honey collection site of Bagmati Province, Nepal \n\nSample \nNo. \n\nHarvested Time Types of Honey Honeybee \nSpecies \n\nHarvested Location \n\nYear Season \n\nS1 2019 Spring Honeydew Honey Apis \nlaboriosa \n\nUttargaya R.M. \u2013 1, Karyangmaryang, Rasuwa \n[39.1 Km Perimeter, 71.9 sq. Km Area] \n\nS2 2021 Spring General Nectar \nHoney \n\nApis \nlaboriosa \n\nUttargaya R.M. \u2013 2, Thulogaun, Rasuwa \n[28\u00b0 1'28.36\"N, 85\u00b0 9'50.73\"E] \n\nS3 2021 Autumn General Nectar \nHoney \n\nApis \nlaboriosa \n\nUttargaya R.M. \u2013 2, Thulogaun, Rasuwa \n[28\u00b0 1'28.36\"N, 85\u00b0 9'50.73\"E] \n\nS4 2021 Autumn Diploknema \nbutyraceae Nectar \n\nHoney \n\nApis \nmellifera \n\nRakshirang R.M., Silinge, Makwanpur \n[42.7 Km Perimeter, 48.8 sq. Km Area] \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n34 \n\u00a9NJB, BSN   34 \n\nand it was raised to a certain height above the honey \n\nsurface to allow for free-flow of the honey samples. The \n\nflowing nature of honey was then observed. Honey \n\nsamples, which flow in a continuous thread-like pattern, \n\nare considered to be pure [7]. \n\nPhysicochemical Properties \n\nColour \nColour tone of honey was noted, which also revealed \n\nthe identity and nature of various floral sources and \n\nhelped in the differentiation of types of honey. \n\npH \nFirstly, the pH meter (Model: pH 211 microprocessor, \n\ninstrument manufacturer: Hanna Instruments) was \n\ncalibrated using Qualigen (Fisher\u2019s scientific) buffer \n\ntablets of pH 4.0, 7.0, and 9.2. As specified in the label \n\nof the buffer tablets, each buffer tablet was dissolved in \n\ndistilled water to the volume mark of 100 ml volumetric \n\nflask. After the device has been calibrated, each honey \n\nsample was checked for its pH readings. The pH range \n\nof unadulterated honey should lie between the range of \n\n3.5 to 5.5 [8].", "start_char_idx": 4745, "end_char_idx": 7778, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b553a290-208e-4e75-acb7-55fec05a293b": {"__data__": {"id_": "b553a290-208e-4e75-acb7-55fec05a293b", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "671ee396-7393-4c6e-84c9-711c16515cb5", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "8b357ed3170a74aabeb57878b0f6e40494723c46a297a8e82eec07cac8d81e6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "64efb1d7-8464-49ab-81c2-94b448959740", "node_type": "1", "metadata": {}, "hash": "8b82a020f2b63e155424e386c6cdab9864c70ef2c94d640ec5a5d0633f46a03e", "class_name": "RelatedNodeInfo"}}, "text": "Rheological Properties \nTo make uniform treatment on viscosity, overall \n\nsamples were maintained at 41\u00b11\u2103 by using an electric \n\nair-heater. Viscosity was analyzed by viscometer \n\n(Model: DV-III Ultra Programmable Rheometer, \n\nInstrument Manufacturer: Brookfield) attaching \n\nspindle size of 63, the rheological properties of Honey \n\nwere studied [1]. \n\nOptical Rotation \nFor this experiment, 1.0% (w/v) of the Honey sample in \n\ndistilled water was prepared at first. To the prepared \n\nsample, using blank correction as distilled water; the \n\nprepared sample was filled in the tube of polarimeter \n\n(Model: BK-P2S, Instrument Manufacturer: Biobase \n\nBiodustry [Shandong] Co. Ltd.) and its triplicate \n\nreading was taken. Honeydew honey should exhibit \n\npositive optical rotation while nectar honey exhibit \n\nnegative optical rotation to the incident plane-polarized \n\nlight [6]. By using the following equation, specific \n\noptical rotation was computed:- \n\n[\ud835\udefc]\ud835\udf06\n\ud835\udc47 =\n\n\ud835\udefc\ud835\udc42\ud835\udc4f\ud835\udc60\ud835\udc52\ud835\udc5f\ud835\udc63\ud835\udc52\ud835\udc51\n\ud835\udc4f \u00d7 \ud835\udc50\n\n \n\nWhere, [\ud835\udefc]\ud835\udf06\n\ud835\udc47  = Specific Rotation in degree at T\u2103 and \n\nLight Wave-length (\ud835\udf06) \n\n \ud835\udefc\ud835\udc42\ud835\udc4f\ud835\udc60\ud835\udc52\ud835\udc5f\ud835\udc63\ud835\udc52\ud835\udc51 = Observed Rotation in degree \n\n b =  Path-length in the decimeter \n\n c =  Concentration in gm/mL \n\nRelative Density \nUsing the pycnometer (Glassware Manufacturer: Jain \n\nScientific Glass Works [JSGW]), Digital Analytical \n\nBalance (Instrument Manufacturer: Bel Engineering), \n\nand laboratory thermometer, the density of our four (4) \n\nhoney samples were determined. The room \n\ntemperature of the lab during the time of the \n\nExperiment was also measured with the help of a \n\nLaboratory Thermometer. After that, an empty \n\npycnometer was taken and its mass was noted using \n\ndigital analytical balance. Now, it was filled with the \n\nhoney sample and again its mass was weighed. The \n\ndensity of uncontaminated Honey typically ranges \n\nbetween 1.38 and 1.45 gm/ml [9]. \n\n\ud835\udc37\ud835\udc52\ud835\udc5b\ud835\udc60\ud835\udc56\ud835\udc61\ud835\udc66(\ud835\udf0c) =\n\ud835\udc4a\ud835\udc61\ud835\udc39\ud835\udc56\ud835\udc59\ud835\udc59\ud835\udc52\ud835\udc51 \u2212 \ud835\udc4a\ud835\udc61\ud835\udc38\ud835\udc5a\ud835\udc5d\ud835\udc61\ud835\udc66\n\n25\n \n\nWhere,  WtFilled =the mass of Pycnometer after sample \n\nfilling \n\nWtEmpty =the mass of the empty Pycnometer \n\nbefore filling \n\nRefractive Index \nThe refractive index of the honey samples was \n\nmeasured by using Abbe Refractometer (Model: SN \n\n4040, Instrument Manufacturer: Guru Nanak \n\nInstruments, New Delhi). Sample holding prism of the \n\ninstrument was cleaned well by rinsing it, with the help \n\nof ethanol and soft tissue paper. After that, a drop of \n\nhoney sample was then loaded into the lower Sample \n\nprism. Now, the upper sample prism was interlocked \n\nwith the lower sample prism which facilitated contact \n\nbetween the two Sample prisms and ultimately formed \n\na film layer of honey sample. By viewing through the \n\neye-piece, coarse scale adjustment and fine scale \n\nadjustment knobs were rotated and readings were \n\nnoted. Refractive index of unspoiled honey typically \n\nranges from 1.474 to 1.504 indicating the presence of \n\nwater content in honey from 25% to 13% respectively \n\n[10]. \n\nPollen Contents \nHoney sample was prepared in a conical flask and it \n\nwas left to shake in incubator shaker (Manufacturer: \n\nBiobase Biodustry [Shandong] Co. Ltd.) at 100 \n\nRevolution per Minute (RPM) for 15 Minutes at 45\u2103.", "start_char_idx": 7781, "end_char_idx": 10899, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "64efb1d7-8464-49ab-81c2-94b448959740": {"__data__": {"id_": "64efb1d7-8464-49ab-81c2-94b448959740", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b553a290-208e-4e75-acb7-55fec05a293b", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "953f2a0bc3c64306d4f674c0da06ce9766e9b8051de781c45c36dbfd5c03492f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "43503965-5420-4826-bd74-72cf32131eed", "node_type": "1", "metadata": {}, "hash": "02b88c45dda45488fc985de8a613b13f6100e842030c2365385a2d10a8e7d812", "class_name": "RelatedNodeInfo"}}, "text": "The stock solution was then poured into the \n\ncentrifugation tube. After that, it was allowed to \n\ncentrifuge at 5000 RPM for 10 minutes in Centrifugation \n\nApparatus (Model: NF 200, Manufacturer: N\u00fcve \n\nLaboratories). The settled precipitate was scraped out \n\nand finally, it was observed in the microscope for \n\ndetermination of its shape and size.  \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n35 \n\u00a9NJB, BSN   35 \n\nQualitative Screening Test of Phyto-\n\nchemical Classes \nPlant metabolites were screened according to \n\npreviously established methods [11-13] \n\nTotal Phenolic Content \nTotal phenolic content was determined by Folin-\n\nCiocalteu (Thermo Fisher\u2019s Scientific India Pvt. Ltd.) \n\nmethod, according to previously published  method \n\n[12].The honey stock solution was prepared at 10% in \n\nwater.  A portion of 1 ml of the honey and 0.8 ml of 10 \n\n% aqueous reagent and followed by 2 ml of 15% sodium \n\ncarbonate was added. Final volume was made by \n\nadding water. Mixture was incubated for 2 hrs, and \n\nabsorbance was measured at 765 nm against the blank. \n\nA standard curve of gallic acid was prepare for \n\nquantification, using a concentration range of 250, 500, \n\n750, 1000 and 1250 ppm and result were expressed as \n\nmg Gallic acid/Kg honey. Calculation of TPC was done \n\nusing the following formula expressed in mg Gallic \n\nAcid Equivalent (GAE) per Kg of Honey sample, \n\n\ud835\udc47\ud835\udc43\ud835\udc36 =\n\ud835\udc3a\ud835\udc34\ud835\udc38(\ud835\udc5a\ud835\udc54/\ud835\udc59) \u00d7 \ud835\udc49(\ud835\udc5a\ud835\udc59) \u00d7 10\u22123 (\ud835\udc59/\ud835\udc5a\ud835\udc59) \u00d7 \ud835\udc37\ud835\udc53\n\n\ud835\udc4a\ud835\udc61\ud835\udc46\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52 (\ud835\udc54\ud835\udc5a) \u00d7 10\n\u22123(\ud835\udc58\ud835\udc54/\ud835\udc54\ud835\udc5a)\n\n \n\nwhere TPC =Total Phenolic Content (in mg GAE/Kg \n\nHoney) \n\nGAE = Gallic Acid Equivalent (in mg/l) \n\nV = Total Volume of Methanol Extract (in ml) \n\nDf = Dilution Factor \n\nTotal Flavonoid Content \nTotal Flavonoids content in honey was determined by a \n\ncalorimetric method according to previous method. \n\nBriefly, 1.0 gm of honey sample was taken, which was \n\ndissolved in 10 ml 80% Ethanol to make sample stock \n\nsolution. The sample stock solution was then allowed to \n\nincubate in incubator shaker (Biobase Biodustry \n\n[Shandong] Co. Ltd.) at 100 RPM for 15 Minutes at 45\u2103. \n\nTotal 1.0 ml of supernatant sample stock solution was \n\npipette out to which 0.2 ml of 10% (w/v) aqueous \n\nAluminum Chloride was then added and subsequently, \n\n0.2 ml of 1 M. Aqueous Potassium Acetate and 3 ml of \n\n80% Ethanol was added. Finally, volume make-up was \n\ndone to the mark adding sufficient Distilled Water and \n\nabsorbance reading was measured at the wavelength of \n\n415 nm against Blank Solution from the standard stock \n\nsolution.", "start_char_idx": 10902, "end_char_idx": 13426, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "43503965-5420-4826-bd74-72cf32131eed": {"__data__": {"id_": "43503965-5420-4826-bd74-72cf32131eed", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "64efb1d7-8464-49ab-81c2-94b448959740", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "33782709abb6f18e17e09600f54605f494082b8155b62af42d74c5f238fda8e7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2fa4aa28-09e1-4df8-b194-c21746ecdb08", "node_type": "1", "metadata": {}, "hash": "ec40f7edc5e2d66cfd880b6561a4819e4f48682d1378571830986dd968a4b7ff", "class_name": "RelatedNodeInfo"}}, "text": "[12].TFC was done using the following \n\nformula expressed in mg Quercetin Equivalent per Kg \n\nof honey; \n\n\ud835\udc47\ud835\udc39\ud835\udc36 =\n\ud835\udc44\ud835\udc38(\ud835\udc5a\ud835\udc54/\ud835\udc59) \u00d7 \ud835\udc49(\ud835\udc5a\ud835\udc59) \u00d7 10\u22123 (\ud835\udc59/\ud835\udc5a\ud835\udc59) \u00d7 \ud835\udc37\ud835\udc53\n\n\ud835\udc4a\ud835\udc61\ud835\udc46\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52 (\ud835\udc54\ud835\udc5a) \u00d7 10\n\u22123(\ud835\udc58\ud835\udc54/\ud835\udc54\ud835\udc5a)\n\n \n\nwhere, TFC is Total Phenolic Content (in mg \n\nQuercetin/Kg Honey) \n\nQE is Quercetin Equivalent (in mg/l) \n\nV is Total Volume of Ethanol Extract (in ml) \n\nDf is Dilution Factor \n\nAnti-Oxidant DPPH Assay \n\nDPPH assay was estimated using the 2,2-diphenyl-1-\n\npicrylhydrazyl hydrate radical (DPPH) (Glentham Life \n\nScience Ltd, UK)) according to previous method of \n\nKa\u010d\u00e1niov\u00e1[14 - 15]. The honey samples were diluted in \n\nMethanol at concentrations of 400, 800, 1200, 1600 and \n\n2000 ppm solutions and from each dilution 0.3 ml was \n\nmixed with DPPH. The mixtures were vortexed, left in \n\ndark room temperature for 60 min and the absorbance \n\nwas measured at 517 nm under UV-Visible \n\nspectrophotometer (UV 1800, Manufacturer: Shimadzu \n\nScientific Instruments) correcting baseline Blank \n\ncorrection by Methanol. The Inhibition % is given by the \n\nrelation \n\n\ud835\udc3c\ud835\udc5b\u210e\ud835\udc56\ud835\udc4f\ud835\udc56\ud835\udc61\ud835\udc56\ud835\udc5c\ud835\udc5b % =  \n\ud835\udc34\ud835\udc36\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59 \u2212 \ud835\udc34\ud835\udc46\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52\n\n\ud835\udc34\ud835\udc36\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59\n\u00d7 100% \n\nWhere AControl = mean Absorbance reading of 0 ppm \n\nsolution against Methanol as Blank \n\nASample =e mean Absorbance reading of 400, 800, \n\n1200, 1600, and 2000 ppm solutions \n\nResults and Discussions \nPreliminary Purity Test \n\nSand Sinking Test \nS1 took shortest period of time among all the samples \n\nwhile S4 took longer period of time to sink through \n\nSand-hills. Various time (in Seconds) taken by samples \n\nto sink through the Sand surface is as shown in Table 2. \n\nTable 2. Time of Sand Sinking Test \n\nS.N. Sample Time (in Seconds) \n\n01. S1 62.33\u00b12.08 \n02. S2 197.33\u00b12.52 \n03. S3 262.33\u00b12.517 \n04. S4 900\u00b10 \n\nWater Sinking Test \nAll of our honey samples went down to the bottom of \n\nthe cup without mixing up with the water except when \n\nstirred, which showed that all our honey samples \n\ncomplied the Water Sinking Test as mentioned in \n\nliteratures mentioned above. \n\nAir Flow Test \nAll the honey samples went down like a thread without \n\nbreaking ranging 0.5 to 3 seconds as a continuous \n\nthread, which also showed that all our honey samples", "start_char_idx": 13426, "end_char_idx": 15572, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2fa4aa28-09e1-4df8-b194-c21746ecdb08": {"__data__": {"id_": "2fa4aa28-09e1-4df8-b194-c21746ecdb08", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "43503965-5420-4826-bd74-72cf32131eed", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "207a1f9afee81155271ee5e70fce48810421dcae5c395637afa90534fbd8d8e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "73b8b207-2cd4-4bf5-9f5a-af2e54917a0b", "node_type": "1", "metadata": {}, "hash": "e242b0295203801af51f55440485d1d1cac5e4e4338bba3563633d6cfbcc7b66", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n36 \n\u00a9NJB, BSN   36 \n\ncomplied for the Air Flow Test as mentioned in \n\nliteratures mentioned above. \n\nConfirmatory Purity Test \n\nColour \nFrom colour shade analysis of Honey Samples with that \n\nof reference shade of Yellow colour, our different honey \n\nsamples demonstrated varieties of colour as listed in \n\nTable 3. \n\nTable 3. Different Shades of honey as per base Yellow \ncolour \n\nS.N. Sample Shade of Yellow Colour \n\n1. S1 Amber \n2. S2 Light Amber \n3. S3 White \n4. S4 Extra-Light White \n\npH \nPresence of carbohydrate in dominant amount is the \n\nreason to which Honey shows slightly acidic nature. \n\nDue to the reason, Honey has a good Anti-Microbial \n\npotency. Among four samples, S3 showed highly acidic \n\nwith pH of (4.467\u00b10.0306) where S1 showed lowest pH \n\nwith the value of (5.05 \u00b1 0.02). Various values of pH \n\nshown by different honey samples are tabulated down \n\nin Table 4. \n\nTable 4. Various pH readings of different Honey Samples \n\nS.N. Sample pH \n\n1. S1 5.05\u00b10.02 \n2. S2 4.833\u00b10.0379 \n3. S3 4.467\u00b10.0306 \n4. S4 4.767\u00b10.0416 \n\nRheological Properties \nAmong the samples, only S1 showed Newtonian type of \n\nflow property, while rest of the samples showed \n\nPseudo-plastic flow property upon checking \n\nRheological properties of Honey Samples using \n\nBrookfield Viscometer. Plot of Rotation per Minute \n\n(RPM) Versus Torque% and RPM Versus Viscosity (in \n\ncenti-Poise) of different Honey Samples  as shown in \n\nfigure 3-6. \n\n \n\nFigure 3. RPM Versus Torque% and RPM Versus Viscosity (cP) \n\n(i.e. Rheological Properties of S1) \n\n \n\nFigure 4. RPM Versus Torque% and RPM Versus Viscosity (cP) \n\n(i.e. Rheological Properties of S2) \n\n \n\n \n\n \n \n\n320\n\n345\n\n370\n\n395\n\n0 100 200 300\n\nV\nis\n\nco\nsi\n\nty\n (\n\ncP\n)\n\nRPM\n\nS2\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n0 50 100 150\n\nT\no\n\nrq\nu\n\ne\n %\n\nRPM\n\nS3\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n0 50 100 150\n\nT\no\n\nrq\nu\n\ne\n %\n\nRPM\n\nS1\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n0 100 200 300\n\nT\no\n\nrq\nu\n\ne\n %\n\nRPM\n\nS2\n\n0\n\n50\n\n100\n\n150\n\n200\n\n250\n\n300\n\n0 50 100 150\n\nV\nis\n\nco\nsi\n\nty\n (\n\ncP\n)\n\nRPM\n\nS1", "start_char_idx": 15577, "end_char_idx": 17648, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "73b8b207-2cd4-4bf5-9f5a-af2e54917a0b": {"__data__": {"id_": "73b8b207-2cd4-4bf5-9f5a-af2e54917a0b", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2fa4aa28-09e1-4df8-b194-c21746ecdb08", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "7947604b6d77b0a4b2115ee7017a45be2892f9ca138dd7c316cb90996c93f4a9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9270529e-87ee-49c3-a649-eec9bcbaa4c4", "node_type": "1", "metadata": {}, "hash": "2e9115818c8d8f391296abd71d7672d331e6c323a8aa541664bada16fd1e7842", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n37 \n\u00a9NJB, BSN   37 \n\n \nFigure 5. RPM Versus Torque% and RPM Versus Viscosity (cP) \n\n(i.e. Rheological Properties of S3) \n\n \nFigure 6. RPM Versus Torque% and RPM Versus Viscosity (cP) \n\n(i.e. Rheological Properties of S4) \n\nSpecific Optical Rotation \n\nFrom the literatures, it is established fact that \n\nHoneydew Honey and Adulterated Honey only shows \n\npositive Specific Optical Rotation, while Nectar Honey \n\nshows negative Specific Optical Rotation to the plane \n\npolarized light. S2 showed highest angle of rotation \n\namong the four samples while S1 showed positive angle \n\nof rotation. Various values of degree of Rotation are \n\nshown in Table 5. \n\nSpecific Gravity \nHoney is denser than water with 1.3 to 1.4 folds. S3 and \n\nS1 are the respectively highest and lowest dense Honey \n\nSamples among the samples involved in this study. \n\nTable 6. shows different values of Specific Gravity \n\nwhich was obtained during analysis. \n\nTable 5. Specific Optical Rotations of different \nSamples at Concentration of 1.0% (w/v) in Water. \n\nS.N. Sample Specific Optical Rotation \n\n1. S1 (+)5.75\u00b10.4684 \n2. S2 (-)12.71\u00b10.234 \n3. S3 (-)8.79\u00b10.3098 \n4. S4 (-)2.299\u00b10.3098 \n\nTable 6. Specific Gravity of samples observed at Room \nTemperature of 14\u2103. \n\nS.N. Sample Specific Gravity \n\n1. S1 1.35\u00b10.00017 \n2. S2 1.404\u00b10.0016 \n3. S3 1.409\u00b10.00022 \n4. S4 1.376\u00b10.00497 \n\nRefractive Index and Moisture Content \nMoisture Content in Honey Samples is indicated by the \n\nRefractive Index values of Honey Samples. S1 has \n\nhighest Refractive Index and thus Moisture Content \n\nand S4 has lowest Refractive Index and Moisture \n\nContent upon analysis of four different samples. Table \n\n7 is listed with different observations of Refractive \n\nIndex and Moisture Content of all four honey samples \n\ninvolved in the study. \n\nTable 7. Refractive Index and Moisture Content (in %) of \n\nSamples \n\nS.N. Sample Refractive Index Moisture Content \n(in %) \n\n01. S1 1.473 25 \n02. S2 1.476 24.2 \n03. S3 1.476 24.2 \n04. S4 1.489 19 \n\nFigure 7. Microscopic View of Pollen Content at 400x \n\nMagnification of S1(1), ,S2(2), S3(3) andS4(4). \n\nMicroscopic Studies of Pollen Contents \nUpon viewing microscopic slides of different honey \n\nsamples, we found out the spring season harvested \n\nhoney containing the same kind of pollen and it was \n\ndifferent from autumn harvested sample. For the case \n\nof S4, as it is nectar honey of Diploknema butyraceae; the \n\nhoney sample exhibit different kind of pollen contents \n\nin it, which is totally different from all of the honey \n\nsamples. Figure 7 contains the pictures of Microscopic \n\nSlides of different Honey Samples.  \n\n100\n\n150\n\n200\n\n250\n\n300\n\n0 50 100 150\n\nV\nis\n\nco\nsi\n\nty\n (\n\ncP\n)\n\nRPM\n\nS3\n\n0\n\n20\n\n40\n\n60\n\n80\n\n0 50 100 150\n\nT\no\n\nrq\nu\n\ne\n %\n\nRPM\n\nS4\n\n640\n\n660\n\n680\n\n700\n\n720\n\n740\n\n0 50 100 150\n\nV\nis\n\nco\nsi\n\nty\n (\n\ncP\n)\n\nRPM\n\nS4", "start_char_idx": 17652, "end_char_idx": 20564, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9270529e-87ee-49c3-a649-eec9bcbaa4c4": {"__data__": {"id_": "9270529e-87ee-49c3-a649-eec9bcbaa4c4", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "73b8b207-2cd4-4bf5-9f5a-af2e54917a0b", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "28d6eb1a014a5e07acbfb958e8f405802f32cf780a317dbe8f95cacff888d8dd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bfc14c23-a20f-4146-8aa9-64c1b41fd4e5", "node_type": "1", "metadata": {}, "hash": "4a750455282f6b48797bf8b7943b013952182496e7370b5a3c32f4fae05ae670", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n38 \n\u00a9NJB, BSN   38 \n\nSecondary Metabolites \nUpon performing qualitative screening of secondary \n\nmetabolites in honey samples; there was positive test \n\nfor flavanoids, glycosides, saponins, tannins, reducing \n\nsugar, terpenoids, amino acids and proteins. There was \n\nnegative test for alkaloids and phlobatanins. Table 8 \n\nshows the summary of results for all four honey \n\nsamples as obtained from qualitative screening of \n\nsecondary metabolites. \n\nTable 8. Qualitative Screening of Secondary Metabolites in \ndifferent Honey Samples \n\nS.N. Name of Test S1 S2 S3 S4 \n\n01. Mayer\u2019s Test for \n\nAlkaloid \n\n- - - - \n\n02. Flavanoid Test +++ +++ +++ +++ \n\n03. Browntoger\u2019s Test for \n\nGlycosides \n\n+ + + + \n\n04. Foam Test of Saponins ++ ++ ++ ++ \n\n05. Ferric Chloride Test for \n\nTannins \n\n+ + + + \n\n06. Benedict\u2019s Test for \n\nReducing Sugar \n\n++ +++ ++ +++ \n\n07. Xanthoproteic Test for \n\nAmino Acids and \n\nProtein \n\n+++ +++ +++ +++ \n\n08. Terpenoids Test +++ +++ +++ +++ \n\n09. Phlobatannins Test - - - - \n- Negative, + Weak Positive, ++ Moderate Positive, +++ Strong \nPositive \n\nQuantitative Determination of Secondary \n\nMetabolites \nTPC and TFC \nUpon plotting of standard calibration plot of Gallic acid \n\nfor TPC and Quercetin for TFC, we obtained following \n\ngraphs as shown in figures with R2 of 0.9658 and 0.981 \n\nrespectively for the linear regression keeping \n\nConcentration (in ppm) along X-axis and mean \n\nabsorbance along Y-axis. Mean absorbance is the \n\naverage of absorbance reading taken from triplicate \n\ndata. \n\n \nFigure 8. Standard Calibration Curve of Gallic Acid for TPC \n\n \n\n \n\nFigure 9. Standard Calibration Curve of Quercetin for TFC \n\nUsing above Standard Calibration Curve and linear \n\nRegression equation of line, TPC and TFC was found to \n\nbe highest in S1 and lowest in S3. Table 9 shows the \n\nresults of estimated TPC and TFC of different Honey \n\nSamples. \n\nTable 9. Quantitative Determination of Secondary \nMetabolites \n\nS.N. Sample TPC (gm GAE/ \nKg Honey) \n\nTFC (gm QE/ \nKg Honey) \n\n01. S1 6.862884943 0.3531322456 \n02. S2 1.626269056 0.1607248345 \n03. S3 1.042708154 0.01675598334 \n04. S4 2.615476423 0.1883074849 \n\nTPC = Total Phenolic Content and TFC = Total Flavanoid Content \n\nFigure 10. Concentration Versus Inhibition% Graph from \n\nDPPH Assay (Anti-Oxidant of different samples at varying \n\nConcentration) \n\nTable 10. Results of Anti-Oxidant Assays (IC50 Values and \nTimes Potency with the respect to Standard) \n\nS.N. Samples IC50 (in ppm) Times Potency \n\n01. Quercetin  346.9112586 1.000 \n02. S1 649.6465517 0.534 \n03. S2 2353.956344 0.1475 \n04. S3 9867.161765 0.0353 \n05.", "start_char_idx": 20568, "end_char_idx": 23247, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bfc14c23-a20f-4146-8aa9-64c1b41fd4e5": {"__data__": {"id_": "bfc14c23-a20f-4146-8aa9-64c1b41fd4e5", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9270529e-87ee-49c3-a649-eec9bcbaa4c4", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "1a59a11196699ff482409659b0eaa1271457b91dc032c57570df9382f3ec1b94", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ce62407e-183f-4f66-8339-4e02f7324c3c", "node_type": "1", "metadata": {}, "hash": "a80ead66fd76dcbcf5335295beeb636f10899474bc088e4f527c5c84812c3d24", "class_name": "RelatedNodeInfo"}}, "text": "S3 9867.161765 0.0353 \n05. S4 1390.792215 0.2497 \n\ny = 0.00338x + 0.09117\nR\u00b2 = 0.96580\n\n0\n\n0.2\n\n0.4\n\n0.6\n\n0.8\n\n1\n\n1.2\n\n0 100 200 300\n\nM\ne\na\n\nn\n A\n\nb\nso\n\nrb\na\n\nn\nce\n\nConcentration (ppm)\n\nTPC\n\ny = 0.0426x + 0.0519\nR\u00b2 = 0.9811\n\n0\n\n0.1\n\n0.2\n\n0.3\n\n0.4\n\n0.5\n\n0.6\n\n0 5 10 15\n\nA\nb\n\nso\nrb\n\na\nn\n\nce\n\nConcentration (ppm)\n\nTFC\n\ny = 0.00348x + 47.73923\nR\u00b2 = 0.93397\n\ny = 0.00733x + 32.74550\nR\u00b2 = 0.96365\n\ny = 0.00272x + 23.16132\nR\u00b2 = 0.91245\n\ny = 0.01824x + 24.63195\nR\u00b2 = 0.93571\n\ny = 0.02076x + 42.78935\nR\u00b2 = 0.97686\n\n20\n\n30\n\n40\n\n50\n\n60\n\n70\n\n80\n\n90\n\n300 1300\n\nIn\nh\n\nib\nit\n\nio\nn\n\n %\n\nInhibition Concentration [ppm]\n\nDPPH Assay\n\nS1 S2 S3 S4 Quercitin Extract from Allium cepa\n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):3 2 - 3 9      Bajgain et al.  \n\n39 \n\u00a9NJB, BSN   39 \n\nAnti-Oxidant DPPH Assay \nUpon plotting, Concentration (in ppm) Versus % \n\nInhibition of different Honey Samples and similarly, \n\nStandard of Quercetin Extract extracted from Allium \n\ncepa, we obtained various IC50value from the plot, \n\nthrough which we became able to compare between the \n\ntimes potency value of Anti-Oxidant ability of different \n\nsamples to that with Standard Quercetin Extract. From \n\nthe observation, we found out that S1 had maximum \n\npotency with 0.534 times and S3 has minimum potency \n\nwith 0.0353 times of standard Quercetin Extract from \n\nAllium Cepa. Figure 10 is the plot of inhibition \n\nconcentration (in ppm) versus % inhibition of different \n\nSamples and Standards involved in the study. Table 10 \n\nis the result of IC50 value and times potency of different \n\nhoney samples obtained from the calculation. \n\nConclusions \nDifferent tests performed during this research complied \n\nwith the various standard research articles published in \n\ndifferent journals. Potency of honey is affected by the \n\ntypes of honey and seasons at which it has been \n\nharvested. Honey with lower anti-oxidant potency can \n\nbe used in formulation of topical cosmetological \n\nproducts or as daily dietary supplement. Honey with \n\nhigher values of anti-oxidant potency can be used in \n\ncombination with other different active ingredients for \n\nthe formulation of different therapeutic products. \n\nConflict of Interest \nThe author declares no conflict of interest.  \n\nAuthors Contribution  \nAll authors have equal contribution \n\nReferences \n1.  Bambang N, Ikhsan M, Tensiska, Sukri N, Mahani, Rheological \n\nProperties of Honey and its Application on Honey Flow \nSimulation through Vertical Tube. IOP Conf Ser Earth Environ Sci. \n2019 Oct 1;334(1):012041.  \n\n2.  Kitnya N, Prabhudev MV, Bhatta CP, Pham TH, Nidup T, Megu \nK, et al, Geographical distribution of the giant honey bee. 2020;15.", "start_char_idx": 23221, "end_char_idx": 25880, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ce62407e-183f-4f66-8339-4e02f7324c3c": {"__data__": {"id_": "ce62407e-183f-4f66-8339-4e02f7324c3c", "embedding": null, "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-228", "node_type": "4", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "733f5fbcadbdb8c85c604bec17f379cfb588091b6f0f951aeedaaa7b47ed84fc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bfc14c23-a20f-4146-8aa9-64c1b41fd4e5", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "8ca6c3a7246e3f8c6aa9371d1f1a407e08249633499b6ac3bb8869ec3cc6b2b3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "df543969-55f4-4b3d-870d-c35ef592f8eb", "node_type": "1", "metadata": {}, "hash": "9b3c4dbf6b28f18fb50f4dda47d9736beb4ff437e18986a6d2dff8e86254f65e", "class_name": "RelatedNodeInfo"}}, "text": "2020;15. \nZooKeys 951: 67 - 81. doi: 10.3897/zookeys.951.49855 \n\n3.  Jansen SA, Kleerekooper I, Hofman ZL, Kappen IF, Stary-\nWeinzinger A, van der Heyden MA. Grayanotoxin poisoning: \n'mad honey disease' and beyond. Cardiovasc Toxicol. 2012 \nSep;12(3):208-215 \n\n4.  Khalil MI, Sulaiman SA, Boukraa L, Antioxidant Properties of \nHoney and Its Role in Preventing Health Disorder. :11. The Open \nNutraceuticals Journal 2010, 3,  6 -16. \n\n5.  Nwankwo, CM, Ezekoye, CC, Igbokwe SO, Phytochemical \nscreening and antimicrobial activity of apiary honey produced by \nhoney bee (Apis mellifera) on clinical strains of Staphylococcus \n\naureus, Escherichia coli and Candida albicans. Afr J Biotechnol, \n2014 Jun 4;13(23):2367\u201372.  \n\n6.  Dinkov D, A scientific note on the specific optical rotation of three \nhoney types from Bulgaria. :2. Page 319 - 320 \n\n7.  Asokan, S. and Jayanthi, 2017, \u201cPhytochemical analysis of various \nhoney samples obtained from Theni district, South India\", \nInternational Journal of Current Research, 9, (01), 45387-45390. \n\n8.  Kivima E, Tanilas K, Martverk K, Rosenvald S, Timberg L, Laos \nK. The Composition, Physicochemical Properties, Antioxidant \nActivity, and Sensory Properties of Estonian Honeys. Foods. 2021 \nMarch 1;10(3):511.  \n\n9.  Tomasik P, Chemical and Functional Properties of Food \nSaccharides, Chapter 6, Page No 74, ISBN 978-0-203-49572-8. \nInternational Standard Book No-13:978-0-203-49572-8 \n\n10. Stefan Bogdanov, Honey as Nutrient and Functional Food, in \nBook of Honey, Chapter 8, Bee Product Science, 2016, 1-47. \n\n11. Amabye TG, Phytochemical and biochemical compostion of wild \nhoney a case study in Estern zone areas in tigray Ethiopia. MOJ \nFood Process Technol. 2017;4(3):88\u201294. DOI: \n10.15406/mojfpt.2017.04.00094   \n\n12. \u00d6zk\u00f6k A, D\u2019arcy B, Sorkun K, Total Phenolic Acid and Total \nFlavonoid Content of Turkish Pine Honeydew Honey. J \nApiProduct ApiMedical Sci. 2010 Apr 1;2(2):65\u201371.  \n\n13. Adalina Y, Kusmiati E, Pudjiani M, Phytochemical test and \nphysical chemical properties of rubber honey from three types of \nbees ( Apis mellifera, Apis dorsata and Trigona Itama ). IOP Conf Ser \nMater Sci Eng. 2020 Sep 23;935:012007.  \n\n14. Subroto E, Lembong E, Filianty F, Indiarto R, Primalia G, \nTheodora HC, et al, The Analysis Techniques Of Amino Acid And \nProtein In Food And Agricultural Products. 2020;9,29-36.  \n\n15. Ka\u010d\u00e1niov\u00e1 et al, Antimicrobial and Antiradical Activity of \nSlovakian Honeydew Honey Samples. Journal of Microbiology, \nBiotechnology and Food Sciences 2011/12 1 (3) 354 - 368.", "start_char_idx": 25872, "end_char_idx": 28414, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "df543969-55f4-4b3d-870d-c35ef592f8eb": {"__data__": {"id_": "df543969-55f4-4b3d-870d-c35ef592f8eb", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ce62407e-183f-4f66-8339-4e02f7324c3c", "node_type": "1", "metadata": {"identifier": "njb-228", "author": "Bajgain, Abhishek; Dev Neupane, Bashu; Sarraf, Diwakar; Karmacharya, Jwalant; Ranjitkar, Saksham; Shrestha, Rajan; Gyawali, Rajendra", "title": "Quality Evaluation of Apis laboriosa and Apis mellifera Honey Collected from Bagmati Province, Nepal", "date": "2022-07-30", "file": "njb-228.pdf"}, "hash": "272a48385bb9a3153ea2d915f0f443e0ce6c0d9b4d1c0363d8508af5eb15f340", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9611008e-04c1-40a1-920b-9eaa09e6d79f", "node_type": "1", "metadata": {}, "hash": "30d6aa03391da6e766f52a8002282020f31260346b39bc7c84727a37087538c6", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0  (1): 4 0 - 4 4   Research article  DOI: https://doi.org/10.54796/njb.v10i1.229 \n \n\n\u00a9NJB, BSN   40 \n\nScreening of Carbapenem Resistance Klebsiella pneumoniae and its MIC \nagainst Imipenem   \nSarada Saud1, Ashwani Agrawal1, Soniya Pokhrel1, Sushma Subedi1, Sanjit Shrestha2, and Niroj Man Amatya 1  \n1Department of Medical Microbiology, Nobel College, Sinamangal, Kathmandu, Nepal Affiliated to Pokhara \n\nUniversity. \n2Pathology Department, B & B Hospital, Lalitpur, Nepal. \n\nReceived: 26 Feb 2022; Revised: 20 Jul 2022; Accepted: 26 Jul 2022; Published online: 30 Jul 2022 \n\nAbstract \nKlebsiella pneumoniae is a common opportunistic pathogen causing a wide range of infections; pneumonia, urinary tract \ninfection, bacteremia, and liver abscesses. It infects primarily immunocompromised and immunocompetent individuals. It \npresents itself as an antibiotic-resistant bacterium, especially for third-generation cephalosporins and carbapenems, creating \nserious global challenges. Therefore, this cross-sectional study was conducted in B & B Hospital, Lalitpur to screen the \ndistribution of carbapenem resistance K. pneumoniae through ertapenem and to assess the minimum inhibitory concentration \nof imipenem for screened carbapenem resistance K. pneumoniae. From 3447 different clinical samples collected according to \nstandard guidelines, K. pneumoniae was identified using standard microbiological techniques; staining and a panel of \nbiochemical tests. The antibiotic susceptibility test of the isolates was performed by the Kirby-Bauer disc diffusion method as \nper CLSI 2018 guidelines. The screening of carbapenem resistance was assessed by using ertapenem disc and the MIC of \nimipenem for carbapenem resistance and intermediate was performed using an Epsilometer. A total of 85 K. pneumoniae were \nidentified and their antibiotic susceptibility test revealed that ceftriaxone was the least effective antibiotic. The number of \nMDR, carbapenem-resistant and intermediate isolates was 51, 46, and 3, respectively. The MIC of imipenem through an \nEpsilometer from ertapenem resistant and intermediate revealed that 31, 5, and 13 isolates were resistant, intermediate, and \nsensitive respectively. These findings showed the inconsistency in the detection of carbapenem-resistant isolates in routine \nmicrobiology laboratories and further support the other tests for the detection of carbapenem resistance as suggested by CLSI. \n\nKeywords: Carbapenem-resistant Klebsiella pneumoniae, Ertapenem, MIC \n\nCorresponding author, e-mail: mahaju@gmail.com \n\nIntroduction \nKlebsiella pneumoniae, an important member of the \n\nEnterobacteriaceae family; residing in the \n\ngastrointestinal tract of us, is not only the most clinically \n\nisolated opportunistic pathogen from immune-\n\ncompromised individuals, neonates, critically ill patients, \n\nor patients with other risk factors in healthcare settings \n\n[1] but also from immunocompetent individuals causing \n\na wide range of infections mostly urinary tract infection, \n\npyogenic liver abscess, necrotizing pneumonia or other \n\nlife-threatening infections [2]. Management of its \n\ninfection becomes complicated after it is found to be not \n\nsusceptible to the third-generation cephalosporin \n\nantibiotics, including monobactams [3].", "start_char_idx": 48, "end_char_idx": 3363, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9611008e-04c1-40a1-920b-9eaa09e6d79f": {"__data__": {"id_": "9611008e-04c1-40a1-920b-9eaa09e6d79f", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "df543969-55f4-4b3d-870d-c35ef592f8eb", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "ef1268c0cb15acc699f9aac2ffbb4c78222043a437308ca7d749932da3f85ca1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ac121847-ce68-49e6-8cc9-d784c4229ed3", "node_type": "1", "metadata": {}, "hash": "e39dfe9dbc752c7f4fb5e3966a3f20d63ef5934451ec334ee8153fa105b36251", "class_name": "RelatedNodeInfo"}}, "text": "This is further \n\naggravated by the non-response of carbapenem \n\nantibiotics through either the expression of \n\ncarbapenemase enzymes that make bacteria almost \n\nresistant to a \u03b2-lactam group antibiotic [4, 5] or alteration \n\nof permeability due to loss of porin or overexpression of \n\nthe efflux pump [5, 6]. Therefore, WHO prioritizes \n\nextended-spectrum \u03b2-lactamase [ESBL] and carbapenem-\n\nresistant Klebsiella pneumoniae [CRKP] as a critical public \n\nhealth threat [7]. The epidemiological distribution of \n\nCRKP fluctuates in all countries [8] with significantly \n\nhigher morbidity and mortality rate than those of \n\ncarbapenem susceptible K. pneumoniae, which initiates \n\ndevastating public health conditions [9]. \n\nThis bacterium notoriety gained its name among \n\nantibiotic-resistant bacteria. The European Antimicrobial \n\nResistance Surveillance Network (EARS-Net) showed \n\nthat from 2005 the non-susceptibility rate of this \n\nbacterium had increased significantly against third-\n\ngeneration cephalosporins, aminoglycosides, \n\nfluoroquinolones, and carbapenems that have greater \n\nvariation in different countries of the European Union \n\n[10] and other parts of the world, increasing global public \n\nhealth concerns [11]. Hence, WHO recognized this \n\nbacterium  with Acinetobacter baumannii, Pseudomonas \n\naeruginosa as a WHO Priority Pathogen list for \u201cResearch \n\nand Development\u201d of New Antibiotics [7]. Therefore, this \n\nstudy was carried out to find out the frequency of CRKP \n\nalong with their antibiotic susceptibility profile and MIC \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0003-3398-7422\nmailto:mahaju@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):4 0 - 4 4      Saud et al.  \n\n\u00a9NJB, BSN   41 \n\nof imipenem for screened carbapenem-resistant and \n\nintermediate isolates. \n\nMaterials and Methods \nIt is a hospital-based prospective cross-sectional study; \n\ncarried out in the Microbiology Department of B and B \n\nHospital, Nepal, from 15 July 2018 to 15 October 2018. The \n\ntarget group of this study was irrespective of sex, all age \n\ngroups of patients attending the hospital for medical \n\ntreatment. All collected data were entered and analyzed \n\nusing SPSS V17.0. Ethical consent was obtained from the \n\nNobel Institutional Review Committee (IRC). \n\nBacterial isolation and identification  \nSamples (blood, pus, urine, respiratory specimen, \n\ncatheter tips, and joint fluid) were collected aseptically \n\naccording to standard microbiological guidelines [12]. \n\nGood quality specimens were accepted, while unlabeled \n\nor mislabeled specimens, dry swabs, specimens that leak \n\nfrom a container, delay in specimen transport, and \n\ninappropriately stored samples were excluded from this \n\nstudy. All samples, except blood, were cultured on blood \n\nagar and MacConkey agar; incubated at 370C overnight, \n\nand identified as K. pneumoniae using Gram staining and \n\nconventional biochemical tests (Catalase, Oxidase, indole \n\ntest, motility, citrate utilization, triple sugar iron \n\nutilization and Urease Test) [13]. The reagents and \n\nculture media were purchased from HiMedia \n\nLaboratories, India. The BD\u2122 BACTEC\u2122 FX40 \n\nAutomated Blood Culture System was used for blood \n\nculture, and a positive culture bottle was further sub-\n\ncultured on blood agar and MacConkey as previously for \n\nidentification [13]. \n\nAntibiotic susceptibility test \nThe antibiotic susceptibility test was performed \n\nfollowing CLSI guidelines 2018 through the disc \n\ndiffusion method.", "start_char_idx": 3364, "end_char_idx": 7019, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ac121847-ce68-49e6-8cc9-d784c4229ed3": {"__data__": {"id_": "ac121847-ce68-49e6-8cc9-d784c4229ed3", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9611008e-04c1-40a1-920b-9eaa09e6d79f", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "0eda1972fa5157409add7fd899bb58191fd7ad59c0031d6e31b1c61e22b42428", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "366ba797-0cd0-498a-b4cc-a818d9b4180b", "node_type": "1", "metadata": {}, "hash": "926c601db40b15c2f5e5cf37836c57dfffa709d4bbed4d74490fa81e9fb24414", "class_name": "RelatedNodeInfo"}}, "text": "Eleven different antibiotics (HiMedia \n\nLaboratories, India); amikacin (30 \u00b5g), cefepime (30 \u00b5g), \n\nceftriaxone (30 \u00b5g), ciprofloxacin (5 \u00b5g), \n\nchloramphenicol (30 \u00b5g), cotrimoxazole (25 \u00b5g), \n\ngentamicin (10 \u00b5g), nitrofurantoin (300 \u00b5g), piperacillin-\n\ntazobactam (100/10 \u00b5g), and ofloxacin (5\u00b5g) were tested \n\nin isolated Klebsiella pneumoniae. The bacterium showing \n\nresistance to at least one antibiotic from three or more \n\nthan three different classes was categorized as multidrug \n\nresistance [14]. The resistance to carbapenem was \n\nscreened using ertapenem (10\u03bcg) disc. According to zone \n\nsize diameter, isolates were differentiated as sensitive, \n\nintermediate, and resistant with zone size inhibition \u226522 \n\nmm, 19-21mm, and \u226418 mm, respectively [15]. The \n\nminimum inhibitory concentration (MIC) of the \n\nimipenem was tested for isolates showing intermediate \n\nor resistance to carbapenem using the Imipenem Ezy \n\nMICTM strip following the manufacture instructions \n\n(HiMedia Laboratories, India). The MIC of imipenem \n\nwas interpreted and the isolates were differentiated as \n\nsensitive (\u22641 \u00b5g/ml), intermediate (2 \u00b5g/ml), and \n\nresistant (\u22654\u00b5g/ml) [15]. For quality control of the MIC \n\ntest strip, carbapenem susceptible Escherichia coli ATCC \n\n25922 was used.  \n\nResults \nPatient Characterization \nA total of 3447 specimens were received in the trimester \n\nperiod in which 771 samples showed culture-positive \n\nfrom which 815 bacteria were isolated. Among 815 \n\nbacterial isolates, 85 isolates were confirmed as Klebsiella \n\npneumoniae. The rest data are shown in Table 1. \n\nTable 1. Clinical and social demography of patients \n\nS.N. Status of patients Number (%) \n\n1 OPD 15 (17.65%) \n2 Inpatients 70 (82.35%) \n\n   \n\nS.N. Sex Number \n\n1 Male 50 (58.82%) \n2 Female 35 (41.18%) \n\n   \n\nS.N. Age group Number \n\n1 Below 20 12 (14.11%) \n2 20-30 14 (16.47%) \n3 30-40 12 (14.11%) \n4 40-50 9 (10.59%) \n5 50-60 14 (16.47%) \n6 60 and above  24 (28.24%) \n\n \nAntibiotic susceptibility test  \nThe antibiotic susceptibility test showed that ceftriaxone \n\n(59, 69.42%) was the most non-susceptible antibiotic, \n\nfollowed by ciprofloxacin (49, 57.65%) and gentamicin \n\n(48, 56.47%). Furthermore, 60% (51 of 85) isolates were \n\nmultidrug-resistant. The remaining data are shown in \n\nTable 2.  \n\nCarbapenem-resistant  \nThe screening of carbapenem resistance showed that in \n\n85 K. pneumoniae isolates, the carbapenem resistance and \n\nintermediate isolates were 46 (54.13%) and three (3.52%) \n\nrespectively. The MIC test of both intermediate and \n\nresistant isolates was done as suggested by the CLSI \n\nguideline 2018. Among the 49 isolates, the MIC test of \n\nimipenem showed that 31 isolates were resistant, five \n\nintermediate, and 13 sensitives. Furthermore, in 85 \n\nisolates, 51 isolates were MDR K. pneumoniae isolates, in \n\nwhich 45 isolates were resistant to carbapenem and six \n\nisolates were susceptible to carbapenem.  \n\n \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):4 0 - 4 4      Saud et al.", "start_char_idx": 7020, "end_char_idx": 10026, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "366ba797-0cd0-498a-b4cc-a818d9b4180b": {"__data__": {"id_": "366ba797-0cd0-498a-b4cc-a818d9b4180b", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ac121847-ce68-49e6-8cc9-d784c4229ed3", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "9f756eb4d542a398390e506661874537e1cc69bd274cb96e15a3d803343947cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "655a1308-6ce8-422f-a6ad-839114b2831a", "node_type": "1", "metadata": {}, "hash": "3fbeb3f8de0f82503bc9681a0a694246e490ef9b3859a8ccd1cd111c63c30345", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN   42 \n\n \n\nFigure 1. MIC Test \n\nDiscussion \nKlebsiella pneumoniae is an opportunistic pathogen \n\nresponsible for causing various community-acquired and \n\nhealthcare-associated infections. Furthermore, the \n\ninfection caused by this bacterium cannot be neglected, \n\nas it is included in ESKAPE pathogens and the increasing \n\nincidence of CRKP strains attracts attention to clinicians \n\nand other stakeholders. \n\nIn our study, the highest percentage of K. pneumoniae was \n\nobtained in the inpatient department than others. This \n\nhigher incidence of K. pneumoniae in long-term \n\nhospitalized patients may be related to the immune \n\nstatus of the patients, as the bacterium was isolated from \n\nthe surgery unit with the use of invasive devices and \n\nadministration of immunosuppressive drugs. In hospital \n\nsettings, the transmission of the pathogen increases \n\ndrastically because the colonization rate increases with an \n\nextended stay in the hospital and prolonged antibiotic \n\ntherapy. A similar study carried out in the United States \n\nalso claimed a higher incidence of K. pneumoniae \n\ninfections in long-term acute care hospitals than in the \n\nshort-term hospital stay [16]. \n\nAntibiotic resistance is a common problem in K. \n\npneumoniae. It is naturally resistant to the penicillin group \n\nof antibiotics [17] or acquires antibiotic resistance genes \n\nfrom mobile genetic cassettes called integrons, often \n\ncarried out by transposons and transferable plasmids that \n\ntransmit horizontally to receptor cells, integrated on \n\nplasmids or chromosomes through homologous \n\nrecombination, expressing its fitness in the presence of \n\nantibiotics [18]. In this study, we evaluated 11 different \n\nantibiotics, in which amikacin, gentamicin, ciprofloxacin, \n\nofloxacin, ceftriaxone, ertapenem were tested in all \n\nisolates, and the rest antibiotics were tested either as \n\nsecond-line antibiotics or depending on a source of \n\nclinical samples. Our results showed a mixed antibiotic \n\nresistance profile compared to others in terms of \n\nantibiotic use, time period, bacterium source and \n\ncountry.  \n\nApproximately 48.24% and 56.47% of the isolates were \n\nnot susceptible to aminoglycosides, amikacin, and \n\ngentamicin, respectively. A range of studies shows a \n\nwide variation in resistance pictures that range from 1% \n\nto 86% for gentamicin. A study in the EU/EEA region \n\nshowed that its resistance per cent ranges from below 1% \n\nto greater than 50 [19]. A comparative study from France \n\nand Algeria revealed that its resistance was 28% and 86%, \n\nrespectively, [20] while in a study from Iran and India, \n\nthe resistance rate was found to be 24% and 37.5%, \n\nrespectively [21, 22]. A slightly lower percentage, 41%, \n\nwas observed in Nepal [23] which is nearly equal to our \n\nstudy. Cephalosporins, such as ceftriaxone, are \n\nfrequently used antibiotics for K. pneumoniae until they \n\nbecome ESBL producers. Our study showed that nearly \n\n70% of the isolates were resistant to ceftriaxone. A similar \n\nfinding was described from Ethiopia [24] and a four-year \n\nconsecutive study from Greece [19]. Ciprofloxacin and \n\nTable 2. Antibiotic Susceptibility Pattern of K. pneumoniae \n\nS.N.", "start_char_idx": 10030, "end_char_idx": 13226, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "655a1308-6ce8-422f-a6ad-839114b2831a": {"__data__": {"id_": "655a1308-6ce8-422f-a6ad-839114b2831a", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "366ba797-0cd0-498a-b4cc-a818d9b4180b", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "01b2bfe04e2ae993c3a6ce0b673dabf3354a6bee27756042059631403ce3164e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cfba4f85-c936-4e81-b44a-3e77e8741503", "node_type": "1", "metadata": {}, "hash": "3a1ee98e45f5fee17f7475e06b84f90808dacc1461a9035b934c026ad4cc4bd6", "class_name": "RelatedNodeInfo"}}, "text": "Antibiotic Susceptibility Pattern of K. pneumoniae \n\nS.N. \nAntibiotic Susceptibility Pattern Total Isolates \n\nAntibiotics used Sensitive (%) Intermediate (%) Resistant (%)  \n\n1 Amikacin  24  (28.23) 20 (23.53) 41 (48.24) 85 \n\n2 Gentamicin 34  (40) 3 (3.53) 48 (56.47) 85 \n\n3 Ciprofloxacin 28 (32.94) 8 (9.41) 49 (57.65) 85 \n\n4 Ofloxacin 33 (38.82) 5 (5.88) 47 (55.30) 85 \n\n5 Ceftriaxone 24 (28.23) 2 (2.35) 59 (69.42) 85 \n\n6 Ertapenem 36 (42.35) 3 (3.52) 46 (54.13) 85 \n\n7 Cefepime 11 (16.18) 8 (11.76) 49 (72.06) 68 \n\n8 Piperacillin-tazobactam 9 (14.06) 12 (18.75) 43 (67.19) 64 \n\n9 Chloramphenicol 38 (61.30) 11(17.74) 13 (20.96) 62 \n\n10 Cotrimoxazole 28 (50) 1 (1.78) 27 (48.22) 56 \n\n11 Nitrofurantoin 6 (13) 6 (13) 34 (74) 46 \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):4 0 - 4 4      Saud et al.  \n\n\u00a9NJB, BSN   43 \n\nofloxacin are alternative antibiotics of choice if the \n\nisolates are ESBL producers. The non-susceptibility rates \n\nfor ciprofloxacin and ofloxacin were 57.65 % and 55.3 % \n\nin our study. Similar to aminoglycosides, a wider range \n\nof variations in the non-susceptibility of quinolones was \n\nobserved, ranging from less than 1% to more than 90%. \n\nThe study of Bulgaria, Italy, and Romania was in line \n\nwith our study, while from Germany, Denmark, Iran, and \n\nIndia, the non-susceptibility rate was lower than in our \n\nstudy [19, 21, 22]. A few studies from Nepal showed that \n\nthe resistance rate is more than 85 %, which is quite much \n\nhigher than ours [23, 25]. In general, different studies \n\nconducted at different times revealed significant \n\nvariability in antibiotic resistance patterns. Such a type of \n\nvariation was also observed in other antibiotics \n\nmentioned in Table 2, which might be due to the low \n\nnumber of sample studies or how meticulously \n\nantibiotics were used in that country to mitigate \n\nantibiotic resistance problems. Hence, resistance to these \n\nfirst-line agents represents an unprecedented challenge \n\nto clinicians, scientists, and healthcare systems. \n\nCarbapenems (imipenem, meropenem, and ertapenem) \n\nare the antibiotics of choice for those K. pneumoniae that \n\nare MDR and ESBL producers [26]. The phenotypic lab \n\ndetection of carbapenem resistance is quite confusing and \n\nfor confirmation of carbapenemase producer, different \n\nadditional tests should be performed [27]. Therefore, \n\nCLSI recommends that ertapenem non-susceptibility is \n\nthe most sensitive indicator for carbapenemase \n\nproducers, and additional tests should not be performed \n\nfor purposes other than epidemiological or infection \n\ncontrol after breaking point evaluation of ertapenem, \n\nmeropenem, and imipenem [15]. In our study, 49 (both \n\nintermediate and resistant) isolates showed positive \n\nscreening through non-susceptibility to ertapenem.", "start_char_idx": 13169, "end_char_idx": 15959, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cfba4f85-c936-4e81-b44a-3e77e8741503": {"__data__": {"id_": "cfba4f85-c936-4e81-b44a-3e77e8741503", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "655a1308-6ce8-422f-a6ad-839114b2831a", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "53ed9fc6f098262362c3aee497707fe9897c67753a1530c0166e76f7ed736ebb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0781e037-ce7c-4da1-a6f7-2ce51b884ad0", "node_type": "1", "metadata": {}, "hash": "e1933fdc1ce8d923d23c1f6204aea97ae1463e0537fbe307de2622aacb7c66ee", "class_name": "RelatedNodeInfo"}}, "text": "This \n\nsuggests that resistance to carbapenem arises due to the \n\nformation of carbapenemase enzymes of classes A, B and \n\nD of the Amber class of \u03b2-lactamase, restricting the \n\ntreatment option [27]. The CRKP was subjected to the E-\n\ntest to determine the MIC of the imipenem. The test \n\nresults showed that susceptible, intermediate and \n\nresistant were 13 [MIC \u22641\u00b5g/ml), 5 [4\u00b5g/ml \n\n\u2264MIC\u22651\u00b5g/ml) and 31 [MIC \u22654 \u03bcg/ml), respectively. \n\nThis implies that results from phenotypic methods can \n\nvary, as suggested by CLSI.  \n\nConclusions \nThe fast-growing antibiotic-resistant Klebsiella pneumoniae \n\nis a global problem, including in Nepal, such that the \n\nchoice of an effective antibiotic is now chaotic. The \n\ncontinued emergence of CRKP has wreaked havoc on its \n\nroutine diagnosis, as well as on its therapeutic treatment \n\noptions. Susceptibility testing [disc diffusion and MIC) \n\nprovides valuable information for therapeutic inference \n\nbut does not adequately address carbapenem resistance, \n\nwhich is significant for infection control and \n\nepidemiological evidence necessary to curb the spread of \n\ncarbapenem-resistant Enterobacteriaceae. \n\nAuthors' Contributions \nThe laboratory works were performed by Sarada Saud, \n\nAshwani Agrawal, Soniya Pokhrel and Sushma Subedi. \n\nResearch conceptualization, protocol selection and \n\npreliminary manuscript preparation were done by \n\nSarada Saud, Ashwani Agrawal, Soniya Pokhrel, Sushma \n\nSubedi, Sanjit Shrestha and Niroj Man Amatya. Final \n\nmanuscript preparation was done by Niroj Man Amatya. \n\nThe study was supervised under the guidance of Sanjit \n\nShrestha and Niroj Man Amatya. All authors read and \n\napproved the final manuscript. \n\nCompeting Interests \nOn behalf of all authors, the corresponding author states \n\nthat there is no competing interest. \n\nFinancial disclosure \nIt is a self-funded pilot study and no funding is granted. \n\nAcknowledgements  \nWe would like to thank all the participants attained in \n\nthis study and the Pathology Department of the B and B \n\nHospital, Nepal. \n\nEthics approval  \nThe ethical consent was procured from Nobel \n\nInstitutional Review Committee [IRC) with reference \n\nnumber BCH IRC 161/2018.  \n\nAdditional disclosure \nThis manuscript has been already published in preprint \n\nform with title \u201cScreening ofKlebsiella pneumoniaefor \n\nCarbapenem Resistance and MIC of Imipenem\u201d with \n\nweblink: https://www.researchsquare.com/article/rs-\n\n36584/v1 \n\nReference \n1. Broberg CA, Palacios M, Miller VL. Klebsiella: a long way to go \n\ntowards understanding this enigmatic jet-setter. F1000Prime Rep. \n2014;6:64. \n\n2. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the offense \nwith a strong defense. Microbiol Mol Biol Rev. 2016;80[3):629-61  \n\n3. WHO. Antimicrobial resistance: global report on surveillance. \nGeneva, Switzerland: World Health Organization; 2014. \n\n4. Queenan AM, Bush K. Carbapenemases: the versatile \u03b2-lactamases. \nClinical microbiology reviews. 2007;20(3):440-58  \n\n5. Mart\u00ednez-Mart\u00ednez L, Pascual A, Hern\u00e1ndez-All\u00e9s S, Alvarez-D\u00edaz \nD, Su\u00e1rez AI, Tran J, et al. Roles of \u03b2-lactamases and porins in \nactivities of carbapenems and cephalosporins against Klebsiella \n\n\n\nNepal J Biotechnol. 2 0 2 2  J u l ; 1 0  (1):4 0 - 4 4      Saud et al.  \n\n\u00a9NJB, BSN   44 \n\npneumoniae. Antimicrobial agents and chemotherapy. \n1999;43(7):1669-73  \n\n6.", "start_char_idx": 15960, "end_char_idx": 19317, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0781e037-ce7c-4da1-a6f7-2ce51b884ad0": {"__data__": {"id_": "0781e037-ce7c-4da1-a6f7-2ce51b884ad0", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cfba4f85-c936-4e81-b44a-3e77e8741503", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "bf450fb5595832c7cc3653f2d84c235af206a91679a58ecc121f18dddf664250", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e4f55432-02ce-436e-a36d-a20c672e8c72", "node_type": "1", "metadata": {}, "hash": "c1066da1beaf29d820fae2c2e37709194fd8c49003d6194917e0bbc42e49afe9", "class_name": "RelatedNodeInfo"}}, "text": "1999;43(7):1669-73  \n\n6. Padilla E, Llobet E, Dom\u00e9nech-S\u00e1nchez A, Mart\u00ednez-Mart\u00ednez L, \nBengoechea JA, Albert\u00ed S. Klebsiella pneumoniae AcrAB efflux \npump contributes to antimicrobial resistance and virulence. \nAntimicrobial agents and chemotherapy. 2010;54(1):177-83  \n\n7. WHO. Global priority list of antibiotic-resistant bacteria to guide \nresearch, discovery, and development of new antibiotics. February \n27, 2017. 2018. \n\n8. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, \nCormican M, et al. Clinical epidemiology of the global expansion of \nKlebsiella pneumoniae carbapenemases. The Lancet infectious \ndiseases. 2013;13(9):785-96  \n\n9. Xu L, Sun X, Ma X. Systematic review and meta-analysis of mortality \nof patients infected with carbapenem-resistant Klebsiella \npneumoniae. Annals of clinical microbiology and antimicrobials. \n2017;16(1):18. \n\n10. European Centre for Disease Prevention and Control. Surveillance \nAtlas of Infectious Diseases: ECDC;  [Available from: \nhttp://atlas.ecdc.europa.eu/public/index.aspx?Instance. \n\n11. Nordmann P, Poirel L. The difficult-to-control spread of \ncarbapenemase producers among Enterobacteriaceae worldwide. \nClinical Microbiology and Infection. 2014;20(9):821-30  \n\n12. Leber AL. Clinical microbiology procedures handbook. \nWashington, DC, USA: American Society for Microbiology; 2016. \n\n13. Tille P. Bailey & Scott's diagnostic microbiology: Elsevier Health \nSciences; 2015. \n\n14. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, \nGiske CG, et al. Multidrug\u2010resistant, extensively drug\u2010resistant and \npandrug\u2010resistant bacteria: an international expert proposal for \ninterim standard definitions for acquired resistance. Clinical \nmicrobiology and infection. 2012;18(3):268-81  \n\n15. CLSI. Performance standards for antimicrobial susceptibility \ntesting. Wayne, PA: Clinical and Laboratory Standards Institute; \n2018. \n\n16. Lin MY, Lyles-Banks RD, Lolans K, Hines DW, Spear JB, Petrak R, \net al. The importance of long-term acute care hospitals in the \nregional epidemiology of Klebsiella pneumoniae carbapenemase\u2013\nproducing Enterobacteriaceae. Clinical infectious diseases. \n2013;57(9):1246-52  \n\n17. Mandell G, Dolin R, Bennett J. Mandell, Douglas, and Bennett's \nprinciples and practice of infectious diseases: Elsevier; 2009. \n\n18. Partridge SR, Kwong SM, Firth N, Jensen SO. Mobile genetic \nelements associated with antimicrobial resistance. Clinical \nmicrobiology reviews. 2018;31(4):e00088-17  \n\n19. European Centre for Disease PC. Surveillance of antimicrobial \nresistance in Europe 2016. Annual Report of the European \nAntimicrobial Resistance Surveillance Network (EARS-Net). 2017. \n\n20. Berrazeg M, Diene SM, Drissi M, Kempf M, Richet H, Landraud L, \net al. Biotyping of multidrug-resistant Klebsiella pneumoniae \nclinical isolates from France and Algeria using MALDI-TOF MS. \nPloS one. 2013;8(4):e61428. \n\n21. Estabraghi E, Salehi TZ, Aminiq K, Jamshidian M. Molecular \nidentification of extended-spectrum \u03b2-lactamase and integron \ngenes in Klebsiella pneumonia.", "start_char_idx": 19293, "end_char_idx": 22346, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e4f55432-02ce-436e-a36d-a20c672e8c72": {"__data__": {"id_": "e4f55432-02ce-436e-a36d-a20c672e8c72", "embedding": null, "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-229", "node_type": "4", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "004ac4ad3516e7572d761531f8e4ad9879b8040e166d1ddd2d34f20efe2f978f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0781e037-ce7c-4da1-a6f7-2ce51b884ad0", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "559574980435c4aca2391a5a0731733e394d6ebad89991d63174d5be687704fb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f717aea1-eaaf-4d0a-a1ea-57de1eb0ef5b", "node_type": "1", "metadata": {}, "hash": "3cc8b5954458b31168c22eaab53a5559dc32a173718763427b5055d06f2526e9", "class_name": "RelatedNodeInfo"}}, "text": "Journal of the Nepal Medical \nAssociation. 2016;54(202 ). \n\n22. Shilpa K, Thomas R, Ramyashree A. Isolation and Antimicrobial \nsensitivity pattern of Klebsiella pneumoniae from sputum samples \nin a tertiary care hospital. Int J Biomed Adv Res. 2016;7(2):53-7. \n\n23. Subedi S, Maharjan J, Shrestha B. Antibiotic Susceptibility Test of \nKlebsiella pneumoniae and K. oxytoca Isolated from Different \nClinical Samples and Perform Random Amplified Polymorphic \nDNA among K. pneumoniae. Microbiology Research Journal \nInternational. 2016:1-11. \n\n24. Gashe F, Mulisa E, Mekonnen M, Zeleke G. Antimicrobial \nresistance profile of different clinical isolates against third-\ngeneration cephalosporins. Journal of pharmaceutics. 2018;2018  \n\n25. Parajuli NP, Acharya SP, Mishra SK, Parajuli K, Rijal BP, Pokhrel \nBM. High burden of antimicrobial resistance among gram negative \nbacteria causing healthcare associated infections in a critical care \nunit of Nepal. Antimicrobial Resistance & Infection Control. \n2017;6(1):67  \n\n26. Paterson DL. Recommendation for treatment of severe infections \ncaused by Enterobacteriaceae producing extended-spectrum \u03b2-\nlactamases (ESBLs). Clinical Microbiology and Infection. \n2000;6(9):460-3. \n\n27. Miller S, Humphries RM. Clinical laboratory detection of \ncarbapenem-resistant and carbapenemase-producing \nEnterobacteriaceae. Expert review of anti-infective therapy. \n2016;14(8):705-17  \n\n \n\nhttp://atlas.ecdc.europa.eu/public/index.aspx?Instance", "start_char_idx": 22347, "end_char_idx": 23819, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f717aea1-eaaf-4d0a-a1ea-57de1eb0ef5b": {"__data__": {"id_": "f717aea1-eaaf-4d0a-a1ea-57de1eb0ef5b", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e4f55432-02ce-436e-a36d-a20c672e8c72", "node_type": "1", "metadata": {"identifier": "njb-229", "author": "Saud, Sarada; Agrawal, Ashwani; Pokhrel, Soniya; Subedi, Sushma; Shrestha, Sanjit; Man Amatya, Niroj", "title": "Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem", "date": "2022-07-30", "file": "njb-229.pdf"}, "hash": "f196bbdffb38d9b9225e4b44a563f90a41cb824bb361db3db429ea10cfdee469", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "49bd0bc9-54ce-4aed-bd13-af6d6e7e1424", "node_type": "1", "metadata": {}, "hash": "371ea678393217e29ed0471eb3714255f49fcd7e585722cec122058564f76b68", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:  43-46  ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n \n\u00a9NJB, Biotechnology Society of Nepal    43                Nepjol.info/index.php/njb \n\n \n\nPrevalence of Non-Albicans Candida Among the Patients Attending \n\na Tertiary Care Hospital in Kathmandu, Nepal \nManisha Sharma1, Narayan Dutt Pant2*, Pratikshya Pandey3 \n\n1Department of Microbiology, Kathmandu medical college, Kathmandu, Nepal. \n2Medical Microbiologist, Department of Microbiology, Grande International Hospital, Dhapasi, Kathmandu, Nepal. \n\n3Quality Control Officer, Department of Microbiology, Mark Formulations Pvt. Ltd., Kathmandu, Nepal. \n\nAbstract \nThe main objective of this study was to determine the prevalence of non-albicans Candida among the \n\npatients attending a tertiary care hospital in Kathmandu, Nepal. Candida spp. isolated from different \n\nclinical samples (sputum, urine, vaginal swab, blood, endotracheal (ET) secretion, pus) from 250 patients \n\nbetween the period of February 2013 and December 2015 were included in the study. Of those 250 \n\npatients, 20% were immunocompromised. Sabouraud dextrose agar was used for the isolation of \n\nCandida spp. and the identification was performed on the basis of colony morphology, Gram\u2019s stain, \n\nIndia ink preparation, germ tube test, temperature tolerance test, characteristic color change in \n\nCHROMagar, chlamydospore production, sugar fermentation test and sugar assimilation test. \n\nOut of total 300 Candida spp., majority were isolated from sputum (43.33%) followed by urine (40%) and \n\nvaginal swab (6.67%). Of total 151 (50.33%) non-albicans Candida, the most common species isolated \n\nwere C. tropicalis (62.25%) followed by C. glabrata (23.84%). High prevalence of non-albicans Candida \n\namong the patients attending a hospital in Kathmandu, Nepal was noted. \n\nKey words: Candida, non-albicans Candida, Nepal \n\n*Corresponding Author \nEmail: ndpant1987@gmail.com \n\nIntroduction \nIncidence of fungal infection has increased \n\nsignificantly over the past few years, causing \n\nconsiderable morbidity and mortality, mainly \n\naffecting immunocompromised patients. The \n\nemergence of fungal infections as the worldwide \n\nhealth care problems may be attributed to the \n\nextensive use of broad spectrum antibiotics and \n\nimmune-suppressive agents, as well as an increase in \n\nthe population of immunocompromised people [1]. \n\nCandida is one of the commonest fungal pathogens \n\nand may cause infection in immunocompetent as well \n\nas immunocompromised person [2]. Due to presence \n\nof Candida spp. as normal flora of mucosal surfaces, \n\nthey mostly cause endogenous infections [3]. Candida \n\nalbicans is considered as the commonest species of \n\nCandida responsible for causing various infections [4]. \n\nBut in recent years there have been significant \n\nincrease in infections caused by the species of the \n\nnon-albicans Candida [5]. Only small numbers of \n\nspecies among the 150 species of Candida are well \n\nestablished as human pathogens [6]. Among them, \n\nCandida glabrata, Candida parapsilosis and Candida \n\ntropicalis are non-albicans Candida of increasing \n\nclinical significance [5]. Most of non-albicans Candida \n\nlike C. glabrata, C. krusei, C. tropicalis and C. \n\nparapsilosis, which are of clinical significance are \n\nknown to show resistance toward certain commonly \n\nused antifungal agents [7, 8]. So, for optimizing the \n\ntreatment of the infections caused by Candida spp., it \n\nis necessary to identify the Candida spp.", "start_char_idx": 47, "end_char_idx": 3604, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "49bd0bc9-54ce-4aed-bd13-af6d6e7e1424": {"__data__": {"id_": "49bd0bc9-54ce-4aed-bd13-af6d6e7e1424", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f717aea1-eaaf-4d0a-a1ea-57de1eb0ef5b", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "332718dbfef4596bddbe0deb6bd68a22fd81ad2b6243b9a8dfccb0fa6e64637f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ce989b95-728a-429e-859c-f9bb445eda6d", "node_type": "1", "metadata": {}, "hash": "9b04407c877df5e0ffcff3bf9daa3f7ed54330b2069c49b1964bc124a3bed653", "class_name": "RelatedNodeInfo"}}, "text": "up to species \n\nlevel, even if it is not possible to perform antifungal \n\nsusceptibility testing [9]. Further the isolation of the \n\nCandida spp. from the clinical samples like sputum, \n\nurine, vaginal swab etc. does not necessarily suggest \n\nthe infection and clinical correlation or confirmation \n\nof infection by alternative methods is necessary. \n\nHowever, the high rate of isolation of Candida spp. \n\nfrom clinical specimens (mainly from debilitated \n\npatients) suggests the possibility of high rate of \n\nendogenous infections by these organisms. In Nepal \n\nthere are limited data regarding the rate of isolation \n\nof non-albicans Candida from different clinical \n\nspecimens. So, in this study we determined the", "start_char_idx": 3605, "end_char_idx": 4321, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ce989b95-728a-429e-859c-f9bb445eda6d": {"__data__": {"id_": "ce989b95-728a-429e-859c-f9bb445eda6d", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "49bd0bc9-54ce-4aed-bd13-af6d6e7e1424", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "b3d8be08a19d6cc447929aa28bc481294e35f1b30fc3e02a25bc6711bade41ca", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "500a196c-2f8a-411f-b386-61dab85cacbf", "node_type": "1", "metadata": {}, "hash": "b665180b5b73825aa062d30aba5af354a617a9c62fc54446aa539ffbdfa29521", "class_name": "RelatedNodeInfo"}}, "text": "So, in this study we determined the \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 43-46  Sharma et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal    44                Nepjol.info/index.php/njb \n\n \n\nprevalence of non-albicans Candida among the \n\npatients attending a tertiary care hospital. \n\nMaterials and Methods \nCandida spp. isolated from different clinical samples \n\n(sputum, urine, vaginal swab, blood, endotracheal \n\nsecretion, pus) from 250 patients between the period \n\nof February 2013 and December 2015, were included \n\nin the study. Of those 250 patients, 20% were \n\nimmunocompromised. The clinical specimens were \n\ncollected using standard techniques [10]. For the \n\nisolation of the Candida spp. the samples were \n\ninoculated on sabouraud dextrose agar and were \n\nincubated aerobically at 35oC for 48 hrs. Easily \n\nemulsifiable, white, opaque, dome or flat shaped \n\ncolonies were subjected to further identification by \n\nGram\u2019s stain and India ink preparation. The Candida \n\nspp. were identified up to species level by using the \n\nfollowing methods: \n\nGerm tube test: This test was used for the \n\npreliminary identification of the Candida albicans. A \n\nsmall inoculum of the yeast cells from pure culture \n\nwas suspended in 0.5 ml of sheep serum and was \n\nincubated at 37oC for three hours. Then a drop of the \n\nincubated serum was observed under microscope \n\nusing 40X objective. The isolates were identified as \n\ngerm tube producing or germ tube non-producing. \n\nTemperature tolerance: This test was also used for \n\nthe presumptive identification for Candida albicans. \n\nThe isolates were cultured on sabouraud dextrose \n\nagar and incubated aerobically at 45\u00b0C for 72 hours \n\nand observed for any growth if present. \n\nCHROMagar Candida: CHROMagar Candida \n\n(HiMedia, Mumbai, India) was used for presumptive \n\nidentification of various Candida species. The pure \n\nculture was seeded into CHROMagar media and \n\nincubated at 35\u00b0C for 48 hours. The media was \n\nobserved for characteristic color change.  \n\nChlamydospore production: It was used for the \n\npreliminary confirmatory identification of Candida \n\nalbicans. The isolates were inoculated on corn meal \n\nagar by slide culture technique and incubated at 25\u00b0C \n\nfor 72 hours and observed for chlamydospore \n\nproduction using lactophenol cotton blue stain. \n\nSugar fermentation test: Six percent solution of \n\ndextrose, maltose, lactose and sucrose with basal \n\nmedia were used for the test. \n\nSugar assimilation test: Sugars used for this test were \n\nglucose, lactose, maltose, sucrose and galactose. \n\nEthics statement: Our study was in compliance with \n\nHelsinki declaration. \n\nResults \nBetween February 2013 and December 2015, 300 \n\nCandida spp. were isolated from various clinical \n\nsamples. Majority of the Candida spp. were isolated \n\nfrom sputum (43.33%) followed by urine (40%) and \n\nvaginal swab (6.67%) (Figure 1). \n\n \n\nFigure 1: Sample wise distribution of Candida spp. \n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\n140\n\nNo. of samples\n\n130\n120\n\n20 15\n10 5\n\nTable 1: Candida spp. isolated from different clinical samples. \n\nCandida spp. Sputum Urine Vaginal \nswab \n\nET secretion Blood Pus Total \n\nC. albicans 65 56 10 8 6 4 149 \n\nC. tropicalis 43 40 6 2 2 1 94 \n\nC. glabrata 10 16 3 5 2 _ 36 \n\nC. kefyr 7 6 _ _ _ _ 13 \n\nC. krusei 3 _ 1 _ _ _ 4 \nC. dubliniensis 2 2 _ _ _ _ 4 \n\nTotal 130 120 20 15 10 5 300", "start_char_idx": 4286, "end_char_idx": 7679, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "500a196c-2f8a-411f-b386-61dab85cacbf": {"__data__": {"id_": "500a196c-2f8a-411f-b386-61dab85cacbf", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ce989b95-728a-429e-859c-f9bb445eda6d", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "6088bbf7e5e3f338aa7fd63064d4ab585931f64a7209997705548439403d6dd3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "58516d72-af83-46a0-a130-9dcaff595ca0", "node_type": "1", "metadata": {}, "hash": "5f28274f0008641dcc81bcd999d81aafec0884e64553c8d15d01eacb264c382b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 43-46  Sharma et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal    45                Nepjol.info/index.php/njb \n\n \n\nAmong total 300 Candida spp. isolated, 149 (49.67%) \n\nwere Candida albicans and 151 (50.33%) were non-\n\nalbicans Candida. Out of 151 non-albicans Candida \n\nspp., C. tropicalis (62.25%) was the most common non-\n\nalbicans Candida followed by C. glabrata (23.84%) \n\n(Table 1). \n\nDiscussion \nIn spite of their ubiquitous presence, only a few fungi \n\nare considered pathogenic. Skin, nail and hair are the \n\ncommonest sites of fungal infections. But recently \n\nthere have been increased numbers of systemic fungal \n\ninfections due to the use of broad spectrum \n\nantibiotics, increase in the numbers of \n\nimmunocompromised persons (due to conditions like \n\nlymphomas, leukemia, organ transplantation, human \n\nimmunodeficiency virus (HIV) infection and use of \n\nimmunosuppressive drugs) [11]. Among the fungal \n\npathogens, Candida is a leading cause of a variety of \n\nhuman infections [2]. \n\nIn our study the rate of isolation of non-albicans \n\nCandida (among all Candida spp.) was > 50%; which \n\nwas in accordance to the result reported by \n\nDeorukhkar et al. (63.3%) [2]. In the present study, \n\namong total 130 Candida spp. isolated from sputum, \n\nthe rate of isolation of C. albicans was 50% followed \n\nby C. tropicalis (33.076%) and C. glabrata (7.69%). \n\nSimilar to our observation, in a study by Jha et al., the \n\ncommonest species of Candida isolated from sputum \n\nwas found to be C. albicans (70%) followed by Candida \n\ntropicalis (13.33%) [3]. However, the isolation of the \n\nCandida spp. from sputum in our study did not \n\nnecessarily suggest the infection and clinical \n\ncorrelation or confirmation of infection by alternative \n\nmethod was necessary. But the isolation of the \n\nCandida spp. from the immunocompromised people \n\n(having predisposing risk factors) suggests the higher \n\nrisk of endogenous infection. The main risk factors \n\nresponsible for increased numbers of respiratory tract \n\ninfections by Candida spp. are smoking, chronic \n\nobstructive pulmonary disease, tuberculosis, \n\nmalnutrition, malignancy, diabetes mellitus, human \n\nimmunodeficiency virus infection and prolonged use \n\nof antibiotics [3].  \n\nIn urine samples, the majority of Candida spp. isolated \n\nwere C. albicans (46.67%) followed by C. tropicalis \n\n(33.3%) and C. glabrata (19.2%). Among all Candida \n\nspp. the rate of isolation of non-albicans Candida from \n\nurine was 53.33%. Our finding was in accordance \n\nwith the findings of Deorukhkar et al.  [2] and \n\nKauffmann [12], in which >50% of the Candida spp. \n\nisolated from urine samples were non-albicans \n\nCandida.  Along with the well adaptability of the non-\n\nalbicans Candida spp. for urinary tract infection, it is \n\nmore difficult to treat the infection caused by them in \n\ncomparison to that caused by C. albicans [2]. Old age, \n\ndiabetes mellitus, pregnancy and urinary \n\ncatheterization are the predisposing factor for urinary \n\ntract infection by Candida spp.  \n\nIn a study by Helmy, 14 % of the cases of \n\nvulvovaginal candidiasis were due to non-albicans \n\nCandida [8] but in our study 50% of the Candida spp. \n\nisolated from vaginal swab were non-albicans \n\nCandida.", "start_char_idx": 7684, "end_char_idx": 10989, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "58516d72-af83-46a0-a130-9dcaff595ca0": {"__data__": {"id_": "58516d72-af83-46a0-a130-9dcaff595ca0", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "500a196c-2f8a-411f-b386-61dab85cacbf", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "43175734f82191c6b61b552072321fc4b4eace564e534f494335c88336a251e9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "52218fe2-27c1-46e2-b2ad-e9e1b0eacafb", "node_type": "1", "metadata": {}, "hash": "a94604a357b4751309fa171b44e55a9d2b7e7e462870de4874659dc8ad240460", "class_name": "RelatedNodeInfo"}}, "text": "isolated from vaginal swab were non-albicans \n\nCandida. However, in both studies the C. tropicalis \n\nfollowed by C. glabrata were the most common \n\nspecies of Candida   isolated from vaginal swab. In the \n\nstudy done by Deorukhkar et al. the predominant \n\nCandida spp. responsible for causing vulvovaginal \n\ncandidiasis were found to be C. glabrata followed \n\nby C. tropicalis [2]. \n\nAlthough, the isolation of the Candida spp. from urine \n\nand vaginal swab does not necessarily suggest the \n\ninfection; the high rate of isolation of these organisms \n\nfrom patients having risk factors for infection by \n\nCandida spp. could not be neglected. In our study 20 \n\n% of the patients were immunocompromised.  \n\nAll the patients from whom Candida spp. were \n\nisolated from endotracheal secretion were from \n\nintensive care unit and were intubated. And non-\n\nalbicans Candida isolated from their endotracheal \n\nsecretion were 43.75% of all the Candida spp. isolated \n\nfrom endotracheal secretion. This indicates that the \n\nintubation is a very important risk factor for \n\nacquiring yeast infection. \n\nIn our study, 40% of the non-albicans Candida were \n\nisolated from the cases of candidemia. The \n\npredominant non-albicans Candida isolated from \n\nblood were C. glabrata followed C. tropicalis. Among \n\nall Candida spp., we found C. albicans to be the \n\nmost predominant cause of candidemia. But in \n\nthe study by Deorukhkar et al. C. glabrata was the \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 43-46  Sharma et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal    46                Nepjol.info/index.php/njb \n\n \n\ncommonest species isolated from the cases of \n\ncandidemia [2]. Despite the isolation of C. glabrata \n\n(mortality rate due to infection by which is higher) \n\nfrom only 20% of the cases of the candidemia, in our \n\nstudy the mortality rate for the cases of candidemia \n\nwas 80% [2]. \n\nIn our study rate of isolation of Candida spp. was \n\nhigher among patients from intensive care unit and \n\nthose having history of treatment with broad \n\nspectrum antibiotics (for long period of time). The use \n\nof broad spectrum antibiotics upsets the balance of \n\nthe normal bacterial flora and results in infection by \n\nCandida spp.  [2]. Further the patients from intensive \n\ncare unit are debilitated. \n\nDue to adaptation of the Candida spp. to various \n\nhabitats including medical devices, the incidence of \n\nhospital acquired infection due to Candida spp. has \n\nincreased [2]. Candida spp. have ability to form \n\nbiofilm and in the study by Deorukhkar et al. the \n\nbiofilm forming capability has been found to be \n\nhigher in C. tropicalis as compared to C. albicans. This \n\nattribute can help non-albicans Candida like C. \n\ntropicalis to show higher drug resistance [2]. Further, \n\nsome non-albicans Candida are intrinsically resistant \n\nto some antifungals mainly in context of \n\nopportunistic infections in immunocompromised [4]. \n\nConclusion \nHigh prevalence of non-albicans Candida among the \n\npatients attending a hospital in Kathmandu, Nepal \n\nwas noted. From our study, it can be concluded that \n\nnon-albicans Candida may be of high clinical \n\nsignificance mainly in case of the patients with \n\npredisposing risk factors. \n\nAuthor\u2019s Contribution  \nNDP and MS designed the study, performed the \n\nresearch work, analysed the data and prepared the \n\nfinal manuscript. PP contributed in analyzing of the \n\ndata. \n\nCompeting Interests  \nThe authors declare that they have no competing \n\ninterests. \n\nAcknowledgement  \nThe authors would like to thank all who contributed \n\ndirectly or indirectly in carrying out of this research. \n\nReferences  \n1.", "start_char_idx": 10934, "end_char_idx": 14602, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "52218fe2-27c1-46e2-b2ad-e9e1b0eacafb": {"__data__": {"id_": "52218fe2-27c1-46e2-b2ad-e9e1b0eacafb", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "58516d72-af83-46a0-a130-9dcaff595ca0", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "bff85bc297cf5aa03fad7470a109d429131b31cb63220ab67c05ed4ec802d179", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7bf8e78d-9dda-4f5f-87b8-d4455f2ddace", "node_type": "1", "metadata": {}, "hash": "874ea702b0074165fa479520d457d2678304d200e41cb1e378c95b39d86f8c31", "class_name": "RelatedNodeInfo"}}, "text": "References  \n1. Agrawal V, Bhagwat AM, Vishalakshi V, Gode V, Sawant CS: \n\nExploring the potential of chromogenic medium for the \n\nidentification of medically important yeast species other \n\nthan candida. Int J Pharm Pharm Sci. 2014, 6(3):291-294. \n\n2. Deorukhkar SC, Saini S, Mathew S. Non-albicans Candida \n\nInfection: An Emerging Threat. Interdiscip Perspect Infect \n\nDis. 2014; 2014:615958. \n\n3. Jha BK, Dey S, Tamang MD, Joshy ME, Shivananda PG, \n\nBrahmadatan KN: Characterization of candida species \n\nisolated from cases of lower respiratory tract infection. \n\nKUMJ. 2006, 4(3):290-294. \n\n4. Kangogo MC, Wanyoike MW, Revathi G, Bii CC: Phenotypic \n\ncharacterization of Candida albicans from clinical sources in \n\nNairobi, Kenya. Afr J Health Sci. 2011, 19:21-25. \n\n5. Sheth CC, Johnson E, Baker ME, Haynes K, M\u00fchlschlegel FA: \n\nPhenotypic identification of Candida albicans by growth on \n\nchocolate agar. Med Mycol. 2005, 43(8):735-738. \n\n6. Sharma RK, Chaudhary SK, Srinivasa H: Speciation of \n\nCandida Isolated in Significant Count from Urine Samples. \n\nJGMC. 2014, 7(2):23-26. \n\n7.  Raju SB, Rajappa S: Isolation and Identification \n\nof Candida from the Oral Cavity. ISRN Dent. 2011, \n\n2011:487921. \n\n8. Helmy MM: Phenotypic analysis of Candida Species \n\nAssociated with Vulvovaginal Candidiasis. Egypt J Med \n\nMicrobiol. 2012, 21(1):109-115. \n\n9. Cendejas-Bueno E, Gomez-Lopez A, Mellado E, Rodriguez-\n\nTudela JL: Cuenca-Estrella M. Identification of pathogenic \nrare yeast species in clinical samples: comparison between \n\nphenotypical and molecular methods. J Clin Microbiol. 2010, \n\n48(5): 1895-1899. \n\n10. Engbaeck K et al: Specimen collection and transport for \n\nmicrobiological investigation. WHO Regional Publications, \n\n1995. WHO Regional Office for the Eastern Mediterranean, PO \n\nBox 7608, Nasr City, Cairo, 11371, Egypt. ISBN 92\u20139021\u2013196\u20132. \n\n11. Kannan P, Janaki C, Selvi GS: Prevalence of dermatophytes \n\nand other fungal agents isolated from clinical samples. \n\nIndian J Med Microbiol. 2006, 24 (3):212-215.  \n\n12. Kauffman CA: Candiduria. Clin Infect Dis. 2005, 41(6):S371\u2013\n\nS376.", "start_char_idx": 14587, "end_char_idx": 16692, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7bf8e78d-9dda-4f5f-87b8-d4455f2ddace": {"__data__": {"id_": "7bf8e78d-9dda-4f5f-87b8-d4455f2ddace", "embedding": null, "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-23", "node_type": "4", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "814f7a121bdb249b0881a65d641934b575f6972c8247d78124c9424d38996711", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "52218fe2-27c1-46e2-b2ad-e9e1b0eacafb", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "69a620f38741f721bc5b8912ec9c28cef8bc8b10431fba692ebba19bb2557360", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5a0e0aa4-ab62-4f08-9b19-82f66f764125", "node_type": "1", "metadata": {}, "hash": "107363b6f64ecaf6d9b65671c2e94e3db87416c0a7a738a7a8cfc5299610e5e4", "class_name": "RelatedNodeInfo"}}, "text": "http://www.ncbi.nlm.nih.gov/pubmed/?term=Saini%20S%5BAuthor%5D&cauthor=true&cauthor_uid=25404942\nhttp://www.ncbi.nlm.nih.gov/pubmed/25404942\nhttp://www.ncbi.nlm.nih.gov/pubmed/25404942\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Sheth%20CC%5BAuthor%5D&cauthor=true&cauthor_uid=16422305\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Johnson%20E%5BAuthor%5D&cauthor=true&cauthor_uid=16422305\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Baker%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=16422305\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Haynes%20K%5BAuthor%5D&cauthor=true&cauthor_uid=16422305\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=M%C3%BChlschlegel%20FA%5BAuthor%5D&cauthor=true&cauthor_uid=16422305\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Phenotypic+identification+of+Candida+albicans+by+growth+on+chocolate+agar\nhttp://www.hindawi.com/13852918/\nhttp://www.hindawi.com/27615462/\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Isolation+and+Identification+of+Candida+from+the+Oral+Cavity+%2CInternational+Scholarly+Research+Notices\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Cendejas-Bueno%20E%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Gomez-Lopez%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Mellado%20E%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Rodriguez-Tudela%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Rodriguez-Tudela%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Cuenca-Estrella%20M%5BAuthor%5D&cauthor=true&cauthor_uid=20237094\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Identification+of+Pathogenic+Rare+Yeast+Species+in+Clinical+Samples%3A+Comparison+between+Phenotypical+and+Molecular+Methods%E2%96%BF", "start_char_idx": 16722, "end_char_idx": 18544, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5a0e0aa4-ab62-4f08-9b19-82f66f764125": {"__data__": {"id_": "5a0e0aa4-ab62-4f08-9b19-82f66f764125", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7bf8e78d-9dda-4f5f-87b8-d4455f2ddace", "node_type": "1", "metadata": {"identifier": "njb-23", "author": "Sharma, Manisha; Pant, Narayan Dutt; Pandey, Pratikshya", "title": "Prevalence of Non-Albicans Candida Among the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal", "date": "2016-12-31", "file": "njb-23.pdf"}, "hash": "e57815b60ba3a97cafbe7d6df3b750d3e7a647c6dcebfd618264e9db20e77aa5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3ac9c24a-6f09-4bf6-9ba7-ac292f931f98", "node_type": "1", "metadata": {}, "hash": "dc0497cca48e862101f6204af747ccc757fbb50922162c0651e248012ed4e134", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0  (2): 45-51 Research article DOI: https://doi.org/10.54796/njb.v10i2.239 \n\n\u00a9NJB, BSN 45 \n\nAssessment of Phytochemicals, Antioxidant Activity, Total Phenolic \nand Flavonoid Contents of Selected Nepalese Medicinal Plants \nDeepak Kumar Shrestha  , Basanta Kumar Sapkota , Krishna Prasad Sharma  \n\nTribhuvan University, Department of Chemistry, Butwal Multiple Campus, Butwal   \n\nReceived: 31 Aug 2022; Revised: 24 Dec 2022; Accepted: 25 Dec 2022; Published online: 31 Dec 2022 \n\nAbstract \nSeveral medicinal plants have been used from the traditional period of times to cure different diseases but there is little \nscientific evidence. The phytochemicals of plants can reduce cardiovascular and other diseases. The present study analyzed \nthe five different medicinal plants of the Gulmi and Rupandehi districts of Nepal using in vitro studies. They were Crateva \nUnilocularis, Aegle marmelos, Nyctanthes arbor-tristis, Urtica dioica and Justicia adhatoda. The antioxidant potential of the \nmethanolic extract of plants was evaluated by DPPH radical scavenging assay, total phenolic content was determined by \nusing the Folin-Ciocalteu method and total flavonoid content was determined by using the Aluminium chloride colorimetric \nmethod. Results revealed that the methanolic extract of plants contained phytochemicals such as alkaloids, flavonoids, \npolyphenols, saponins, quinones, terpenoids, etc. The extract of Nyctanthes arbor-tristi showed the highest % of radical \nscavenging activity up to 64.931\u00b10.032% with an IC50 value of 70.506\u00b11.55\u00b5g/ml followed by Aegle marmelos and the lowest \nin Urtica dioica. Nyctanthes arbor-tristis revealed the highest TPC (97.647\u00b17.01mgGAE/g) and lowest in Urtica dioica. Crateva \nunilocularis had  the highest TFC 31.99\u00b12.345mgQE/g and followed by Nyctanthes arbortristis and lowest in Justicia adhatoda. \nThese parameters were analyzed from the period 5 September 2021 to 10 October 2021. \n\nKeywords: Antioxidant, Flavonoid, Phenolic compound, 2,2-Diphenyl-1-picrylhydrazyl. \n\n Corresponding author, email: shresthadeepak854@gmail.com \n\nIntroduction \nNepal is situated in South Asia. It is a land-locked \n\ncountry that occupies 0.03 % and 0.3% land area of the \n\nWorld and Asia respectively and has total area 1,47,181 \n\nsq. km [1,2]. Nepal is ranked 25th and 11th positions in \n\nbiodiversity richness in the world and Asia, respectively. \n\nNepal is enriched with several climatic conditions, \n\ngeographical variations, and an immense variety of \n\nmedicinal plants has contributed about 10% medicinal \n\nplants of the expected 7000 species of flowering plants \n\n[2,3]. Gulmi district, a part of province no. 5 of Nepal, \n\nwhich altitude ranges from 465 m to 2690 m [4]. \n\nRupandehi District is also part of Lumbini Province, its \n\naltitude ranges from 100m to 1229m from sea level. \n\nNatural products are the major sources of new drug \n\ndiscovery. About 90% of Nepalese populations, residing \n\nin rural areas are still using medicinal plants for their \n\nprimary health care. Despite the widespread use of \n\nmedicinal plants in Nepal, there are limited studies on \n\nphytoconstituents and their antioxidant activity [2]. \n\nSynthetic drugs in the long run, show harmful side effects \n\nand they are expensive too.", "start_char_idx": 48, "end_char_idx": 3336, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3ac9c24a-6f09-4bf6-9ba7-ac292f931f98": {"__data__": {"id_": "3ac9c24a-6f09-4bf6-9ba7-ac292f931f98", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5a0e0aa4-ab62-4f08-9b19-82f66f764125", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "2b9f051573b9f580f78e576566b148e14c9f4c1d9f042629052a36e26ff4ad38", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7a25cccf-0a05-41e1-91d6-d8717e16b566", "node_type": "1", "metadata": {}, "hash": "9530b9d145a4017d6bf58a7b4e76ccea15e74eed7a279df312adbee2545d9e06", "class_name": "RelatedNodeInfo"}}, "text": "Synthetic drugs in the long run, show harmful side effects \n\nand they are expensive too. Hence, drug development \n\nfrom the natural product is a promising field. Therefore, \n\nit is urgent to identify phytochemicals and explore the \n\nantioxidant with a quantitative estimation of flavonoid \n\nand phenolic content in the natural resources of Nepal. \n\nHence, the present study mainly focused on medicinal \n\nplants found in Gulmi and Rupandehi districts. The \n\noxidative stress inside body produces chronic diseases \n\nsuch as diabetes, heart disease, and cancer [5]. In \n\noxidative stress, the balance between the formation of \n\nreactive oxygen species (ROS) and the amount of \n\nantioxidants in the body is destroyed which causes \n\ndamage to cell components such as proteins, lipids, and \n\nnucleic acid and eventually leads to cell death [6]. ROS \n\nand reactive nitrogen species (RNS) are the main sources \n\nof free radicals which lead to serious disorders such as \n\nAlzheimer's disease, Parkinson's disease, and Strokes [7]. \n\nAlthough NO and superoxide radicals are involved in \n\nhost defense, overproduction of these two radicals \n\ncontributes to the pathogenesis of inflammatory diseases \n\n[8]. Antioxidants are compounds that hinder oxidative \n\nprocesses and thereby delay or prevent oxidative stress \n\n[9]. Increasing intake of antioxidants can prevent diseases \n\nand lower health problems. In the present context, many \n\nantioxidants are manufactured synthetically such as \n\nbutylated hydroxyanisole (BHA), butylated \n\nhydroxytoluene (BHT), tertiary butylated \n\nhydroxyquinone (TBHQ), and gallic acid ester. These \n\nsynthetically produced antioxidants possess certain side \n\neffects and toxic effects. Natural products, mainly \n\nobtained from dietary sources provide a large number of \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-4185-0103\nmailto:shresthadeepak854@gmail.com\nhttps://orcid.org/0000-0002-9085-539X\nhttps://orcid.org/0000-0003-3071-4648\nmailto:shresthadeepak854@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    46 \n\nantioxidants that decreases oxidative injury [1]. Plants \n\ncontain many phytochemicals that are useful sources of \n\nnatural antioxidants, such as phenolic diterpenes, \n\nflavonoids, tannins, alkaloids and phenolic acids etc [10]. \n\nPolyphenols are the strong antioxidant in plant extracts \n\n[11]. Flavonoids are highly effective scavengers of most \n\noxidizing molecules, including singlet oxygen and \n\nvarious free radicals. As per [12] by GC-MS analysis for \n\ndifferent extract of bark of      Crateva religosa suggest that \n\nthis plant has vast variety of photochemical which can be \n\nused for various ailments and drug formulations in \n\nfuture. The ethnic claims of this plant were also verified \n\nby the present study. As per  [13] the phytochemical \n\nanalysis of methanolic extracts of all nine medicinal \n\nplants of Kavre district, displayed the presence of various \n\nsecondary metabolites such as alkaloids, flavonoids, \n\npolyphenols, saponins, and quinones [13]. The \n\nmethanolic extract of S. pinnata showed the highest \n\npercentage of radical scavenging activity up to 87.94\u00b11.88 \n\nwith 50% inhibitory concentration (IC50) 17.51\u00b11.27 \n\n\u03bcg/mL [13]. Moreover, S. pinnata displayed the highest \n\ntotal phenolic content (TPC) 48.26\u00b11.23 mg GAE/g while \n\nthe highest flavonoid content was displayed by Melia \n\nazedarach 41.07\u00b11.53 mg QE/g [13],[14].", "start_char_idx": 3248, "end_char_idx": 6847, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7a25cccf-0a05-41e1-91d6-d8717e16b566": {"__data__": {"id_": "7a25cccf-0a05-41e1-91d6-d8717e16b566", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3ac9c24a-6f09-4bf6-9ba7-ac292f931f98", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "8ca1acc6d4fbff2d77595acd383540f7d0a81f33aaf8192880fcb3ac88c6a058", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "916a8fef-af11-42f4-bb1b-348b2262e1e7", "node_type": "1", "metadata": {}, "hash": "4d2f294dd3eea0546b2fc3edb3910b39f7a2a5127d7cb4c3ebf84613e3c6a9ca", "class_name": "RelatedNodeInfo"}}, "text": "As per [15] \n\nbioactive properties were found strongly on 4 selected \n\ntraditionally used medicinal plants with strong enzyme \n\ninhibition potential where \u03b1-Glucosidase and \u03b1-amylase \n\ninhibitory activities were investigated using in vitro \n\nmodel followed up by antioxidant and antimicrobial \n\nactivities. The study showed that ethyl acetate fraction of \n\nMelastoma melabathrium (IC50 9.1 \u00b1 0.3 \u00b5g/mL) and water \n\nfraction Acacia catechu (IC50 9.0 \u00b1 0.6 \u00b5g/mL) exhibit \n\nstrong \u03b1-glucosidase inhibition. Furthermore, to identify \n\nthe metabolites within the fractions, they employed high-\n\nresolution mass spectrometry (LC-HRMS) and annotated \n\n17 known metabolites [15]. As per [16] hydro-alcoholic \n\nextract of Urtica dioica shows positive results for \n\nantioxidant activity with IC50 value of 88.33 \u00b1 2.88 \u00b5g/ml \n\nwhile standard ascorbic acid showed IC50 value of 2.8 \u00b1 \n\n0.62 \u00b5g/ml [13]. \n\nExtract of Nyctanthes arbor-tristis exhibited various \n\npharmacological activities like hepatoprotective, \n\nantileishmanial, antipyretic, antihistaminic, antimalarial, \n\nantibacterial, anti-inflammatory, antioxidant, antiviral, \n\nand antifungal etc. because of the presence of glycosides \n\nand phenolic compounds [17]. Gnidia glauca and D. \n\nbulbifera contained significant amounts of \n\nphytochemicals with antioxidative properties [18]. \n\nSimilarly, the highest radical scavenging activity of \n\nDioscorea bulbifera fruit powder was found in vacuum \n\ndrying ranged from 65.36% to 81.33% of the \n\nconcentration of 200 \u03bcg/ml to1000 \u03bcg/ml, [19]. In vitro \n\nantioxidant activity of the Aegle marmelos plant extract \n\nrevealed that both the extracts showed good antioxidant \n\npower with IC50 value ranges of 37.11\u00b13.50 to \n\n158.99\u00b159.46 \u00b5g/ml for aqueous extract and 35.02\u00b18.10 to \n\n283.06 \u00b1 135.80\u00b5g/ml and 35.02\u00b18.10 to 283.06 \u00b1 \n\n135.80\u00b5g/ml for alcoholic extract [20]. Polyphenols, \n\nespecially flavonoids are the strong antioxidant in plant \n\nextracts [21]. Different extract of Justicia adhatoda by ultra \n\nUHPLC analysis revealed the presence of polyphenolic \n\ncompounds and flavonoids which might be responsible \n\nfor bioprotective activity. Among the five fractions \n\n(hexane, chloroform, ethyl acetate, n-butanol and \n\naqueous), n-butanol and ethyl acetate exhibited \n\nsignificant antioxidant activity with minimum IC50 value \n\n(< 105.33 \u00b5g/ml) [22].  The present study was to analyze \n\nsecondary metabolites, total phenolic, flavonoid \n\ncontents, and antioxidant activity of five different \n\nmedicinal plants. It helps to find scientific pieces of \n\nevidence of the medicinal value of plants and also help to \n\nfurther investigation.  \n\nMethods and materials \nAssembling and identifying plant samples \nDifferent parts of five different selected medicinal plants \n\nCrateva unilocularis from Gulmi district and other four \n\nplants viz Aegle marmelos, Nyctanthes arbotristis \n\n,Urticadiocia, Justicia adhatoda were collected from \n\ndifferent region of Rupandehi (Table 1). They were \n\ncollected in the period of 1 march 2021 to 2 April 2021. \n\nBotanist Dr. Ananta Gopal Singh of BMC identified the \n\nplants. \n\nStatistical evaluation \nThere were three copies of each experiment run. The \n\nmean \u00b1 standard deviation (SD) was used to express the \n\nresults. Microsoft Excel 2016 was used for all statistical \n\nanalysis. The IC50 values was calculated by using \n\nMicrosoft Excel 2016. \n\nExtract preparation after drying \nThe plant's raw fruits and leaves were gathered locally \n\nand processed.", "start_char_idx": 6848, "end_char_idx": 10334, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "916a8fef-af11-42f4-bb1b-348b2262e1e7": {"__data__": {"id_": "916a8fef-af11-42f4-bb1b-348b2262e1e7", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7a25cccf-0a05-41e1-91d6-d8717e16b566", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "efe5e14445c44a1c52c128a52e61ab40d2905e7331deebc7e1b19bde2c2043e7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e40c5046-fad6-4bfb-92c6-417cfd29d247", "node_type": "1", "metadata": {}, "hash": "e2ff93ba0496f7bb8ce47619b25f295d19c5c3121b20454bb0298a5da07fb7ac", "class_name": "RelatedNodeInfo"}}, "text": "Extract preparation after drying \nThe plant's raw fruits and leaves were gathered locally \n\nand processed. The components of the gathered plant \n\nwere cleaned, then dried at room temperature in the \n\nTable 1: Chosen medicinal plants list \n\nName of plants Local Name  Utilized Parts  Family \n\nAegle marmelos       Bel  Raw fruits Rutaceae \n\nNyctanthes arbor-tristis      Parijat Leaves Oleaceae \n\nUrtica dioica       Sisnu Leaves Urticaceae \n\nCrateva unilocularis     Siplikan Buds and leaves Capparaceae \n\nJusticia adhatoda       Asuro leaves Acanthaceae \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    47 \n\nshade. They were ground into a powder using a \n\nmechanical grinder, stored at a low temperature in a \n\nclean plastic bag. By using the cold percolation process, \n\nextract was created. 400 ml of methanol and 150 g of dried \n\npowdered components were combined in separate, clean, \n\ndry conical flasks. Extraction was carried out in a flask \n\nthat was firmly sealed for 72 hours while being frequently \n\nshaken and filtered. The resulting residue was once more \n\nsteeped in methanol. Until the sample's methanol became \n\ncolorless, the procedure was repeated. The rotary \n\nevaporator was used to concentrate the thusly produced \n\nfiltrate. Different plants' solid methanolic extracts were \n\nprepared and kept in a refrigerator at 4\u00b0C until analysis \n\n[13]. \n\nTable 2. Methods used for data analysis \n\nParameters Methods employed \n\nAntioxidant activity DPPH radical Scavenging assay \n\nTotal Phenolic content  Folin-Ciocalteu method \n\nTotal Flavonoid \ncontent \n\nAluminium chloride \ncolorimetric method \n\nUV visible \nSpetrophotometer \n\n Aczel Pvt. Ltd, Model no: \nAUV-8S ID: 210702206 \n\nChemicals and reagents needed \nAnalytical-grade chemicals were employed throughout. \n\nFehling's solution, alpha-Naphthol, FeCl3, NaHCO3, \n\nBi(NO3)3, KI, HgCl2, Picric acid, dimethyl sulfide, sodium \n\nhydrogen phosphate, sodium carbonate, sodium \n\nchloride, sodium hydroxide, sodium nitroprusside, and \n\ndistilled water are a few examples of substances that fall \n\nunder this category. The 2,2-diphenyl-1-picrylhydrazyl \n\n(DPPH), gallic acid, quercetin, and the Folin-Ciocalteu \n\nreagent were bought from Qualigens Fine Chemicals in \n\nIndia. Meyer's reagent, Dragendorff's reagent, Molisch's \n\nreagent, and other reagents and solvents used in \n\nphytochemical analysis were made in the lab using \n\nchemicals of the laboratory reagent grade. \n\nRequired apparatus \nHot air oven, mechanical grinder, digital weighing \n\nbalance, cuvettes, burettes, pipettes, micropipettes, \n\nthermometer, condensers, beakers, conical flasks, test \n\ntubes, reagents bottles, stands, vial tubes, round bottom \n\nflasks, Rotary evaporator with water bath was used for \n\nthe evaporation of solvents. Using a UV-visible \n\nspectrophotometer, absorbance for the DPPH assay and \n\nabsorbance for total phenolic and flavonoid content were \n\ndetermined. \n\nPhytochemical analysis \nThe method used for phytochemical screening was based \n\non protocol put forward as per [23]. Basically \n\nphytochemical screening helps to identify secondary \n\nmetabolites (bioactive compounds) present in plants. The \n\nanalysis was done by the color reaction using different \n\nspecific reagents [24]. The qualitative results are \n\nexpressed as (+) for the presence and (\u2212) for the absence \n\nof phytochemicals.", "start_char_idx": 10228, "end_char_idx": 13615, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e40c5046-fad6-4bfb-92c6-417cfd29d247": {"__data__": {"id_": "e40c5046-fad6-4bfb-92c6-417cfd29d247", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "916a8fef-af11-42f4-bb1b-348b2262e1e7", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "cc249e4bb36a65e267c8d8fe14675e28f5251e1f4d52dbb6e85888ddc22dbd7a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0a490c06-7771-4de1-b5d0-461bbae11b6c", "node_type": "1", "metadata": {}, "hash": "157456aadd5b35f78137740de39f7e67b856edd65faa6c213614b4f7352970e0", "class_name": "RelatedNodeInfo"}}, "text": "The phytochemical screening was \n\ncarried out to test for Alkaloids (Meyer\u2019s test, \n\nDragendorff\u2019s test), Coumarins, Flavonoids, Quinones, \n\nPolyphenols, Glycosides, Reducing sugar, Saponins, \n\nTannins, and carbohydrates (Molish\u2019s test) etc. \n\nrespectively \n\nAntioxidant activity measurement \n\nDPPH radical scavenging activity \nThe protocol developed as per [25] and [26] was used to \n\nconduct the 2,2-diphenyl-1-picrylhydrazyl (DPPH) \n\nradical scavenging experiment. This test is a \n\nstraightforward, popular, and widely accepted method \n\nto assess the antioxidant potential of plant extracts. Each \n\nplant sample's extract solution was diluted to a different \n\nconcentration (10 \u03bcg/ml to 100 \u03bcg/ml) and mixed with 2 \n\nml of DPPH solution (60 \u03bcM). For a 30-minute reaction, \n\nthe mixture was left to stand in full darkness. Finally, a \n\nUV spectrophotometer was used to assess each plant \n\nsample's absorbance at 517 nm. Each sample's radical \n\nscavenging activity was determined using the formula \n\nbelow: \n\nRadical scavenging (%) = [(A0 - A1 / A0) \u00d7 100%] \n\nWhere A0 is the absorbance of the control and A1 is the \n\nsample extract's absorbance. The test solution without the \n\nsample is the control. The standard was ascorbic acid. A \n\nsimilar process was used with ascorbic acid solutions that \n\nranged in concentration from 10 to 100 \u03bcg/ml. Each \n\nsample's antioxidant activity was quantified using its IC50 \n\n(the concentration needed to suppress the production of \n\nDPPH radicals by 50%) value. The sample's effective \n\nconcentration was defined as the 50% inhibitory \n\nTable 3. Phytochemical screening of methanolic extract of \ndifferent plant samples. \n\nPhytochemicals A.M N.A U.D C.U J.A \n\nAlkaloids + + + + ++ \nTerpenoids + + + + + \nCoumarins + + + + + \nFlavonoids + + - + ++ \nQuinones + + + + + \nPolyphenols + + - + + \nGlycosides + + - + + \nReducing sugar + + - - - - \nSaponins + + - - - \nTannins + + - + + \nCarbohydrates + + - + - - \n\nA.M= Aegle marmelos, N.A= Nyctanthes arbortristis, U.D= Urtica \ndioica, C.U= Crateva unilocularis, J.A= Justicia adhatoda, and  (-) for \nabsence & (+) for presence. \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    48 \n\nconcentration (IC50) value, which was needed to \n\neffectively scavenge 50% of the DPPH free radicals. By \n\ngraphing the extract concentration versus the \n\ncorresponding scavenging action, the inhibition curve \n\nwas used to determine the IC50 values. \n\nTotal phenolic content determination \nUsing gallic acid as a reference and the oxidation-\n\nreduction reaction as a basis, the total phenolic content of \n\nthe extract was calculated using the Folin-Ciocalteu \n\ntechnique. The method described as per [13] was used to \n\ndetermine the total phenolic content, with a few changes \n\n[5]. 5 ml of 10% Folin-Ciocalteu reagent were mixed with \n\n1 ml of crude extract. 4 ml of 7% (w/v) sodium carbonate \n\nwas mixed and shook after standing for 5 minutes. After \n\n40 minutes of incubation, the mixture's absorbance at 760 \n\nnm was measured. The entire experiment was run in \n\ntriplicate. Gallic acid was used as the standard in the \n\ncreation of the calibration curve.", "start_char_idx": 13616, "end_char_idx": 16784, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0a490c06-7771-4de1-b5d0-461bbae11b6c": {"__data__": {"id_": "0a490c06-7771-4de1-b5d0-461bbae11b6c", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e40c5046-fad6-4bfb-92c6-417cfd29d247", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "0fc0c13ef4795e6c46da959e9cfad6c8b2d04649f0583c2b8dd8dde33cd9c6bd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e7ab06b-bcc8-4386-8bff-8218a9cde9a1", "node_type": "1", "metadata": {}, "hash": "b52d09231c6328167b194f11e270e6c43773c1e55e8a9c66216fc1110829978b", "class_name": "RelatedNodeInfo"}}, "text": "Gallic acid was used as the standard in the \n\ncreation of the calibration curve. The calibration curve \n\nwas used to quantify the total phenolic content, and the \n\nresults were represented as mg of gallic acid equivalent \n\n(GAE) per gram dry weight of extract using formula, \n\nTPC= C\u00d7V/M where V= Volume of extract in ml, \n\nM=Weight of plant extract in mg, and C=Concentration \n\nof Galic Acid Obtained from Calibration Curve in \n\nmg/ml. The gallic acid calibration curve was used to \n\nderive the linear correlation coefficient (R2) value and \n\nregression equation. Each extract's concentration was \n\ndetermined using the regression equation. The TPC was \n\ndetermined using the calculated value of each extract's \n\nconcentration. \n\nTotal flavonoid content determination \nWith a small modification of protocol as per [11] and [27], \n\nthe aluminum chloride colorimetric method was \n\nemployed to determine the flavonoid concentration.   4 \n\nml of distilled water and readily added 0.3 ml of 5% \n\nsodium nitrite were combined with 1 ml of each extract \n\nsolution. 0.3 ml of 10% aluminum chloride was added \n\nafter 5 minutes, and the mixture was let to stand for 6 \n\nminutes. After shaking well, 2 ml of 1 M sodium \n\nhydroxide and 2.4 ml of distilled water were added, \n\nbringing the total volume to 10 ml. The absorbance was \n\nthen measured at 510 nm using a UV spectrophotometer. \n\nThe standard used to create the calibration curve was \n\nquercetin. From the calibration curve, the total flavonoid \n\nconcentration was determined and results were \n\nrepresented as mg of quercetin equivalent (QE) per gram \n\ndry extract weight. \n\nResults  \nExtraction yield value \nThe extract of the selected plant samples was prepared in \n\nmethanolic solvent by cold percolation method. The yield \n\npercent of the plant extract is given in Table 4.  \n\nTable 4: Methanolic extract of different plant samples' yield \npercentages. \n\n  Name of Plants  Yield% of extract \n\n(in methanol) \n\nAegle marmelos 12.42% \n\nNyctanthes arbo-tristis 10.28% \n\nUrtica dioica 11.42% \n\nCrateva unilocularis 10.14% \n\nJusticia adhatoda 12.4% \n\nNote: Extract Yield Percent is equal to [(Weight of Dry \n\nExtract/Weight of Crude Plant Sample)      100%]. \n\nVariations in the scavenging of DPPH radicals \nFigure 1 displays the percentage of DPPH radical \n\nscavenging activity of various samples at various \n\nconcentrations in methanolic solvent, whereas Table 4 \n\ndisplays the IC50 value for the DPPH radical scavenging \n\nactivity. With the least amount of inhibitory \n\nconcentration and the highest DPPH radical scavenging \n\nactivity (IC50) value was found Nyctanthes arbor-tristis \n\n(IC50 value 70.506\u00b11.55 \u00b5g/ml), Aegle marmelos has \n\n99.872\u00b11.27, Justicia adhatoda has103.146\u00b11.33, Crateva \n\nunilocularis has 127.672\u00b12.61 whereas Urtica dioica (IC50 \n\nvalue 179.103\u00b13.58 \u00b5g/ml) Has relatively little DPPH \n\nradical scavenging efficacy in comparison to ascorbic \n\nacid standard (IC50 value \n\n55.40\u00b10.003 \u00b5g/ml).The \n\nantioxidants are the \n\nchemical elements found \n\nin plants that can \n\neffectively quench the \n\nstable purple DPPH \n\nradical, converting it to \n\nthe more visible yellow \n\nDPPH. \n\n \n\n \n\nTable 5.", "start_char_idx": 16704, "end_char_idx": 19865, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e7ab06b-bcc8-4386-8bff-8218a9cde9a1": {"__data__": {"id_": "9e7ab06b-bcc8-4386-8bff-8218a9cde9a1", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0a490c06-7771-4de1-b5d0-461bbae11b6c", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "eb02c09f01f624266f3541b3fc9d4c9556222ddea1b47b22703022fb2f019ef7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "16d3f6b9-572e-4c02-a8fd-c7d189bde8e5", "node_type": "1", "metadata": {}, "hash": "82c22e0028f9fb9ee2c31a13b414715733f6fe5ae9cab5e5dba8f82fcca2ae5e", "class_name": "RelatedNodeInfo"}}, "text": "Table 5. Antioxidant activity of Ascorbic acid \n\nS.N Concentration (ppm) \nAbsorbance \n\nMean \u00b1 SD % Scavenging \nA1 A2 A3 \n\n1 10 0.445 0.441 0.452 0.446 \u00b1 0.005 9.712 \u00b1 0.132 \n\n2 20 0.397 0.401 0.403 0.400 \u00b1 0.003 19.01 \u00b1 0.013 \n\n3 40 0.312 0.322 0.323 0.319 \u00b1 0.006 35.38 \u00b1 0.002 \n\n4 60 0.209 0.208 0.216 0.211 \u00b1 0.004 57.12 \u00b1 0.003 \n\n5 80 0.140 0.148 0.144 0.144 \u00b1 0.004 71.42 \u00b1 0.012 \n\n6 100 0.054 0.057 0.057 0.056 \u00b1 0.001 88.47 \u00b1 0.013 \n\nIC50 55.40 Blank  0.494\u00b10.001 \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    49 \n\n \n\nTable 6. percentage Scavenging (Antioxidant) activity of methanolic extract of different plants {result expressed as the mean \n\u00b1SD (n=3) \n\nPlants \nConcentration \n\n(ppm)  \n10 20 40 60 80 100 \n\nNyctanthes \narbortristis \n\nMean% \nScavenging \n\n5.697\u00b1.089 26.031 \u00b10.12 32.514 \u00b10.33 44.007 \u00b10.45 57.563 \u00b10.33 64.931\u00b10.032 \n\nAegle marmelos \nMean % \n\nScavenging \n1.305\u00b12.3 3.343\u00b11.023 6.534\u00b10.45 14.021\u00b10.12 30.931\u00b10.22 63.033\u00b10.14 \n\nCrateva \nunilocularis \n\nMean % \nScavenging \n\n1.089\u00b11.8 2.008\u00b11.4 4.144\u00b10.45 9.509\u00b10.34 21.034\u00b10.01 48.310\u00b10.22 \n\nUrtica dioica \nMean% \n\nScavenging \n0.495\u00b12.4 1.450\u00b11.44 3.432\u00b10.89 9.098\u00b10.76 19.231\u00b10.33 40.789\u00b10.02 \n\nJusticia \nadhatoda \n\nMean % \nScavenging \n\n2.024\u00b11.34 5.048\u00b10.98 9.09\u00b10.42 14.56\u00b10.56 29.123\u00b10.32 60.756\u00b10.13 \n\nFigure 1. Percentage Scavenging of DPPH free radicals by \nmethanolic extract of plants with reference to ascorbic \nacid, results expressed as the mean \u00b1 standard deviation \n(n=3) at a concentration of 10, 20, 40, 60, 80, and 100 \n\u00b5g/ml. \nTable 7. Ascorbic acid and methanolic extract of several \n\nplant samples' DPPH radical scavenging activity, expressed \n\nin terms of IC50 value. \n\n Name of plant extracts IC50(\u00b5g/mL), mean \u00b1 SD \n\n  1. Ascorbic acid (standard) 55.40 \u00b10.003 \n\n  2. Aegle marmelos 99.872 \u00b11.27 \n\n  5. Nyctanthes arbortristis 70.506 \u00b11.55 \n\n  6. Urtica dioica 179.103 \u00b13.58 \n\n  7. Crateva unilocularis 127.673 \u00b12.61 \n\n  8.", "start_char_idx": 19857, "end_char_idx": 21819, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "16d3f6b9-572e-4c02-a8fd-c7d189bde8e5": {"__data__": {"id_": "16d3f6b9-572e-4c02-a8fd-c7d189bde8e5", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e7ab06b-bcc8-4386-8bff-8218a9cde9a1", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "892831de71890756f2f8dfda2a65a597762f2b7f939dadd7aea81a83af857faf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e933179e-7668-49c6-8d36-cbdfe0ad4963", "node_type": "1", "metadata": {}, "hash": "bf00fd8a6176bdae0f2defebc18117df4ceebb022a179dfcb13b44e2311fa1da", "class_name": "RelatedNodeInfo"}}, "text": "Justicia adhatoda 103.146 \u00b11.33 \n\nVariation of Total phenolic content \n\nBy applying the Folin-Ciocalteu method and using gallic \n\nacid as a reference, the total phenolic content of \n\nmethanolic extracts was calculated [13,5]. At 760 nm, the \n\nhighest absorption was noted. Concentrated gallic acid \n\nsolution (10-100 \u00b5g/ml) confirmed at 760 nm with a \n\nregression coefficient (R2) of 0.9698 to Beer's Law (Figure \n\n2). With the aid of a calibration curve using gallic acid as \n\nthe reference, the total phenolic content was determined \n\nand expressed as mg GAE/g dry extract weight, total \n\nphenolic content was found highest in Nyctanthes \n\narbortristis (97.647\u00b17.01) mg GAE/g followed by Aegle \n\nmarmelos (88.559.647\u00b16.71), Crateva unilocularis \n\n(83.333\u00b12.73),and lowest in Justicia adhatoda \n\n(64.126\u00b11.368) respectively and was found lowest in \n\nUrtica dioica (24.36\u00b11.33 mg GAE/g).  \n\n \n\nTable 8. Total phenolic content of different plant extract \nName of \n\nplants \nAbsorbance TPC (mgGAE/g) \n\n A1 A2 A3 C1 C2 C3 Mean\u00b1SD \n\n   A.M 1.645 1.646 1.624 88.919 88.973 87.78 88.559\u00b1.6.715 \n\n   N.A 1.846 1.664 1.915 99.7831 89.8459 103.514 97.647\u00b17.01 \n   U.D 0.473 0.424 0.455 25.5676 22.9189 24.5946 24.36\u00b11.33 \n\n   C.U \n\n   J.A \n\n1.533 \n\n1.215 \n\n1.496 \n\n1.167 \n\n1.596 \n\n1.177 \n\n82.8649 \n\n65.67 \n\n80.8649 \n\n63.081 \n\n83.333 \n\n63.621 \n\n83.333\u00b12.73 \n\n64.126\u00b11.368 \n\nGAE: Gallic acid equivalent, SD: Standard deviation,A.M: Aegle marmelos,  \nN.A: Nyctanthes arbortristis,U.D: Urtica dioica, C.U= Crateva \nunilocularis,J.A= Justicia adhatoda, TPC: Total phenolic content, A1 A2, A3= \nAbsorbances triplicate, C1, C2, C3= TPC triplicates                              \n \n\n \nFigure 2: Calibration curve of gallic acid.  \n\ny = 0.0376x\nR\u00b2 = 0.9923\n\n0\n\n0.5\n\n1\n\n1.5\n\n2\n\n2.5\n\n3\n\n3.5\n\n0 20 40 60 80 100\n\nA\nb\nso\nrb\na\nn\nce\n\nconcentration(\u00b5g/ml)\n\nSeries1\n\nLinear (Series1)\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\np\ne\n\nrc\ne\n\nn\nta\n\ng\ne\n\n i\nn\n\nh\nib\n\nit\nio\n\nn\n\nPlant samples\n\nDPPH assay\n\nSeries1 Series2 Series3 Series4 Series5 Series6\n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    50 \n\nVariation in Total amount of Flavonoids  \nUsing quercetin as a reference, the total flavonoid content \n\nof methanolic extracts was determined using the \n\naluminum chloride colorimetric technique. With a \n\nregression coefficient (R2) of 0.9729, the quercetin \n\nsolution (10\u2013100 \u00b5g/ml) obeyed Beer's Law at 510 nm \n\n(Figure 3).", "start_char_idx": 21820, "end_char_idx": 24245, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e933179e-7668-49c6-8d36-cbdfe0ad4963": {"__data__": {"id_": "e933179e-7668-49c6-8d36-cbdfe0ad4963", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "16d3f6b9-572e-4c02-a8fd-c7d189bde8e5", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "76f7e84b2adb26880ea753ec029e7eee1219c85013e3b90be0274bdbba0bb56b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "28e4aa2c-0154-438f-aa6a-df2a231ce61d", "node_type": "1", "metadata": {}, "hash": "9e7aff378640e9a0b5bc2fc98d11ba2c627f1eaf65c139da831544529b5c673e", "class_name": "RelatedNodeInfo"}}, "text": "Total flavonoid content was found highest in \n\nCrateva unilocularis (31.99\u00b12.345) QE/g, followed by \n\nNyctanthes arbortritis (17.62\u00b13.225) then Aegle marmelos \n\n(10.998\u00b10.134), Urtica dioica (10.518\u00b15.231) and lowest in \n\nJusticia adhatoda (9.724\u00b10.103) mg QE/g. \n\nFigure 3. Calibration curve for quercetin \n\nConclusion \nThe DPPH radical scavenging activities and \n\nsubsequently the IC50 values of chosen plants' methanolic \n\nextracts revealed various levels of antioxidant activity. \n\nNyctanthes arbortristis showed highest percent \n\nscavenging, it has IC50 value 70.505\u00b11.55 \u03bcg/ml while the \n\nstandard, ascorbic acid has 55.40\u00b10.003 \u03bcg/ml, followed \n\nby Aegle marmelos. The greater antioxidant property on \n\nthem might be due to the presences of bioactive \n\ncompounds such as phenolic ,terpenoids, flavonoid, \n\nsaponins, tannin, alkaloids etc .The highest TPC was \n\nfound in Nyctanthes arbortristis (97.647\u00b17.01) followed by \n\nA.marmelos and lowest in U.dioica.While highest TFC is \n\nfound in Crateva unilocularis(31.99\u00b12.345) and lowest in \n\nJusticia adhatoda. Hence, these plants contain appreacible \n\namount of antioxidants, TPC and TFC. These results gave \n\nsome scientific proof for these indigenous medicinal \n\nplants. Even while some medical plants have substantial \n\nantioxidant properties, they cannot be used directly as \n\ndrugs. To make these therapeutic plants a possible source \n\nof natural antioxidants, more thorough phytochemical \n\nand pharmacological research must be done. \n\nAuthor\u2019s Contribution   \nD. K. Shrestha and B.K Sapkota designed research and \n\nperformed the experiments. B.K.Sapkota, D. K.Shresth \n\nand K.P. Sharma analysed the data and  K. P. Sharma \n\nreviewed the literature and edited the manuscript. All the \n\nauthors contributed equally in drafting and revising  the \n\nmanuscript. \n\nCompeting Interests  \nWe declare that authors have no conflict of interests of \n\nany kind. \n\nFunding  \nNo financial support for this work was provided by any \n\ninstitution, organisation or agency.All financial resources \n\nwas managed by the authors themselves. Few solvents, \n\nreagents and glasswares and instruments were \n\nsupported by Butwal Multiple Campus, Butwal. \n\n Acknowledgement \nThe authors are grateful to the Department of Chemistry, \n\nButwal Multiple Campus for providing laboratory and \n\ninstrumental facilities, the Campus Chief of BMC, \n\nBotanist Dr. Anant Gopal Singh, and Research \n\nManagement Cell of BMC for providing support to \n\nconduct research successfully. \n\nEthical Approval and Consent: \n All the ethical guidlines from ERB and NHRC are strictly \n\nfollowed. We ensure that there was no use of animal \n\nmodels and hazardous materials in this study.The plants \n\nused in this study were not endangered species or \n\nbanned by the government.", "start_char_idx": 24246, "end_char_idx": 27029, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "28e4aa2c-0154-438f-aa6a-df2a231ce61d": {"__data__": {"id_": "28e4aa2c-0154-438f-aa6a-df2a231ce61d", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e933179e-7668-49c6-8d36-cbdfe0ad4963", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "274f09d02d2b64e6735a2dc4ab8d2d848a56cc06d6117dc140c7d3edd3ec5b58", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "40595127-03e3-452e-b872-f303b7b8ef97", "node_type": "1", "metadata": {}, "hash": "64bc0986babc0a190cb288de7a2db53f762fe4e800d0ba98620756a7c30b38cf", "class_name": "RelatedNodeInfo"}}, "text": "No people other than the \n\nTable 9: Different plant extracts' total flavonoid content \n\nPlants \nAbsorbance TFC (mg QE/g) \n\nA1 A2 A3 C1 C2 C3 Mean \u00b1 SD \nAegle marmelos 0.172 0.171 0.168 11.0968 11.0323 10.838 10.989 \u00b1 0.134 \nNyctanthes arbortristis 0.273 0.323 0.223 17.612 20.838 14.387 17.62 \u00b1 3.225 \nUrtica dioica 0.172 0.171 0.168 11.0468 11.032 10.438 10.518 \u00b1 5.231 \nCrateva unilocularis 0.435 0.505 0.487 28.064 32.580 31.419 31.99 \u00b1 2.345 \n\nJusticia adhatoda 0.157 0.156 0.143 10.1290 9.936 9.108 9.724 \u00b1 0.103 \n\nTFC=Total Flavonoid Content (n=3), SD= Standard deviation, Q.E=Quercetin equivalent  \n\ny = 0.0314x\nR\u00b2 = 0.9903\n\n0\n\n0.5\n\n1\n\n1.5\n\n2\n\n2.5\n\n3\n\n0 20 40 60 80\n\nA\nb\nso\nrb\na\nn\nce\n\nconcentration(\u00b5g/ml)\n\nSeries1 Linear (Series1)\n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :4 5 - 5 1      Shrestha et al.  \n\n\u00a9NJB, BSN    51 \n\nauthors are involved in this research. The harm or \n\ndiscomfort for others during this research was nill. \n\nReferences \n1. Elmasta\u015f M, G\u00fcl\u00e7in I, I\u015fildak \u00d6, K\u00fcfrevio\u011flu \u00d6\u0130, \u0130bao\u011flu K, Aboul-\n\nEnein HY. Radical scavenging activity and antioxidant capacity of \n\nbay leaf extracts. Journal of the Iranian Chemical Society. 2006 \n\nSep;3(3):258-66. \n\n2. GC DB. Study on Biological Activities and Essential oil of Cinnamomum \n\nTamala (Doctoral dissertation, Central Department of Chemistry). \n\n3. Manandhar, N. P. (2002). Plants and people of Nepal. Timber press. \n\n4. Chapagain PS, Aase TH. Changing forest coverage and \n\nunderstanding of deforestation in Nepal Himalayas. Geographical \n\nJournal of Nepal. 2020 Mar 18;13:1-28. \n\n5. Rezaeizadeh A, Zuki AB, Abdollahi M, Goh YM, Noordin MM, \n\nHamid M, Azmi TI. Determination of antioxidant activity in \n\nmethanolic and chloroformic extracts of Momordica charantia. \n\nAfrican Journal of Biotechnology. 2011;10(24):4932-40. \n\n6. Naz\u0131ro\u01e7lu M, Karao\u011flu A, Aksoy AO. Selenium and high dose \n\nvitamin E administration protects cisplatin-induced oxidative \n\ndamage to renal, liver and lens tissues in rats. Toxicology. 2004 Feb \n\n15;195(2-3):221-30. \n\n7. Willcox JK, Ash SL, Catignani GL. Antioxidants and prevention of \n\nchronic disease. Critical reviews in food science and nutrition. 2004 \n\nJul 1;44(4):275-95. \n\n8. Halliwell B. Protection against oxidants in biological systems: the \n\nsuperoxide theory of oxygen toxicity. Free radical in biology and \n\nmedicine. 1989:86-123. \n\n9. Shyur LF, Tsung JH, Chen JH, Chiu CY, Lo CP. Antioxidant \n\nproperties of extracts from medicinal plants popularly used in \n\nTaiwan. International journal of applied science and engineering.", "start_char_idx": 27030, "end_char_idx": 29574, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "40595127-03e3-452e-b872-f303b7b8ef97": {"__data__": {"id_": "40595127-03e3-452e-b872-f303b7b8ef97", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "28e4aa2c-0154-438f-aa6a-df2a231ce61d", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "36618877b5ac8e29afea7931398c33e3a91b777b2bdcbe8478e1624ae7d5ef85", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "348f6830-f93f-471a-87fa-765a099ff9b5", "node_type": "1", "metadata": {}, "hash": "a8d67aa2c0332d0d18481373994a8655459f685f1dd21d855a9dd94ba85e3c0f", "class_name": "RelatedNodeInfo"}}, "text": "International journal of applied science and engineering. \n\n2005 Dec;3(3):195-202. \n\n10. Lee JY, Hwang WI, Lim ST. Antioxidant and anticancer activities of \n\norganic extracts from Platycodon grandiflorum A. De Candolle \n\nroots. Journal of ethnopharmacology. 2004 Aug 1;93(2-3):409-15. \n\n11. Bag, G. C., Devi, P. G., & Bhaigyabati, T. H. (2015). Assessment of \n\ntotal flavonoid content and antioxidant activity of methanolic \n\nrhizome extract of three Hedychium species of Manipur \n\nvalley. International Journal of Pharmaceutical Sciences Review \n\nand Research, 30(1), 154-159. \n\n12. Wagay NA, Khan NA, Rothe SP. Profiling of secondary metabolites \n\nand antimicrobial activity of Crateva religiosa G. Forst. bark-A rare \n\nmedicinal plant of Maharashtra India. Int. J. Biosci. 2017;10(5):343-\n\n54. \n\n13. KHAREL R, SHARMA KR. Evaluation of antioxidant potential and \n\nquantitative estimation of phenolic and flavonoid content in some \n\nselected nepalese medicinal plants. EVALUATION. 2020;13(1). \n\n14. Muhammad A, Rahman M, Kabir AN, Kabir S, Hossain M. \n\nAntibacterial and cytotoxic activities of Spondias pinnata (Linn. f.) \n\nKurz fruit extract.  \n\n15. Aryal B, Niraula P, Khadayat K, Adhikari B, Khatri Chhetri D, \n\nSapkota BK, Bhattarai BR, Aryal N, Parajuli N. Antidiabetic, \n\nantimicrobial, and molecular profiling of selected medicinal plants. \n\nEvidence-based complementary and alternative medicine. 2021 \n\nMay 7;2021. \n\n16. Khare V, Kushwaha P, Verma S, Gupta A, Srivastava S, Rawat AK. \n\nPharmacognostic Evaluation and Antioxidant Activity of Urtica \n\ndioica L. https://doi.org/10.4236/CM.2012.33021 \n\n17. Venkataraman S, Harinya S, Chidiuto DB, Raja RR, Jasmine EN, \n\nJasmine C. Phytochemical Constituents and Pharmacological \n\nactivities of Nyctanthes arbor-tristis. Research Journal of Pharmacy \n\nand Technology. 2019 Oct 12;12(10):4639-43. \n\n18. Ghosh S, Derle A, Ahire M, More P, Jagtap S, Phadatare SD, Patil \n\nAB, Jabgunde AM, Sharma GK, Shinde VS, Pardesi K. \n\nPhytochemical analysis and free radical scavenging activity of \n\nmedicinal plants Gnidia glauca and Dioscorea bulbifera. PLoS One. \n\n2013 Dec 18;8(12):e82529. \n\n19. Wijewardana RM, Nawarathne SB, Wickramasinghe I, \n\nGunawardane CR, Wasala WM, Thilakarathne BM. Retention of \n\nphysicochemical and antioxidant properties of dehydrated bael \n\n(Aegle marmelos) and palmyra (Borassus flabellifer) fruit powders. \n\nProcedia food science. 2016 Jan 1;6:170-5. \n\n20. Rajan S, Gokila M, Jency P, Brindha P, Sujatha RK. Antioxidant and \n\nphytochemical properties of Aegle marmelos fruit pulp. Int J Curr \n\nPharm Res. 2011;3(2):65-70.Reza \n\n21. Bernards MA. Plant natural products: a primer. Canadian Journal \n\nof Zoology. 2010 Jul;88(7):601-14. \n\n22.", "start_char_idx": 29517, "end_char_idx": 32233, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "348f6830-f93f-471a-87fa-765a099ff9b5": {"__data__": {"id_": "348f6830-f93f-471a-87fa-765a099ff9b5", "embedding": null, "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-239", "node_type": "4", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "91a722fbaeabb29f5dce7416ffae04362c3f68441d3f8ed12b9fa47e073d40b7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "40595127-03e3-452e-b872-f303b7b8ef97", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "80f80e67c8a636af91854a49d81c16b989c1b39c9e18b6d93b3897691cf9990c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9f586f56-56e0-45a4-88a4-38670563a4c8", "node_type": "1", "metadata": {}, "hash": "9b5fd1234012caeef2f708e62c72adcc8979b84d4918f2c2c2815a27d1e92600", "class_name": "RelatedNodeInfo"}}, "text": "2010 Jul;88(7):601-14. \n\n22. Kaur A, Kaur D, Arora S. Evaluation of antioxidant and \n\nantimutagenic potential of Justicia adhatoda leaves extract. African \n\nJournal of Biotechnology. 2015;14(21):1807-19. \n\n23. Ciulei I., Phytochem., 2013, 63 (1), 97\u2013104. \n\n24. Shrestha DK. Evaluation of phytochemicals, antibacterial, \n\nantioxidant activity, total flavonoid, and phenolic content of \n\nzanthoxylum armatum found in Palpa district of Nepal. Asian \n\nJournal of Multidimensional Research. 2022;11(6):156-73. \n\n25. Paulsamy, S., & Karthika, K. (2012). Screening of in vitro antioxidant \n\nactivity of methanolic leaf and root extracts of Hypochaeris radicata \n\nL.(Asteraceae). Journal of Applied Pharmaceutical Science, 2(7), 149-\n\n154. \n\n26. Shackelford L, Mentreddy SR, Cedric S. Determination of total \n\nphenolics, flavonoids and antioxidant and chemopreventive \n\npotential of basil (Ocimum basilicum L. and Ocimum tenuiflorum L.). \n\nInternational Journal of Cancer Research. 2009;5(4):130-43. \n\n27. Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total \n\nflavonoid content in propolis by two complementary colorimetric \n\nmethods. Journal of food and drug analysis. 2002 Jul 1;10(3). \n\nhttps://doi.org/10.4236/CM.2012.33021", "start_char_idx": 32205, "end_char_idx": 33429, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9f586f56-56e0-45a4-88a4-38670563a4c8": {"__data__": {"id_": "9f586f56-56e0-45a4-88a4-38670563a4c8", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "348f6830-f93f-471a-87fa-765a099ff9b5", "node_type": "1", "metadata": {"identifier": "njb-239", "author": " Kumar Shrestha, Deepak; Kumar Sapkota, Basanta; Prasad Sharma, Krishna", "title": "Assessment of Phytochemicals, Antioxidant Activity, Total Phenolic and Flavonoid Contents of Selected Nepalese Medicinal Plants", "date": "2022-12-31", "file": "njb-239.pdf"}, "hash": "f9cb96ab2a58e177157a1279ea2f1344bc106f6daa4b42831a87cc2985cbc27c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fc48021e-54a6-4d56-83b6-aaac8034ae94", "node_type": "1", "metadata": {}, "hash": "daa4173503c91440ea4ac98607c1016d4554abb3eec061c096ecbf880f438f20", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 37-42   ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    37        Nepjol.info/index.php/njb \n\nMultiplex Ligation Dependent Probe Amplification Based \nMutation Analysis of Dystrophin Gene in Nepalese Patients \n\nwith Duchenne Muscular Dystrophy \nKushal Shrestha1, Smita Shrestha2, Mr. Saroj Khatiwada3, Bishnu Acharya4, Sulochana Manandhar5, Rohit Kumar \n\nPokharel6,7 * \n1Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. \n2Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. \n\n3Department of Biochemistry, Modern Technical College, Lalitpur, Nepal. \n4Muscular Dystrophy Foundation-Nepal (MDF-Nepal), Lalitpur, Nepal. \n\n5Center for Molecular Dynamics-Nepal (CMDN), Nepal. \n6Muscular Dystrophy Foundation-Nepal (MDF-Nepal), Lalitpur. \n\n7Department of Orthopedics and Trauma surgery, IOM, Tribhuvan University, Kathmandu, Nepal. \n\nAbstract \nDuchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disorders caused due to mutation in \n\ndystrophin gene, leading to progressive muscle weakness. This study was done to identify mutation in \n\ndystrophin gene in Nepalese patients with DMD using Multiplex Ligation Dependent Probe Amplification \n\n(MLPA) assay in Nepal. Twenty one patients from different regions of Nepal, who were clinically diagnosed as \n\nDMD were enrolled in the study. Peripheral blood samples were collected in EDTA vials, gDNA was extracted, \n\nand deletion mutation in the dystrophin gene was analysed using Multiplex Ligation Dependent Probe \n\nAmplification (MLPA) assay. \n\nExon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 DMD cases. The most \n\ncommon exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. The location of \n\ndeletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. Use of \n\nMLPA is useful in detecting copy number changes in DMD proband and suspected carriers in Nepal. \n\nKeywords: Duchene muscular dystrophy, Multiplex ligation dependent probe amplification, Mutation, Nepal. \n\n*Corresponding Author \nEmail: pokharel.rohit@gmail.com \n\n \n\nIntroduction \nDuchenne and Becker muscular dystrophies (DMD & \n\nBMD) are X-linked recessive neuromuscular \n\ndisorders with incidence of 1 in 3,500 and 1 in 30,000 \n\nlive male births, respectively [1]. DMD is \n\ncharacterized by progressive muscle weakness with \n\nonset at 3-5 years of age, leading to loss of \n\nambulation by 10-12 years of age without treatment. \n\nRespiratory, orthopaedic, and cardiac complications \n\nemerge with age, and without intervention, the mean \n\nage at death is around 19 years [2]. \n\nBoth DMD and BMD are caused by mutations in the \n\ndystrophin gene (MIM 300377), one of the largest \n\nknown human genes, spanning about 2.4\u2009Mb of \n\ngenomic DNA [3]. The most common mutations in \n\nthe DMD gene are the deletion or duplication of one \n\nor more exons [3]. Mutations lead to an absence of or \n\ndefect in the dystrophin protein, which results in \n\nprogressive muscle degeneration leading to loss of \n\nindependent ambulation by the age of 13 years.", "start_char_idx": 47, "end_char_idx": 3294, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fc48021e-54a6-4d56-83b6-aaac8034ae94": {"__data__": {"id_": "fc48021e-54a6-4d56-83b6-aaac8034ae94", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9f586f56-56e0-45a4-88a4-38670563a4c8", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "c98a703737a14d4cd5feaea2312618b76b11611c10d4e861c0a59e81b4f045d0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e90b8a2-bdb3-4109-b24f-6219d70bbc78", "node_type": "1", "metadata": {}, "hash": "e3da3ca8de109fc9322efad5b06c10c02cd91b56ba953334cec789e8367b3285", "class_name": "RelatedNodeInfo"}}, "text": "Variable phenotypic expression relates mainly to the \n\ntype of mutation and its effect on the production of \n\ndystrophin [4]. \n\nMuscular dystrophy is diagnosed on the basis of \n\nclinical examination, raised creatine phosphokinase \n\n(CPK) level, muscle biopsy and dystrophin gene \n\nmutation analysis. The genetic tests commonly used \n\nto identify dystrophin mutations are multiplex \n\npolymerase chain reaction (mPCR), multiplex \n\nligation dependent probe amplification (MLPA), \n\nsingle condition amplification/internal primer, and \n\nmultiplex amplifiable probe hybridization [5]. \n\nMutation analysis of dystrophin gene in DMD/BMD \n\nhas been performed in several countries [2, 3]. \n\nHowever, no such studies have been performed in \n\nNepal. MLPA, a simple and cheaper genetic test to \n\ndetect duplication/ deletion mutation, could be a \n\nbetter tool for genetic diagnosis of DMD in Nepalese \n\npatients; thereby assisting in timely diagnosis, \n\ntreatment and management. We conducted present \n\nstudy among clinically diagnosed DMD patients to \n\nfind the mutation pattern in dystrophin gene using \n\nMLPA and test the efficacy of MLPA in identifying \n\nmutation in DMD patients in the Nepalese context. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 37-42  Shrestha et al.  \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    38        Nepjol.info/index.php/njb \n\nMaterials and Methods  \nThe study was conducted among 21 clinically \n\ndiagnosed DMD patients from different regions of \n\nNepal, who were referred to Muscular Dystrophy \n\nFoundation-Nepal (MDF-Nepal). Diagnosis of DMD \n\nwas done on the basis of clinical presentations and \n\nmarkedly elevated serum creatine phosphokinase \n\n(CPK) levels. Proper genetic counselling was done, \n\nand consent was taken from each patient and their \n\nparents. \n\nPeripheral blood samples (5 ml) were collected from \n\neach patient in EDTA vials and transported to \n\nlaboratory of Central Department of Biotechnology, \n\nTribhuvan University, Kirtipur maintaining cold \n\nchain. The samples were stored at -200 C until \n\nanalysis. Genomic DNA was isolated from blood, as \n\nrecommended by the MRC-Holland, by using the \n\nextraction method of the QIAamp DNA mini kit [6]. \n\nThe absorbance of DNA samples were measured at \n\n230 nm, 260 nm and 280 nm using the UV \n\nspectrophotometer to check for purity of DNA \n\nsamples required for MLPA assay. \n\nA commercial MLPA kit (MRC Holland) with probes \n\nof P034 (DMD exons 1-10, 21-30, 41-50, and 61-70) \n\nand P035 (DMD exons 11-20, 31-40, 51-60, and 71-79) \n\nwas used to detect DMD deletion/duplication in \n\nDMD patients according to the manufacturer\u2019s \n\nrecommended protocol (Amsterdam, Netherlands). \n\n50-250 ng of genomic DNA, in a volume of 5 \u00b5L Tris-\n\nEDTA, was denatured at 980 C for 5 min, cooled \n\ndown, and then mixed with MLPA P034 or P035 \n\nprobemix. The mixture was then heated to 950 C for 5 \n\nmin and incubated at 600 C overnight for probe \n\nhybridization. After 16 hours, ligation was \n\nperformed with Ligase-65 enzyme at 540 C for 15 min \n\nand Ligase-65 enzyme was inactivated at 980 C for 5 \n\nmin. Then, PCR amplification was performed with \n\nspecific SALSA FAM PCR primers (5\u2019-\n\nGGGTTCCCTAAGGGTTGGA-3\u2019). After that, the \n\nmixture was separated by capillary electrophoresis \n\nand then analyzed using ABI-310 Genetic Analyzer \n\n[7].", "start_char_idx": 3297, "end_char_idx": 6636, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e90b8a2-bdb3-4109-b24f-6219d70bbc78": {"__data__": {"id_": "9e90b8a2-bdb3-4109-b24f-6219d70bbc78", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fc48021e-54a6-4d56-83b6-aaac8034ae94", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "67b7c2d1dffe5e011af0315ed879a00e6b9a03813321aa88bcb8f3d881c7cb4f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7f90d93f-c071-4846-8892-b3377da626d7", "node_type": "1", "metadata": {}, "hash": "5f71c0a10d72238ab2abfa6a094208a29975c3e7fba0608e0e5cdb8f7741b53b", "class_name": "RelatedNodeInfo"}}, "text": "The Genescan analysis software in the ABI-310 \n\nGenetic Analyzer analyzed the raw data to quantify \n\nthe DNA fragments and determine the size of the \n\nfragments by comparing them to fragments in a size \n\nstandard. The electropherograms of test samples \n\nwere analyzed by comparing with the peak pattern \n\nof the male and female reference samples. The novel \n\nsoftware Coffalyser.net was used to analyze the data \n\nobtained after the capillary electrophoresis run which \n\nwas further confirmed by visual assessment by \n\noverlaying two fragment profiles and comparing the \n\nrelative intensities of fragments. The relative peak \n\nratio (RPR) of every single exon was plotted to its \n\ncorresponding bar chart [8]. Absence of peaks \n\ncorresponding to two or more contiguous exons was \n\ntaken to represent a genuine deletion. The absence of \n\nonly one peak in males, corresponding to a single \n\nexon, were also recorded which are yet to be \n\ninvestigated further by applying the novel methods \n\nas PCR primer flanking the exons in question or by \n\nsequencing. The framedness of the mutation in \n\nprobands were also estimated by using the \n\ndystrophin exonic deletions/duplications reading \n\nframe checker 1.9 as recommended by MRC Holland. \n\nThe data from the study was entered into ms excel \n\nand analyzed using SPSS version 11.0. \n\nResults  \nThe mean age of DMD patients at the time of study \n\nwas 11.4 years (age range from 8-18 years). All the \n\npatients had very high serum CPK level (median: \n\n6245 U/L, range:  2100-32890 U/L). The genomic \n\nDNA extracted from samples had concentration \n\nrange within 27-59 \u03bcg/mL. The ratio of the optical \n\ndensity (OD) of DNA samples at 260 nm and 280 nm \n\nranged from 1.5-2.0 with mean OD 1.76. Among the \n\n21 samples of DMD analyzed by MLPA assay, \n\ndeletions were observed at various exons of \n\ndystrophin gene in 14 samples. Seven samples \n\nhowever did not have any deletion or duplication in \n\nthe exons. Thus, MLPA tool could detect mutations \n\nin about 66.6% (14 of 21 samples). The most prevalent \n\nexonic deletion regions were found to be confined in \n\nthe exon 7-14 and 45-53. Deletion mutations at \n\ndifferent exons observed in samples are shown in \n\ntable 1. Similarly, electropherograms of control \n\nsample and test sample after MLPA assay with \n\nSALSA probe mix P034 and P035 are shown in figure \n\n1. \n\nAs indicated by the dystrophin exonic deletions/ \n\nduplications reading frame checker 1.9, all the 14 \n\nsamples with detection of deletion mutation were \n\ndetected by MLPA assay have out of frame \n\nmutations suggesting that the samples were those of \n\nDMD patients. But the confirmation of the \n\nframedness is yet to be made by gene sequencing, \n\nand the 7 samples with negative MLPA result need \n\nfurther genetic testing like sequencing. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 37-42  Shrestha et al.", "start_char_idx": 6639, "end_char_idx": 9523, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7f90d93f-c071-4846-8892-b3377da626d7": {"__data__": {"id_": "7f90d93f-c071-4846-8892-b3377da626d7", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e90b8a2-bdb3-4109-b24f-6219d70bbc78", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "098623df97c9aba6dad77001e9614a29e2e66a34809677bab334482843530d40", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c06439d1-95a0-4f38-8f79-a199df22e0fb", "node_type": "1", "metadata": {}, "hash": "352a1f18616274fe04e0e2988f5521c09b73912afd0dd329bba5bb7d254e7091", "class_name": "RelatedNodeInfo"}}, "text": "1: 37-42  Shrestha et al.  \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    39        Nepjol.info/index.php/njb \n\n \nTable 1: Exon deletions detected by MLPA assay in \nDMD patients \n\nCase  Age \n(years) \n\nSerum \nCreatine \nkinase level \n(U/L) \n\nExon \ndeletions \n\nPhenotype  \n\n1 5 12122 48-50 DMD \n\n2 7 3412 52 DMD \n\n3 6 14467 10-43 DMD \n\n4 8 1287 52 DMD \n\n5 9 11580 45 DMD \n\n6 5 24200 51-53 DMD \n\n7 14 22000 45-50 DMD \n\n8 5 21887 46-49 DMD \n\n9 12 2700 52 DMD \n\n10 11 2100 51 DMD \n\n11 8 4328 12-14 DMD \n\n12 9 3220 51 DMD \n\n13 6 9123 45 DMD \n\n14 7 6245 7-9 DMD \n\n \nDiscussion \nIn Nepal, DMD/BMD patients are generally \n\ndiagnosed late due to the lack of complete \n\nunderstanding of the disease, poor screening of \n\npaediatric group for muscular dystrophy and lack of \n\ngenetic diagnostic facilities in the country. Most of \n\nthe cases are diagnosed on the basis of history given \n\nby the parents, positive clinical signs of DMD and \n\nvery high CK level in the blood. For the diagnosis at \n\nthe gene level clients usually go to or the sample is \n\nsend outside Nepal, which is not accessible and \n\naffordable to many of the parents. This study to \n\ndetect deletion mutation in DMD using MLPA is first \n\ngenetic pilot study in Nepal.  \n\nAmong the 21 samples of clinically diagnosed DMD \n\npatients, deletion of dystrophin exon was found in 14 \n\nsamples (66.6%). However, 7 samples (33.3%) did not \n\nshow any deletion or duplication. Thus, MLPA was \n\nefficient in accurately confirming mutations in about \n\n67% of all the cases. A number of studies have found \n\nMLPA assay to be highly sensitive diagnostic assay \n\nto identify mutation in DMD and BMD patients [9]. \n\nIn the study to evaluate the efficacy of MLPA \n\ntechnique in comparison with the traditional \n\nmultiplex PCR assay in detection of exon deletions \n\nand duplications of the DMD gene by Lai et al., \n\nMLPA was able to detect all the known deletions and \n\nduplications; it detected four additional mutations \n\nthat had been missed by multiplex PCR  [10]. In a \n\nstudy in China amongst 70 DMD/BMD patients, \n\nMLPA detected exonic deletions in 42 patients (60%), \n\nexonic duplications in 7 patients (10%) and 21 \n\npatients (30%) showed normal results [11]. \n\nIn the present study, only exons deletion was \n\nobserved. No exon duplication was found. We found \n\nmost prevalent exonic deletion regions to be confined \n\nin the exons 7-14 and 45-53. One, 3, 4, 6 and 44 exons \n\ndeletion was observed in 7, 4, 1, 1 and 1 patients \n\nrespectively. No novel mutations were identified in \n\nthis study. In a study in Iranian DMD/BMD patients, \n\n30.9 % of patients had single exon deletion, while \n\ngroup and contiguous exon deletions were identified \n\nin 41% of the patients [12]. The most numerous exon \n\ndeletions included exons 45-50, and two exons 3-5 \n\nand 41-43 duplications (1.4 %) was observed in a \n\nBMD and a DMD patient, respectively [12]. In a \n\nstudy in Polish DMD/BMD patients, Zimowski et al. \n\nidentified 110 deletions, 22 duplication (in one \n\npatient two different duplications were detected) and \n\n2 point mutations.", "start_char_idx": 9498, "end_char_idx": 12575, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c06439d1-95a0-4f38-8f79-a199df22e0fb": {"__data__": {"id_": "c06439d1-95a0-4f38-8f79-a199df22e0fb", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7f90d93f-c071-4846-8892-b3377da626d7", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "00d5763cad19a023a1758790d6d2d3ddd50ef53172aa5985d88d12beaf4bb4e7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "61035d72-9a8a-490c-813e-8174a1040bf2", "node_type": "1", "metadata": {}, "hash": "9a5d05f2c6a70e93b929aee5e1b1f1bba16a9626d3060cd5361b2a3b09bfb120", "class_name": "RelatedNodeInfo"}}, "text": "Deletions involved mainly exons \n\n45-54 and 3-21, whereas most duplication involved \n\nexons 3-18 [13]. It has been found that deletions \n\naccount for approximately 60-65% of mutations and \n\nduplications for 5-10% in DMD/BMD. The \n\nremaining cases are mainly point mutations (30-35% \n\nof the cases). Although deletions encompass all 79 \n\nexons, two deletion hotspots (exons 45-55 and exons \n\n2-19) are recognized [14]. \n\nIdentification of exon deletion is important to design \n\ngene therapy by exon skipping method that is an \n\nemerging therapy for DMD that can transform DMD \n\ninto BMD is based on the recovery of reading frame \n\ninduced by alternative splicing of antisense \n\noligonucleotides [15]. More patients may benefit \n\nfrom individual exon skipping therapies following a \n\ncomprehensive understanding of the correlation \n\nbetween genotypes and phenotypes under the \n\nguidance of large-scale genetic epidemiological \n\nstudies [16]. Similarly identifying disease at earlier \n\nstage could help in better management of patients \n\nand thus better life quality. \n\nThis was the first genetic study on DMD in Nepal. \n\nThe location of deletion in dystrophin gene is \n\napparently non-random with a preponderance found \n\nin the hot spot regions in Nepalese population as \n\nwell. Use of MLPA is useful in detecting copy \n\nnumber changes in DMD proband and suspected \n\ncarriers. \n\n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 37-42  Shrestha et al.  \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    40        Nepjol.info/index.php/njb \n\n \n\nA \n\nB\n\nA \n\nC\n\nA", "start_char_idx": 12576, "end_char_idx": 14155, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "61035d72-9a8a-490c-813e-8174a1040bf2": {"__data__": {"id_": "61035d72-9a8a-490c-813e-8174a1040bf2", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c06439d1-95a0-4f38-8f79-a199df22e0fb", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "c2d3b63c8b3d1e0c04f78d996f2a9d66d4f1a5d286ac0e2485cb5d620b65511f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "98b53d5e-d5a3-43db-b4e0-d38fe2d6cbbe", "node_type": "1", "metadata": {}, "hash": "92cac7b8735ada3126c49d93d1db22a742ec178a900f219a5395375b00de5652", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 37-42  Shrestha et al.  \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    41        Nepjol.info/index.php/njb \n\nFigure 1. Electropherogram of control sample and test sample after MLPA. Fig. 1A and Fig. 1B are for normal control \nsample with P034 and P035 respectively. Fig. 1C and Fig. 1D are for DMD patient with deletion in dystrophin gene with \nP034 and P035 respectively. \n\n \n\nCompeting Interest \nNone  \n\nAcknowledgement \nWe appreciate the parents and DMD boys who gave \n\nconsent to take part in this study. We acknowledge \n\nthe support of MDF-Nepal for counseling parents \n\nand probands, providing the detail information \n\nabout the clients and also bearing the cost of some \n\nchemicals for gDNA extraction and charge of genetic \n\nanalyzer. We express our gratitude to Prof. Masafumi \n\nMatsuo, M.D; Ph.D. from Kobe, Japan for providing \n\nus the MLPA kits, which pioneered the genetic \n\ntesting of DMD cases in Nepal for the first time. We \n\nthank Prof. Tribikram Bhattarai, former head of \n\ndepartment of Biotechnology, Tribhuvan University \n\nand current head Prof. Dr. Rajani Malla for \n\nencouraging and providing the laboratory for the \n\nstudy. We also thank Intrepid Nepal Pvt Ltd.; Centre \n\nfor Molecular Dynamics (CMDN), Thapathali-11, \n\nKathmandu, Nepal, for allowing us to use the \n\nGenetic analyzer at the center. \n\nAuthor\u2019s Contribution \nKS, SS and RKP designed the study and collected \n\nsamples. KS, SS, SK, BA and SM engaged in sample \n\ncollection and conducted laboratory analysis. SK \n\nperformed statistical analysis and wrote the \n\nmanuscript. KS, SS and RKP revised the draft. All \n\nauthors read and approved the final version of the \n\nmanuscript. \n\nReferences \n1. Hegde MR, Chin EL, Mulle JG, Okou DT, Warren \n\nST, Zwick ME: Microarray-based mutation \ndetection in the dystrophin gene. Hum Mutat. \n2008, 29(9):1091-99. \n\n2. Li X, Zhao L, Zhou S, Hu C, Shi Y, Shi W, et al: A \ncomprehensive database of Duchenne and \nBecker muscular dystrophy patients (0-18 years \nold) in East China. Orphanet J Rare Dis. 2015; 10:5. \ndoi: 10.1186/s13023-014-0220-7. \n\n3. Santos R, Gon\u00e7alves A, Oliveira J, Vieira E, Vieira \nJP, Evangelista T, et al: New variants, challenges \nand pitfalls in DMD genotyping: implications in \ndiagnosis, prognosis and therapy. J Hum Genet. \n2014, 59(8):454-64. \n\n4. Bushby K, Finkel R, Birnkrant DJ, Case LE, \nClemens PR, Cripe L, et al: Diagnosis and \nmanagement of Duchenne muscular dystrophy, \npart 1: diagnosis, and pharmacological and \npsychosocial management. Lancet Neurol. 2010, \n9(1):77-93. \n\n5. Flanigan KM, Dunn DM, von Niederhausern A, \nSoltanzadeh P, Gappmaier E, Howard MT, et al: \nMutational spectrum of DMD mutations in \ndystrophinopathy patients: application of \nmodern diagnostic techniques to a large cohort. \nHum Mutat. 2009, 30(12):1657-66. \n\n6. MLPA, General protocol, MRC-Holland, MLPA, \nprotocol version MDP-v002, last update 23-01-\n2012. \n\nD\n\nA \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No.", "start_char_idx": 14160, "end_char_idx": 17199, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "98b53d5e-d5a3-43db-b4e0-d38fe2d6cbbe": {"__data__": {"id_": "98b53d5e-d5a3-43db-b4e0-d38fe2d6cbbe", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "61035d72-9a8a-490c-813e-8174a1040bf2", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "8369b56cc2e528120021a9bd8278e5ab8a14d826a71c6b0813c3018418d254c4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "27226837-3b07-476d-9629-d531b77ebabc", "node_type": "1", "metadata": {}, "hash": "c12eb72305f6da5493326a8d7dbbc639785cb59f56e64f0f4af02e6452899052", "class_name": "RelatedNodeInfo"}}, "text": "2 0 1 6  Vol. 4, No. 1: 37-42  Shrestha et al.  \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    42        Nepjol.info/index.php/njb \n\n7. Chen C, Ma H, Zhang F, Chen L, Xing X, Wang S, \net al: Screening of Duchenne Muscular \nDystrophy (DMD) Mutations and Investigating \nIts Mutational Mechanism in Chinese Patients. \nPLoS ONE. 2014, 9(9):e108038. \n\n8. Wang X, Wang Z, Yan M, Huang S, Chen TJ, \nZhong N: Similarity of DMD gene deletion and \nduplication in the Chinese patients compared to \nglobal populations. Behav Brain Funct. 2008, 4:20. \ndoi: 10.1186/1744-9081-4-20. \n\n9. Stuppia L, Antonucci I, Palka G, Gatta V: Use of \nthe MLPA Assay in the Molecular Diagnosis of \nGene Copy Number Alterations in Human \nGenetic Diseases. Int J Mol Sci. 2012, 13(3): 3245-\n76. \n\n10. Lai KKS, Lo IFM, Tong TMF, Cheng LYL, Lam \nSTS: Detecting exon deletions and duplications \nof the DMD gene using Multiplex Ligation-\ndependent Probe Amplification (MLPA). Clinical \nBiochemistry. 2006, 9 :367-72. \n\n11. Long F, Sun W, Ji X, Li XH, Liu XQ, Jiang WT, Tao \nJ: Clinical application of multiplex ligation-\ndependent probe amplification for the detection \nexonic copy number alterations in the \nDystrophin gene. Zhonghua Yi Xue Yi Chuan Xue \nZa Zhi. 201,28(6):699-704. \n\n12. Zamani GR, Karami F, Mehdizadeh M, Movafagh \nA, Nilipour Y, Zamani M. Analysis of dystrophin \ngene in Iranian Duchenne and Becker muscular \ndystrophies patients and identification of a novel \nmutation. Neurol Sci. 2015. doi:  10.1007/s10072-\n015-2290-2. \n\n13. Zimowski JG, Massalska D, Holding M, Jadczak S, \nFidzia\u0144ska E, Lusakowska A, et al: MLPA based \ndetection of mutations in the dystrophin gene of \n180 Polish families with Duchenne/Becker \nmuscular dystrophy. Neurol Neurochir Pol. 2014, \n48(6):416-22. \n\n14. Nouri N, Fazel-Najafabadi E, Salehi M, \nHosseinzadeh M, Behnam M, Ghazavi MR, et al: \nEvaluation of multiplex ligation-dependent \nprobe amplification analysis versus multiplex \npolymerase chain reaction assays in the detection \nof dystrophin gene rearrangements in an Iranian \npopulation subset. Adv Biomed Res. 2014, 3: 72.  \ndoi: 10.4103/2277-9175.125862. \n\n15. Cirak S, Arechavala-Gomeza V, Guglieri M, Feng \nL, Torelli S, Anthony K, et al: Exon skipping and \ndystrophin restoration in patients with \nDuchenne muscular dystrophy after systemic \nphosphorodiamidate morpholino oligomer \ntreatment: an open-label, phase 2, dose-escalation \nstudy. Lancet. 2011, 378(9791):595-605. \n\n16. Mitrpant C, Fletcher S, Wilton SD: Personalised \ngenetic intervention for Duchenne muscular \ndystrophy: antisense oligomers and exon \nskipping. Curr Mol Pharmacol.", "start_char_idx": 17179, "end_char_idx": 19804, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "27226837-3b07-476d-9629-d531b77ebabc": {"__data__": {"id_": "27226837-3b07-476d-9629-d531b77ebabc", "embedding": null, "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-24", "node_type": "4", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "b1522a7f17a87422e2fa5805677b9996baba08898cd1e98c87cbf9da103d8dc0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "98b53d5e-d5a3-43db-b4e0-d38fe2d6cbbe", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "165dd83cc6ac476a81a6474aaf98b6db8dcd0b6330d46eb65f79ea00157bb598", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "57054d7e-2757-443f-b1c0-1fa3094e0d33", "node_type": "1", "metadata": {}, "hash": "30eb70702c3e7ae48f27643c6de866dbb0bac53ba89294767b82ccfa4bfe495b", "class_name": "RelatedNodeInfo"}}, "text": "Curr Mol Pharmacol. 2009,2(1):110-21", "start_char_idx": 19785, "end_char_idx": 19821, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "57054d7e-2757-443f-b1c0-1fa3094e0d33": {"__data__": {"id_": "57054d7e-2757-443f-b1c0-1fa3094e0d33", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "27226837-3b07-476d-9629-d531b77ebabc", "node_type": "1", "metadata": {"identifier": "njb-24", "author": "Shrestha, Kushal; Shrestha, Smita; Khatiwada, Saroj; Acharya, Bishu; Manandhar, Sulochana; Pokharel, Rohit Kumar", "title": "Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy", "date": "2016-12-31", "file": "njb-24.pdf"}, "hash": "e73808b5a4e43bf48817991f663aa13df302c147114c2c61d7c9b159c4af4429", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2a607c5f-9c64-48a1-b798-181b152dbf23", "node_type": "1", "metadata": {}, "hash": "a3cf30e6a90ebd7e2fb028f8d4e46dcec3c0765e0fb8c619d0bdeb7d3c1b389a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0  (2): 5 2 - 5 6   Research article  DOI: https://doi.org/10.54796/njb.v10i2.240 \n \n\n\u00a9NJB, BSN    52 \n\nComparative Study on Antioxidant Activity of Propolis of Apis mellifera \nfrom Different Regions of Nepal \nRajendra Gyawali  , Aparna Paudel, Babita Lamsal, Pranisha Bhatta, Albina Maharjan, Nina Khaitu, Rajan \n\nShrestha \n\nDepartment of Pharmacy, Herbal Research Laboratory, School of Science, Kathmandu University, Dhulikhel, Kavre, \n\nNepal \n\nReceived: 16 Aug 2022; Revised: 20 Dec 2022; Accepted: 23 Dec 2022; Published online: 31 Dec 2022 \n\nAbstract  \nPropolis is a waxy material obtained from honey bee hives. The physical and chemical property of this product is variable \nbased on the source of hive, plant biodiversity where honeybee feed, season of collection, geographical origin etc. Propolis \nhas several useful chemical compounds, and among them polyphenols are mainly contributing for their broad spectrum of \nmedicinal quality such as antimicrobial, antifungal, antibacterial, and anti-inflammatory activities as well as antioxidant \nproperties. The present study aims to analyze the ethanolic extract of propolis for their phenol and flavonoid content as well \nas its antioxidant characteristics. The samples (SPLs) were collected from farmers of six different locations of Nepal i.e.  Jhapa, \nLalitpur, Kathmandu, Banke and Chitwan districts. Total phenolic content (TPC) and total flavonoid content (TFC) were \nmeasured by Folin Ciocalteu method and the aluminum chloride method respectively expressed as the gallic acid (GAE) and \nquercetin (QE) and equivalent (GAE) per gram. The Diphenyl-Picrylhydrazyl (DPPH) assay method was used to evaluate the \nfree radical scavenging activity. The antioxidant effect of propolis was reported in ascorbic acid equivalent antioxidant \ncapacity per gram of propolis. The highest content of phenolic and flavonoid content was found in sample SPL 2. The range \nof these compounds' concentrations were from 127.36\u00b15.50 mg GAE/gram to 242.7\u00b14.50 mg GAE/gram. Similarly, total \nflavonoid content ranged from 1.3197\u00b10.0261 QE mg/ grams to 5.3921\u00b10.0261 QE mg/ grams. Whereas samples from SPL 2, \nand SPL 5 showed highest antioxidant properties. All samples were found to have strong antioxidant capacity which was \ngreater than standard.  It is concluded that there is no direct correlation on total antioxidant property of propolis with their \ntotal phenolic and total flavonoid content among collected samples. The phenolic characteristics of the samples were variable \nto the geographical location, and plant diversity of their origin.  \n\nKeywords: Nepalese Bee Propolis, Quality, Phenolics, Antioxidant, DPPH assay \n\n Corresponding author, email: ragyawali@gmail.com \n\nIntroduction  \nPropolis is a resinous material, also known as bee glue, \n\nthat bees collect from various plant sources for their \n\nhives. Bees create propolis, by combining saliva, \n\nbeeswax, and exudate obtained from tree buds, sap \n\nflowers, or other botanical sources. It has a variety of \n\nbiological activities and is an well-known natural \n\nremedy. Propolis is used to fill minor gaps, and burr \n\ncomb is used to cover bigger gaps [1]. It possesses the \n\nantibacterial capabilities provide protection against \n\ninfections [2, 3] .", "start_char_idx": 48, "end_char_idx": 3348, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2a607c5f-9c64-48a1-b798-181b152dbf23": {"__data__": {"id_": "2a607c5f-9c64-48a1-b798-181b152dbf23", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "57054d7e-2757-443f-b1c0-1fa3094e0d33", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "590acd61a6bf2a960659775af17071cf9eb04dbabf4b0f7f14ef3ddc9f67ad7e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3b4d541e-4692-4a0b-b692-6ec175013a15", "node_type": "1", "metadata": {}, "hash": "20590798213df8ccbb0e6846633b37f7ba50b43118aa8d894311ced547318f8b", "class_name": "RelatedNodeInfo"}}, "text": "The caffeic acid phenethyl ester is \n\nconsidered as a main constituent to inhibit the nuclear \n\nfactor \u03ba-B, inhibition of cell proliferation, induction of cell \n\ncycle arrest and apoptosis.   \n\nThe different climatic circumstances in which propolis is \n\nproduced affect its chemical components and overall \n\nnature. Geographic origin is associated with variations in \n\nthe chemical composition and consequently in the \n\nmedicinal property of propolis. Propolis contains \n\nphenolic compounds, esters, flavonoids, aromatic \n\naldehydes, resin, wax, and oil [4]. Numerous studies on \n\npropolis have revealed that it contains a variety of \n\nbiologically active constituents as well as unique \n\nproperties such as anti-inflammatory, antimicrobial, \n\nantioxidant, wound healing, and others [5]. The \n\nbiological activity of several beneficial chemicals derived \n\nfrom propolis for antibacterial, antifungal, antiviral, \n\nantiinflammation, anticancer, antioxidant, and other \n\nproperties can be applied in the pharmaceutical and \n\nhealth sectors [6\u20138]. Propolis can be employed as a free \n\nradical scavenger due to its significant oxidation \n\ninhibitory action [9]. The significant biological potency of \n\npropolis is demonstrated by its diverse composition [10]. \n\nMany researchers are working in the propolis because of \n\nits broad range of medicinal value and availability in \n\ndifferent part of the world. \n\nThere is an escalating scientific concern in the impact of \n\ngeographical origin of propolis on their chemical \n\nconstituents, physical characteristics and biological \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0002-5745-0702\nmailto:ragyawali@gmail.com\nmailto:ragyawali@gmail.com\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :5 2 - 5 6      Gyawali et al.  \n\n\u00a9NJB, BSN    53 \n\nactivities. Therefore the primary objective of this study \n\nwas to calculate the flavonoids and phenolics, and \n\nevaluate the antioxidant activities of propolis collected \n\nfrom various locations of Nepal.  \n\nMaterials and methods  \nCollection of propolis  \nBee propolis were collected from Jhapa, Lalitpur, \n\nKathmandu, Banke and Chitwan during June-July 2021. \n\nThe propolis was collected as chunks in propolis traps \n\nevery 15 days. The collected propolis chunks were stored \n\nin an air-tight container under refrigerated condition at \n\n40C for subsequent research purposes. \n\nSample preparation  \nTotal 10 grams of raw pulverized propolis was dissolved \n\nin 100 ml of 70% ethanol and warmed in a water bath. The \n\nmixture was transferred to conical flasks and set on a \n\nrotary shaker with suitable RPM for 72 hours. Thereafter, \n\nWhatman no 1 filter was used to obtain filtrate in conical \n\nflask. The extract was concentrated in rotavapor (Buchi \n\nR-215, Switzerland), 75-90 RPM, under 100 mbar pressure \n\nmaintaining 40\u2103 temperature of water bath (Figure 1). \n\n \nFigure 1. Propolis sample preparation \n\nTotal Phenolic content  \nTotal Phenolic Content (TPC) of developed propolis \n\npowder was estimated with slight modification in \n\npreviously described method [11] . For each sample, 1 ml \n\nof Folin-Ciocalteu reagent was mixed with 0.2 ml of \n\nextract. After 3 minutes, 1 ml of 10% sodium carbonate \n\nwas added, sodium carbonate helped speed up the \n\noxidation reaction of phenol. The resulting mixture was \n\nincubated at room temperature for 30 minutes. \n\nFurthermore, the absorbance at 280 nm was measured in \n\ntriplicates for each sample of propolis. The phenolic \n\ncontent of propolis was recorded in mg of gallic acid \n\nequivalent per gram.", "start_char_idx": 3349, "end_char_idx": 7043, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3b4d541e-4692-4a0b-b692-6ec175013a15": {"__data__": {"id_": "3b4d541e-4692-4a0b-b692-6ec175013a15", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2a607c5f-9c64-48a1-b798-181b152dbf23", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "ed96337223b1540499d30d8281d91027114bf0d09ccaeee06da639eafcf68db4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2638ec02-3eba-4303-81b6-d76810fc5c91", "node_type": "1", "metadata": {}, "hash": "95c6dd2271df901acd1ba95a530d153d1b8d52ca9796a0132ddcf0c81cc3cb3d", "class_name": "RelatedNodeInfo"}}, "text": "The standard curve was generated \n\nwith concentration of 0.8mg/ml of gallic acid. All \n\nsamples were calculated in triplicate.   \n\nTotal Flavonoid content \nTotal flavonoid content was measured with slight \n\nmodification in the procedure of previously described \n\nmethod [12].  For each sample, approximately 1.5 ml of \n\nethanol, 0.1 ml of 10% aluminum       chloride, 0.1 ml of 1 \n\nmol/L potassium acetate and 2.8 ml of water was added \n\nto 0.5 ml of extract. For about 30 minutes, the mixture was \n\nincubated at room temperature. After that, the \n\nabsorbance at 415 nm was measured in UV-Visible \n\nspectrophotometer. Each sample of propolis was \n\nmeasured in triplicates expressing the flavonoid content \n\nin mg of quercetin equivalent per gram of propolis. The \n\nstandard curve was generated with concentration of 0.8 \n\nmg/ml of quercetin.  \n\nDPPH radical scavenging activity assay \nDPPH (2, 2\u2032- diphenyl-1-picrylhydrazyl) assay was used \n\nto measure the free radical scavenging activity of the \n\nfractions as per the method described earlier  [13]. The \n\nstock solution in 100 ml Methanol was prepared by \n\ndissolving 3.94 mg DPPH. Ethanolic extracts of the \n\npropolis samples, and Ascorbic acid of different \n\nconcentrations (i.e., 60, 120, 180, 240 and 300 ppm) were \n\nprepared and reacted with aliquot solution of DPPH in \n\nthe ratio 1:3. The resulting mixture was incubated at \n\nroom temperature for 30 minutes. It was kept in a dark \n\nplace to protect from light. After that, the absorbance at \n\n517 nm was measured in triplicate. Based on the \n\npercentage of DPPH radical scavenged, the scavenging \n\nactivity was calculated using the following equation: \n\n\ud835\udc3c\ud835\udc5b\u210e\ud835\udc56\ud835\udc4f\ud835\udc56\ud835\udc61\ud835\udc56\ud835\udc5c\ud835\udc5b % =  \n\ud835\udc34\ud835\udc36\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59 \u2212 \ud835\udc34\ud835\udc46\ud835\udc4e\ud835\udc5a\ud835\udc5d\ud835\udc59\ud835\udc52\n\n\ud835\udc34\ud835\udc36\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59\n\u00d7 100% \n\nWhere AControl is the mean Absorbance reading of 0 ppm \n\nsolution against Methanol as Blank \n\nASample is the mean Absorbance reading of different \n\nconcentrations of the solutions \n\nResults and discussions \nPropolis from various locations of Nepal were subjected \n\nfor their quality analysis with the prospective of \n\nphenolics and antioxidant property. The electron \n\ndonating capacity of natural products can be estimated \n\nby using 2, 2\u2032-diphenyl-1- picrylhydrazyl radical (DPPH) \n\nreagent. This method is based on scavenging of DPPH \n\nradical through the addition of antioxidant which \n\nreduces the free radical of DPPH leading to its \n\ndecolorization. According to the study's findings, \n\npropolis extract contains phytoconstituents which can \n\ndonate hydrogen to a free radical to scavenge possible \n\ndamage. \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :5 2 - 5 6      Gyawali et al.  \n\n\u00a9NJB, BSN    54 \n\nTotal Phenolic content  \nGallic acid was used to perform the total phenolic content \n\nassay, and the standard curve equation was y = 0.001x + \n\n0.5883, where R2 = 0.9894. The absorbances were put in \n\nthe equation obtained from the Gallic acid standard, and \n\nTPC were determined (Figure 2).", "start_char_idx": 7044, "end_char_idx": 9966, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2638ec02-3eba-4303-81b6-d76810fc5c91": {"__data__": {"id_": "2638ec02-3eba-4303-81b6-d76810fc5c91", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3b4d541e-4692-4a0b-b692-6ec175013a15", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "45387f580b4be455522a5f24039b1426f5bfc380a3e275287c88c6f4ffbaeaf6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9b6f34ce-15a3-45b7-93b0-ae8b0ed97883", "node_type": "1", "metadata": {}, "hash": "09e96c62b3139c7553d97df88047f0348e817dd8de437e9f09079e3b6c6f8fbe", "class_name": "RelatedNodeInfo"}}, "text": "The range of these \n\ncompounds' concentrations was 127.36\u00b15.50 mg \n\nGAE/gram to 242.7\u00b14.50 mg GAE/gram. SPL 2 had the \n\nhighest concentration of phenols, followed by SPL 6. The \n\nlowest concentration was found in SPL 3 and SPL 4. \n\nOthers had average total phenolic content. In propolis \n\nsample, phenolic acids such as benzoic acid, cinnamic \n\nacid, and their derivatives are present. P-hydroxybenzoic \n\nacid, anisic acid, and gallic acid are some of the most \n\nprominent benzoic acid derivatives. Additionally, there \n\nis vanillic acid, salicylic acid, gentisic acid, 3,4-\n\ndimethoxybenzoic acid, protocatechuic acid, and 2-\n\namino-3-methoxybenzoic acid present. [14]. \n\nFigure 2: Total phenolic content of propolis collected from \n\ndifferent area of Nepal. \n\nTotal Flavonoid Content \nQuercetin used as the standard to estimate TFC in the \n\nsamples comparing the standard curve equation. \n\nQuercetin equivalent was calculated in milligram \n\nquercetin equivalent (QE) per gram. The samples were \n\nevaluated based on the quercetin equivalent. Total \n\nflavonoid content ranged from 1.3197\u00b10.0261 QE mg/ \n\ngrams to 5.3921\u00b10.0261 QE mg/ grams. SPL 2 was found \n\nto have the highest content of flavonoids 5.3921\u00b10.0261 \n\nmg QE/gram (Figure 3). Phenolic compounds such as; \n\nOdoratin, 7,3',4'-Trihydroxy-5'-methoxyisoflavonoid, \n\n6,7,3'-Trihydroxy-4'-methoxyisoflavonoid, 7,3'-\n\nDihydroxy-6,5'- methoxyisoflavonoid, Neoflavonoid 1 to \n\nNeoflavonoid 10, (S)-3'-hydroxy-4-methoxydalbergione, \n\n(S)-3',4'-dihydroxy-4-methoxydalbergione were also \n\nreported from Nepalese propolis [15-16].  \n\nFigure 3. Bar graph comparing total flavonoid content in given \n\nsamples \n\nAntioxidant activity  \nThe results from our experimental studies shows that the \n\nantioxidant capacity of the Nepalese originated propolis \n\nsamples are  very strong (Figure 4). The IC50 range of the \n\nsample were from 35 to 218 as compared with ascorbic \n\nacid standard. The sample's antioxidant property has an \n\ninverse relationship with the IC50 value. In other words, \n\nif the IC50 value is lower, the sample will need less to \n\nscavenge the free radical, and vice versa. Free radical \n\nscavenging activity in the sample is caused by the \n\npresence of bioactive components known as antioxidants. \n\nAdditionally, based on the above total phenolic content \n\nand total flavonoid content, SPL 2 contains the highest \n\nTFC and TPC, resulting in the strongest IC50 value. \n\nDPPH\u2022 is a stable purple radical that turns pale yellow \n\nwhen it absorbs free radicals. The antioxidant effect of \n\npropolis was reported in ascorbic acid equivalent \n\nantioxidant capacity (AEAC) per gram of propolis. All \n\npropolis showed an antioxidant capacity greater than 35 \n\nAEAC; 5 samples exceeded 100 AEAC, and sample \n\nnumber 6 exceeded 200 AEAC.  SPL2 has a lower IC50 \n\nvalue, indicating higher antioxidant activity, as less \n\nextract is required to inhibit 50% of the DPPH radical. \n\nPropolis comprises components from several different \n\nchemical families, including flavonoids, four aromatic \n\ncarboxylic acids, and eleven phenolic acid esters, which \n\nare responsible for its antioxidant properties, according \n\nto a prior study. Numerous studies show that the overall \n\nnumber of phenolic compounds is related to antioxidant \n\nactivity [17].", "start_char_idx": 9967, "end_char_idx": 13259, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9b6f34ce-15a3-45b7-93b0-ae8b0ed97883": {"__data__": {"id_": "9b6f34ce-15a3-45b7-93b0-ae8b0ed97883", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2638ec02-3eba-4303-81b6-d76810fc5c91", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "b640b76fc18153c4a1f39771d1341ba33bed0afbee8fa7fe225e98667e5de03c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "225f5b9a-7697-4204-9582-dea58d3b9bb8", "node_type": "1", "metadata": {}, "hash": "6ded9bb3313295fc7bac213e9933deb1e6864d9742bfdce07c8d4fd59b06f24e", "class_name": "RelatedNodeInfo"}}, "text": "The current investigation has also reinforced \n\nthe antioxidant property of Nepalese originated propolis \n\nsamples. The results also supported the assumption that \n\nan increase in the flavonoid concentration can lead to \n\nhigher antioxidant activity. \n\n0\n\n1\n\n2\n\n3\n\n4\n\n5\n\n6\n\nSPL1 SPL2 SPL3 SPL4 SPL5 SPL6\n\nQ\nu\n\ne\nrc\n\ne\nti\n\nn\n e\n\nq\nu\n\niv\na\n\nle\nn\n\nt \n(m\n\ng\n Q\n\nE\n/ \n\ng\n)\n\nPropolis Samples\n\nTotal Flavonoid Content\n\n0\n\n50\n\n100\n\n150\n\n200\n\n250\n\n300\n\nSPL1 SPL2 SPL3 SPL4 SPL5 SPL6\n\nG\na\n\nll\nic\n\n a\nci\n\nd\n e\n\nq\nu\n\niv\na\n\nle\nn\n\nt \n(m\n\ng\n G\n\nA\nE\n\n/\ng\n\n)\n\nPropolis samples\n\nTotal Phenolic Content\n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :5 2 - 5 6      Gyawali et al.  \n\n\u00a9NJB, BSN    55 \n\nIt was observed that bee propolis chemistry and \n\nantioxidant properties need to be compared together \n\nwith their geographical factors and plant species found \n\naround the beehive and bee feeding area having \n\nprominent Nepalese antioxidant herbs [18-20]. The future \n\nresearch on the propolis, can be focused on feeding \n\nexperiments, together with local biodiversity to identify \n\nthe species and behavior on propolis. The chemical \n\ncomposition of the propolis and its biological activities \n\ncan be correlated with their geographical origin of the \n\nsamples.  \n\n \nFigure 4. Comparison of IC50 value of samples. \n\nConclusion \nIt is concluded that the propolis of Apis mellifera from \n\ndifferent regions of Nepal possesses various strengths of \n\nantioxidant property. SPL 2 exhibited the highest levels \n\nof TFC and TPC, but antioxidant property was stronger \n\nin another sample, SPL 5. The concentrations of phenolic \n\ncompounds in sample might be influenced by the \n\nvegetation of bee farming area.  \n\nAuthor\u2019s contribution \nProject supervisors are Dr. Rajendra Gyawali, Dr. Rajan \n\nShrestha. Lab, fieldwork and writing manuscript were \n\ndone by Aparna Paudel, Albina Maharjan, Babita Lamsal, \n\nNina Khaitu and Pranisha Bhatta. Review and final \n\nediting were done by Rajendra Gyawali, Aparna Paudel \n\nand Babita Lamsal. All authors read and approved the \n\nfinal manuscript. \n\nCompeting interest \nNo competing interests were disclosed. \n\nEthical approval and consent \nNot applicable. \n\nAcknowledgement \nThe authors would like to express the gratitude to the \n\nDepartment of Pharmacy at Kathmandu University for \n\nsupplying the essential equipment, resources, and facility \n\nfor this research project. Furthermore, we thank to the \n\nKathmandu University Integrated Rural Development \n\nProject (KU-IRDP) funded by Korean International \n\nCooperation Agency (KOICA) Nepal for the Research \n\nand Business Development (R&BD) program. We are \n\nthankful to Mr Um Bahadur Purja Pun (Sunrise \n\nApiculture Pvt. Ltd) for the collection of the propolis \n\nsamples.  \n\nReferences \n1. Wagh VD. Propolis: A Wonder Bees Product and Its \n\nPharmacological potentials.  Advances in Pharmacological \nSciences.  2013, Article ID 308249 | Doi. 10.1155/2013/308249.  \n\n2. W Greenaway, T Scaysbrook & F R Whatley. The Composition \nand Plant Origins of Propolis: A Report of Work at Oxford, Bee \nWorld, 1990, 71:3, 107-11 Doi. 10.1080/0005772X.1990.11099047 \n\n3. Grange JM, Davey RW. Antibacterial properties of propolis (bee \nglue). J R Soc Med. 1990, 83(3):159-60. Doi.", "start_char_idx": 13260, "end_char_idx": 16488, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "225f5b9a-7697-4204-9582-dea58d3b9bb8": {"__data__": {"id_": "225f5b9a-7697-4204-9582-dea58d3b9bb8", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9b6f34ce-15a3-45b7-93b0-ae8b0ed97883", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "f80a71c9ed2152610b5a07f69a25f5ceceda87e615b1be8558236a8706a55e72", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8b2f748f-1d49-4eb9-811d-5137440c3b55", "node_type": "1", "metadata": {}, "hash": "f091ac55c166539dd5023759ee294b7ac068bf7540c8f7fffa2a65fd60ff52a3", "class_name": "RelatedNodeInfo"}}, "text": "1990, 83(3):159-60. Doi. \n10.1177/014107689008300310.  \n\n4. Pasupuleti VR, Sammugam L, Ramesh N, Gan SH. Honey, \nPropolis, and Royal Jelly: A Comprehensive Review of Their \nBiological Actions and Health Benefits. Oxid Med Cell Longev. \n2017, 2017:1259510. doi: 10.1155/2017/1259510.   \n\n5. Carpes S, Begnini R, Alencar S, Masson M. Study of preparations \nof bee pollen extracts, antioxidant and antibacterial activity. \nCi\u00eancia e Agrotecnologia 2007, 31, 31\u20131818. \n\n6. Wali A, Mushtaq A, Rehman M, Akbar S, Masoodi H. Bee \npropolis (bee's glue): a phytochemistry review. Journal of Critical \nReviews 2017, 4, 9\u201313. Doi. 10.22159/jcr.2017v4i4.20135. \n\n7. Paulino N, Coutinho L, Coutinho J, Vilela G, Silva Leandro V, and \nPaulino A. Antiulcerogenic Effect of Brazilian Propolis \nFormulation in Mice. Pharmacology & Pharmacy, 2015, 6, 580-\n588. Doi: 10.4236/pp.2015.612060. \n\n8. Oki\u0144czyc P, Paluch E, Franiczek R, et al. Antimicrobial activity of \nApis mellifera L. and Trigona sp. propolis from Nepal and its \nphytochemical analysis. Biomedicine & Pharmacotherapy = \nBiomedecine & Pharmacotherapie. 2020,129:110435. Doi. \n10.1016/j.biopha.2020.110435.  \n\n9. Anjm SI, Ullah A, Khan KA, Attaullah M, Khan H, Ali H, Bashir \nMA, Tahir M, Ansari MJ, Ghramh HA, Adgaba N, Dash CK. \nComposition and functional properties of propolis (bee glue): A \nreview. Saudi J Biol Sci. 2019, 26(7):1695-1703. Doi: \n10.1016/j.sjbs.2018.08.013.  \n\n10. Huang S, Zhang CP, Wang K, Li GQ, Hu FL. Recent advances in \nthe chemical composition of propolis. Molecules. 2014, \n26;19(12):19610-32. Doi: 10.3390/molecules191219610.   \n\n11. Vernon L. Singleton, Rudolf Orthofer, Rosa M. Lamuela-\nRavent\u00f3s, [14] Analysis of total phenols and other oxidation \nsubstrates and antioxidants by means of folin-ciocalteu \nreagent,Methods in Enzymology,Academic Press,1999, 299, 152-\n78. Doi. 10.1016/S0076-6879(99)99017-1.  \n\n12. Park YS, Jung ST, Kang SG, Heo BG, Arancibia-Avila P, Toledo F, \nDrzewiecki J, Namiesnik J, Gorinstein S, Antioxidants and \nproteins in ethylene-treated kiwifruits.  Food Chemistry 2008, 107, \n640\u2013648. \n\n13. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical \nmethod to evaluate antioxidant activity, LWT - Food Science and \nTechnology, 1995, 28(1), 25-30. \n\n14. K\u0119dzia B. Chemical composition of polish propolis. Part II. New \nStudies. Post. Fitoter.   2009, 2, 122\u2013128.  \n\n15.", "start_char_idx": 16464, "end_char_idx": 18844, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8b2f748f-1d49-4eb9-811d-5137440c3b55": {"__data__": {"id_": "8b2f748f-1d49-4eb9-811d-5137440c3b55", "embedding": null, "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-240", "node_type": "4", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "5bc4a74320ab88dd375b13ce59e703905872ec68de57cd830db445bc2b638f5d", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "225f5b9a-7697-4204-9582-dea58d3b9bb8", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "7d29e35646688104faf1e246b73ff3da034e9e4cee6cc95a49c20b15312f127f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0cca80e6-53d1-45a9-90dc-f331f934214f", "node_type": "1", "metadata": {}, "hash": "5fb83358a47e17af67d1ba654155ddb1f1b1c23c3dfb8d82bb39e16110f55136", "class_name": "RelatedNodeInfo"}}, "text": "Fitoter.   2009, 2, 122\u2013128.  \n\n15. Shrestha SP, Narukawa Y, Takeda T. Chemical constituents of \nNepalese propolis (II). Chem. Pharm. Bull. 2007, 55, 926\u2013929. \n\n16. Shrestha SP, Narukawa Y, Takeda T. Chemical constituents of \nNepalese propolis: Isolation of new dalbergiones and related \ncompounds. J. Nat. Med. 2007, 61, 73\u201376. \n\n17. Dezmirean DS, sca CPa, Moise AR, Bobi O s. Plant Sources \nResponsible for the Chemical Composition and Main Bioactive \nProperties of Poplar-Type Propolis. Plants, 2021, 10(1), 22. \ndoi: 10.3390/plants10010022. \n\n0 50 100 150 200 250\n\nStd\n\nSPL1\n\nSPL2\n\nSPL3\n\nSPL4\n\nSPL5\n\nSPL6\n\nIC 50 (ppm )\n\nS\na\n\nm\np\n\nle\ns\n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 :5 2 - 5 6      Gyawali et al.  \n\n\u00a9NJB, BSN    56 \n\n18. Bajgain A., Dev Neupane B., Sarraf D., Karmacharya J., Ranjitkar \nS., Shrestha R., & Gyawali R. Quality Evaluation of Apis laboriosa \nand Apis mellifera Honey Collected from Bagmati Province, \nNepal. Nepal Journal of Biotechnology, 2022, 10(1), 32\u201339. Doi. \n10.54796/njb.v10i1.228. \n\n19. Gyawali R, Ghimire A, Khatiwada A, Niraula P, Sharma U,  \nThapa R.  Anti-inflammatory, Anxiolytic and Antioxidant \nProperty of Lactuca sativa L and Formulation of Microspheres \n\nLoaded Sustained Release Anti-inflammatory Gel. Journal of \nNepal Chemical Society, 2020, 41(1), 8\u201315. Doi. \n10.3126/jncs.v41i1.30371 \n\n20. Gyawali R, Poudel N, Shrestha S, Silwal A. Formulation and \nevaluation of antibacterial and antioxidant polyherbal lotion. \nJournal of Institute of Science and Technology, 2016, 21(1):148-\n156.", "start_char_idx": 18809, "end_char_idx": 20346, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0cca80e6-53d1-45a9-90dc-f331f934214f": {"__data__": {"id_": "0cca80e6-53d1-45a9-90dc-f331f934214f", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8b2f748f-1d49-4eb9-811d-5137440c3b55", "node_type": "1", "metadata": {"identifier": "njb-240", "author": "Gyawali, Rajendra; Paudel, Aparna; Lamsal, Babita; Bhatta, Pranisha; Maharjan, Albina; Khaitu, Nina; Shrestha, Rajan", "title": "Comparative Study on Antioxidant Activity of Propolis of Apis mellifera from Different Regions of Nepal", "date": "2022-12-31", "file": "njb-240.pdf"}, "hash": "d214765b6e22209839d9caa514cd61b0f9783e24e6794312a3aa31016589ec0f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2dc04413-49a4-4585-9251-ccc884822cec", "node_type": "1", "metadata": {}, "hash": "f8c49ea714acb53b4f15f601eff7c820a093ea9d3b90360a3f7d72a4156746cf", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0  (2): 77-84  Research article  DOI: https://doi.org/10.54796/njb.v10i2.241  \n\n\u00a9NJB, BSN    77 \n\nEfficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast \nFungus Magnaporthe oryzae triticum in vitro \nM. K. Rehena1, *F. M. Aminuzzaman1  , M. L. Ashrafi1, M.S.M. Chowdhury1, U. A. Habiba1,  \n\nZ. Nazifa1 and M. Ahmed2 \n1Department of Plant Pathology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, \n\nDhaka-1207, Bangladesh,  \n2Scientific Officer, Plant Pathology Division, Bangladesh Rice Research Institute, Gazipur-1701 \n\nReceived: 02 Mar 2022; Revised: 03 Dec 2022; Accepted: 23 Dec 2022; Published online: 31 Dec 2022 \n\nAbstract \nThe present research work was conducted to evaluate the efficacy of eight fresh botanicals namely Azadirachta indica (Neem \nleaf), Allium cepa (Onion bulb), Allium sativum (Garlic), Curcuma longa (Turmeric), Zingiber officinale (Ginger), Allamanda \ncathartica (Allamanda leaf), Nigella sativa (Black cumin) and Aloe vera (Aloe vera) against Magnaporthe oryzae triticum (MoT) \nand solvent, ethanol (95%) was used for the phytochemical extraction of various plant parts. Three concentrations namely 1:1 \n(w/v), 1:0.50 (w/v) and 1:0.25 (w/v) of plant parts and ethanol were used for botanical extraction. The antifungal activity of \nbotanicals against a virulent MoT isolate CHMoT07 was evaluated in-vitro using poison food technique. The lowest mycelia \ngrowth was recorded with Aloe vera (Aloe vera leaf) extracts and Nigella sativa (Black cumin seeds) extracts @ 1:1 w/v and @ \n1:0.25 w/v with growth rate of 3.00 mm and 3.33 mm respectively at 7 days after inoculation, whereas the highest mycelia \ngrowth rate of MoT isolates was recorded in control plates both at 7 DAI and 14 DAI under in-vitro condition. \n\nKeywords: Wheat Blast, Magnaporthe oryzae triticum, botanical extracts, in vitro  \n\n Corresponding author, email: aminsaupp@yahoo.com \n\nIntroduction \nWheat (Triticum aestivum) is recognized as one of the \n\nmost important cereal crops within the world [1]. On the \n\nnational economy, it is typically a human food grain and \n\ncreated optimistic impact globally. Wiese (1987) [2] \n\nmentioned that, wheat provides about 20% of the world \n\nfood calories and nearly 40% of the total world \n\npopulation consumed it as staple food. Origin of wheat is \n\nfrom the Levant region of the Near East however \n\ncurrently cultivated worldwide. Thus, being larger than \n\nany other crop, wheat is grown on more than 701.5 \n\nmillion hectares [3].  In 2017, wheat ranked as the third \n\nmost-produced cereal in the world with the production \n\nof 771.7 million tons [4] through China (131.4 million \n\ntons), India (99.7 million tons), Russia (72.1million tons) \n\nand USA (51.3 million tons) were the four largest wheat \n\nproducers in 2018 [5]. \n\nAccording to the Department of Agricultural Extension, \n\nBangladesh in 2016, total wheat cultivated area in \n\nBangladesh is about 498,000 ha.", "start_char_idx": 48, "end_char_idx": 3044, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2dc04413-49a4-4585-9251-ccc884822cec": {"__data__": {"id_": "2dc04413-49a4-4585-9251-ccc884822cec", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0cca80e6-53d1-45a9-90dc-f331f934214f", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "e93b7ffcd651103d16e2d3476772963885f9908fe532b1964441d36dddb388b1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5d087cb3-91a8-4915-ac54-7162365e25cb", "node_type": "1", "metadata": {}, "hash": "3cc1a0b2cbb09d44f6c1327011ec9c84f8a6d1c68ff7c16c54bf473098bcc276", "class_name": "RelatedNodeInfo"}}, "text": "In the year of 2016 an \n\noutbreak of wheat blast was reported for the first time in \n\nBangladesh particularly in the districts of Kushtia, \n\nMeherpur, Chuadanga, Jhenaidah, Jessore, Barisal, \n\nBhola, Magura, Narail, and Faridpur [6]. It was estimated \n\nthat 15% area were affected around 101,660 ha of \n\ncultivated wheat area by a devastating wheat blast in the \n\nsame year. The incidence and severity of wheat blast \n\nassociated with yield losses among completely different \n\ndistricts varied considerably. The prevalence of wheat \n\nblast was higher in Meherpur (70%) although the very \n\nbest average yield loss (51%) was recorded in Jhenaidah. \n\nThe average yield loss was lower than 51% across \n\ndistricts and in several cases,100% yield losses were \n\nrecorded in individual wheat fields [7].  \n\nThe area beneath wheat cultivation currently extends to \n\nconcerning 1.78 lakh ha and the annual production is \n\nabout 10 lakh metric tons [8].  Some severely infected \n\nfields were burned due to approximately 15% of \n\nBangladesh\u2019s total wheat-affected area, which reduced \n\n15% wheat production in nine infected districts [6, 7, 9]. \n\nIn spite of such a decrease, in 2016 compared to that of \n\n2015, 2.7% of total wheat production in Bangladeshis \n\nincreased (35,000 metric ton [MT]). A rise in total \n\nharvested areas (420,000-425,000 ha) and increasing \n\nyields (3.10-3.14 MT/ha) contributed to the overall wheat \n\nproduction in 2016 [10]. \n\nUse of fungicides against wheat blast wasn\u2019t therefore \n\neffective and chemical application conjointly caused \n\nenvironmental pollution and toxicity to beneficial soil \n\nmicrobes, higher plants furthermore animals and human \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nmailto:aminsaupp@yahoo.com\nhttps://orcid.org/0000-0003-4804-0100\nmailto:aminsaupp@yahoo.com\nmailto:aminsaupp@yahoo.com\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    78 \n\nbeings. It has also enhanced production costs of farmers \n\n[11] and natural biological systems have disrupted over \n\ndecades because of their continual use and sometimes \n\nresulted in the development of fungal resistance besides \n\nproducing undesirable effects on non-target organisms, \n\nand also fostered environmental and human health \n\nconsiderations [12]. Rout and Tewari (2012) [13] stated in \n\ntheir study the potentiality of integrating in the \n\nmanagement of economically important diseases, the \n\nproducts prepared from green plants should be preferred \n\nas they are environmentally safe, non-pollutive, non-\n\ntoxic and non-hazardous to beneficial microorganisms. \n\nAllamanda (Allamanda cathertica) leaves are the \n\npromising source of many antifungal compounds with \n\nmedicinal properties [14]. The antifungal effect of the \n\nextracts of Neem (Azadirachta indica L.), Garlic (Allium \n\nsativum L.) and Calatropis (Calotropis procera L.) against \n\nMagnapothe oryzae triticum (MoT) was evaluated by \n\nKhanzada and Shah [15] following poisoned food \n\ntechnique. They found complete growth inhibition of the \n\ntest fungus in a higher dose of garlic extract in in vitro \n\nbioassay. Development of fungicidal resistance into the \n\npathogens and residual toxicity in soil and in the crop \n\nplants were owing to frequent use of fungicides. On the \n\nother hand, some botanical pesticides and bio-control \n\nagents have tested to be reliable and don\u2019t have any \n\nadverse effect on environment [16, 17]. Mondol et al.", "start_char_idx": 3045, "end_char_idx": 6619, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5d087cb3-91a8-4915-ac54-7162365e25cb": {"__data__": {"id_": "5d087cb3-91a8-4915-ac54-7162365e25cb", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2dc04413-49a4-4585-9251-ccc884822cec", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "e3318a0446a7216cd7233efe51c06f3dfcd75dfa087144f46af8bf662bcbc770", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "912aca74-a3f0-4fd4-8774-34aa669561f3", "node_type": "1", "metadata": {}, "hash": "b3039a45c6b3f3b2222784616e84ec30c8c8b0b10bf6d569a68452a8b57154ed", "class_name": "RelatedNodeInfo"}}, "text": "Mondol et al. 2018 \n\n[18] studied that chitosan (0.4%), salicylic acid (9mM) and \n\nbenzoic acid (9mM) are effective in suppressing the \n\ngrowth of MoT. Chitosan, salicylic acid and benzoic acid \n\nare bio-polymers and not harmful for ecosystem and are \n\ncompletely safe for human health. Therefore, these bio-\n\npolymers can be utilized as alternative to chemical \n\nfungicides for wheat blast management. \n\nTherefore, to control this devastating pathogen MoT \n\necofriendly management with non hazardous botanical \n\nextracts are going to be most effective until crop \n\nimprovement with desired resistance gene against this \n\npathogen in Bangladesh. The present research work was \n\nthus undertaken to find out the antifungal effect of some \n\nbotanical extracts against MoT in vitro. \n\nMaterials and Methods \nThe present investigations were carried out under \n\nlaboratory conditions from January to July, 2019 to \n\nascertain the incidence, severity of wheat blast and in-\n\nvitro evaluation of botanicals against MoT in the \n\nDepartment of Plant Pathology, Sher-e-Bangla \n\nAgricultural University, Dhaka.   \n\n \n\n \n\nFigure 1. Flow chart of isolation, identification, and culture of \nMagnaporthe oryzae triticum on OMA media \n\nDiseased plant samples were collected from infected \n\nwheat fields and preserved at 4\u00b0 C temperature in the \n\nlaboratory of SAU for isolation. Standard blotter method \n\n[19] was followed for the isolation of MoT pathogen from \n\ninfected wheat spikes. The water agar was prepared by \n\nmixing 20 g agar with 1000 ml distilled water, and potato \n\ndextrose agar media was made consisting of 200 g peeled \n\npotatoes, 20 g dextrose, and 20 g agar combined with 1000 \n\nml distilled water were used for the isolation of blast \n\npathogen. Appropriate size (15-20 cm in size) of diseased \n\nspikes infected with pathogen of wheat cultivars were cut \n\naround the area showing the blast lesion and were \n\nsurface sterilized with 1% sodium hypochlorite (NaOCl) \n\nfor 1 minute followed by 3 times washes with sterile \n\ndistilled water. To encourage sporulation, the plant \n\npieces were placed in petri dishes lined with moist filter \n\npapers and incubated at 26\u00b11\u00b0C for 24 hours. After \n\nincubation, these infected spike pieces were examined \n\nunder stereo-dissecting microscope (Motic, China). \n\nAbundant sporulation with grey, dense and bushy \n\nappearance was observed in and around the lesions. \n\nSingle conidium was picked out using a sterile moistened \n\nneedle across the sporulating lesion observing under the \n\nstereo microscope. The conidia were placed on water \n\nagar for further growth experimentation. After 12 hours, \n\nmycelium was visible in petri dish under the stereo \n\nmicroscope and then the hyphal tip was cut and placed \n\nin potato dextrose agar media plates containing \n\nStreptomycin (40 mg/L) and pure culture of MoT were \n\nprepared by incubating there in 26\u00b11\u00b0C. The marginal \n\nmycelial growth that developed subsequently was \n\npicked-up aseptically for sub-culturing until a pure \n\nculture of MoT was obtained. The culture was kept under \n\n12 hrs light and 12 hrs darkness conditions for \n\nsporulation. MoT isolate was identified by three-celled, \n\npyriform, light-colored conidia (Figure 1).  \n\nConidia of \n\nMoT(\u00d7100) \nBleached \n\npanicle \n\nPure culture of \n\nMoT on OMA", "start_char_idx": 6606, "end_char_idx": 9902, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "912aca74-a3f0-4fd4-8774-34aa669561f3": {"__data__": {"id_": "912aca74-a3f0-4fd4-8774-34aa669561f3", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5d087cb3-91a8-4915-ac54-7162365e25cb", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "5bbf18d5426d0f098b35b3a6ead569362d3d143cf70b38ca48287f765ada88df", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "14303a5d-5564-4d69-8f78-f3c53d919355", "node_type": "1", "metadata": {}, "hash": "4d0cdcd4ebed211856c81c06ed7246b261fa1cd242b022c40f644e219c6c2300", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    79 \n\nThe pure culture was maintained by subculturing at an \n\ninterval every 15 days and preserved at low temperature \n\n(4\u2070C) in refrigerator. \n\nFresh plant parts namely Azadirachta indica (Neem leaf), \n\nAllium cepa (Onion bulb), Allium sativum (Garlic), \n\nCurcuma longa (Turmeric), Zingiber officinale (Ginger), \n\nAllamanda cathartica (Allamanda leaf), Nigella sativa (Black \n\ncumin) and Aloe vera (Aloe vera) were used as treatments \n\n(Table 1 & Figure 2). 95% ethanol solvent was used for \n\nthe phytochemical extraction of various plant parts [20]. \n\nThree concentration 1:1 (w/v), 1:0.50 (w/v) and 1:0.25  \n\n (w/v) of ethanol was used for botanical extraction. For \n\n1:1 (w/v) concentration extraction with ethanol, 100g of \n\nplant materials was dissolved in 100 ml ethanol. To avoid \n\nevaporation and subjected to filtration through sterilized \n\nWhatman no.1 filter paper the mixture was kept \n\nundisturbed at room temperature in a sterile flask \n\ncovered with aluminum foil for 24 hrs.  \n\n \n\nFigure 2. Botanicals used in controlling mycelial growth of Magnaporthe oryzae triticum in-vitro. Extracts of A. Allium cepa, B. Allium \nsativum, C. Curcuma longa, D. Zingiber officinale, E. Azadirachta indica, F. Nigella sativa, G. Allamanda cathartica, H. Aloe vera \n\n \n\n \nFigure 3. Botanical extracts used in controlling mycelial growth of Magnaporthe oryzae triticum in-vitro. Extracts of A. Allium cepa, B. \nAllium sativum, C. Curcuma longa, D. Zingiber officinale, E. Azadirachta indica, F. Nigella sativa, G. Allamanda cathartica, H. Aloe vera \n\n \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    80 \n\nAfter filtration, the extract was evaporated in water bath \n\nuntil 100 ml extract was left in the container (Figure 3). \n\nFor 1:0.50 (w/v) and 1:0.25 (w/v) 100 g of plant materials \n\nwere dissolved in 50 ml and 25 ml ethanol, respectively \n\n[20]. Ethanolic extract thus obtained were immediately \n\nevaluated for antifungal activities using the poisoned \n\nfood technique [21].  \n\nPDA plates were amended with different concentration \n\n(1:1 w/v, 1:0.50 w/v, 1:0.25 w/v) of botanical extracts \n\nseparately. The plates were inoculated with 5 mm fungal \n\nblocks with the help of sterilized needle, MoT and these \n\nblocks were transferred to the center of the petri plates. \n\nThe mycelial growth of MoT was recorded at seven days \n\nafter inoculation by measuring the average of two \n\ndiameters at right angles (90 degrees) to one another. \n\nThree replications were maintained for each treatment \n\nand the mean radial mycelial growth was considered for \n\nmeasuring each treatment. The effect of plant extract was \n\ncalculated as percent growth inhibition using the \n\nfollowing formula as adopted by [22, 23]. \n\n% inhibition = (dc \u2013 dt)/dc \u00d7 100  \n\nWhere dc = Average increase in mycelial growth in \n\ncontrol, dt = Average increase in mycelial growth in \n\ntreatment. \n\nStatistical analysis   \nA completely randomized design (CRD) with three \n\nreplications was applied in this experiment.", "start_char_idx": 9907, "end_char_idx": 13010, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "14303a5d-5564-4d69-8f78-f3c53d919355": {"__data__": {"id_": "14303a5d-5564-4d69-8f78-f3c53d919355", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "912aca74-a3f0-4fd4-8774-34aa669561f3", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "72b77564bb58ce82b1772e997682aa8c26c5368db7302a0700244451bf07665b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "02f9901b-772e-4572-9c1b-b2b7b6339567", "node_type": "1", "metadata": {}, "hash": "f61ac95a77a4508572de1f940c742c45a095c94c605a4715e70410da2effca8d", "class_name": "RelatedNodeInfo"}}, "text": "Analysis of \n\ndata of different parameters was subjected to perform by \n\nstatistical analysis using R software version 3.6.0 [24]. \n\nResults \nAll botanicals performed significantly effective against \n\nMoT isolate in lessening mycelial growth compare to \n\ncontrol (Table 2). The result revealed that Aloe vera (Aloe \n\nvera leaf) extracts was found most effective in reducing \n\nthe mycelial growth at 7 days but in 14 days Nigella sativa \n\n(Black cumin seed) extracts and Aloe vera (Aloe vera leaf) \n\nextracts both botanicals significantly reduced the \n\nmycelial growth of MoT.  \n\nTable 2. Efficacy of botanicals on mycelial growth of M. oryzae \n\ntriticum \n\nTreatments \n\nRadial mycelial \ngrowth (mm) \n\n% Growth reduced \nover control \n\n7 DAI 14 DAI 7 DAI 14 DAI \n\nAllium cepa 35.22b 42.00d 28.93 38.93 \n\nAllium sativum 21.67e 52.56b 56.27 23.58 \n\nCurcuma longa 26.78d 47.11c 45.94 31.50 \n\nZingiber officinale 26.78d 36.78f 49.96 46.52 \n\nAzadirachta indica 31.56c 52.00b 36.31 24.39 \n\nNigella sativa 4.11g 10.00g 91.70 85.46 \n\nAllamanda cathartica 26.22d 40.33e 47.09 41.36 \n\nAloe vera 5.22f 8.89g 89.46 87.07 \n\nControl 49.56a 68.78a   \n\nCV (%) 3.68 3.02   \n\nIn a column treatment means with the same letter are not significantly \ndifferent. DAI= Days after inoculation \n\nTable 3. Effect of different concentration levels of botanicals on mycelial \ngrowth of M. oryzae triticum \n\nConcentration of \n\nbotanicals \n\nRadial mycelial growth (mm) \n\n7 DAI 14 DAI \n\n1:0.25 w/v @0.1% 30.37a 47.59a \n\n1:0.50 w/v @0.2% 25.89b 40.74b \n\n1:1 w/v @0.4% 19.44c 31.15c \n\nCV (%) 3.68 3.02 \n\nIn a column treatment means with the same letter are not significantly \n\ndifferent.   \n\nDAI=Days after inoculation \n\n \n\nTable 1.", "start_char_idx": 13011, "end_char_idx": 14707, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "02f9901b-772e-4572-9c1b-b2b7b6339567": {"__data__": {"id_": "02f9901b-772e-4572-9c1b-b2b7b6339567", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "14303a5d-5564-4d69-8f78-f3c53d919355", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "cc9f64933233da5e842105a3e732e73e711edaa7d2b61f820bca934f158bc5a7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "43ace114-a2e6-4240-84a3-d4cd045874aa", "node_type": "1", "metadata": {}, "hash": "fe8874670eb3025fe5444125a05d175a8ab4ddaba6a1a1e0de9fd805b0d16c27", "class_name": "RelatedNodeInfo"}}, "text": "DAI=Days after inoculation \n\n \n\nTable 1. Botanicals used in controlling mycelia growth \n\nof Magnaporthe oryzae triticum in-vitro \n\nName of botanicals \nConcentration \n\nused \nScientific \n\nname \nEnglish name \n\nPlant parts \n\nused \n\nAllium cepa Onion Bulb \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nAllium \n\nsativum \nGarlic Clove \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nCurcuma \n\nlonga \nTurmeric Rhizome \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nZingiber \n\nofficinale \nGinger Rhizome \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nAzadirachta \n\nindica \nNeem Leaf \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nNigella \n\nsativa \nBlack cumin Seed \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nAllamanda \n\ncathartica \nAllamanda Leaf \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nAloe vera Aloe vera Leaf \n\n1:0.25(w/v) \n\n1:0.50 (w/v) \n\n1:1 (w/v) \n\nControl \nNo botanical \n\nextracts", "start_char_idx": 14667, "end_char_idx": 15528, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "43ace114-a2e6-4240-84a3-d4cd045874aa": {"__data__": {"id_": "43ace114-a2e6-4240-84a3-d4cd045874aa", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "02f9901b-772e-4572-9c1b-b2b7b6339567", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "e72b56105de9dbd18bedd1780231d7b7559aab4ba70d5fc89deecc735535ef6e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "436ea7e0-d6d7-4fb6-b827-4c35bed4afb5", "node_type": "1", "metadata": {}, "hash": "fdc644a60b8d5d3d27db28b821a5a6d4a6b50cd887973c25f8e577ad6d1cad0f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    81 \n\nThree concentration level of botanicals (1:0.25 w/v @ \n\n0.1%, 1:0.50 w/v @ 0.2%, 1:1 w/v @ 0.4%) was tested \n\nagainst the pathogen and (1:1) found the best \n\nconcentration in both 7 DAI and 14 DAI (Table 3). With \n\nthe concentration increase of all tested plant extracts, \n\nthe mycelial growth of MoT decreases. \n\nThe growth characteristics like media color, colony color \n\nand shape of colony of MoT on PDA media were noticed \n\nsupplemented with different plant extracts. In case of \n\nPDA media, the colony color of MoT was grey ash centre \n\nand black margin. In contrast, PDA media supplemented \n\nwith Azadirachta indica (Neem leaf) extract was showed \n\nwhite with grey, Allamanda cathartica (Allamanda leaf) \n\nextract, Aloe vera (Aloe vera) leaf extract, and Nigella sativa \n\n (Black cumin) seed extracts showed white colony color. \n\nAllium cepa (Onion bulb) extracts, Allium sativum(Garlic) \n\nextracts and Curcuma longa (Turmeric rhizome) extracts \n\nand Zingiber officinale (Ginger) showed rhizome extract \n\nshowed grey with white color colony.  This result showed \n\nthat PDA media supplemented with totally different \n\nplant extracts have an effect on the colony color of M. \n\noryzae triticum (Figure 4 and Table 4). \n\nAntimicrobial activities of eight botanicals with specific \n\nconcentration were assayed and results on presented in \n\nTable 4. The data revealed that botanicals were found \n\nsignificant in suppression of mycelia growth at higher \n\nconcentration over untreated check in the fungal \n\npathogens.  \n\nAntimicrobial activities of eight botanicals with specific \n\nconcentration were assayed and results on presented in \n\nTable 4. The result reveals that the minimum radial \n\nmycelial growth was recorded from Nigella sativa: 1:1  \n\n \nFigure 4. Mycelial growth, color and appearance of M. oryzae triticum on PDA media supplemented with ethanol \nextracts of different botanicals (7 DAI). A.  Allium cepa, B. Allium sativum, C. Curcuma longa, D. Zingiber officinale, E. \nAzadirachta indica, F. Nigella sativa, G. Allamanda cathartica, H. Aloe vera, I. Control  \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    82 \n\nw/v @ 0.4% (3.00 mm) combination at 7 days culture age \n\nwhich is statistically similar with Aloe vera: 1:1 w/v @ \n\n0.4% (3.33mm) and Nigella sativa: 1:0.50 w/v @ 0.2% (4.33 \n\nmm) combination. Control plates (only PDA: 1:0.50 w/v \n\n@ 0.2% and only PDA: 1:1 w/v @ 0.4%) were always \n\nobserved the highest growth of mycelium. At 14 days \n\nculture age, lowest mycelial growth was observed in Aloe \n\nvera: 1:1 w/v @0.4% combination (6.00 mm). In 2nd lower \n\ngrowth recorder from Aloe vera: 1:0.50 w/v @0.2% (9.00 \n\nmm) and Nigella sativa: 1:1 w/v @0.4% (9.00) \n\ncombination, which were statistically identical to each \n\nother.", "start_char_idx": 15536, "end_char_idx": 18411, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "436ea7e0-d6d7-4fb6-b827-4c35bed4afb5": {"__data__": {"id_": "436ea7e0-d6d7-4fb6-b827-4c35bed4afb5", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "43ace114-a2e6-4240-84a3-d4cd045874aa", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "5d6694783e1fdb4ae6731439752e7e2acc7e496738cf869a9d988394f775a4d1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4a5c8b05-4940-46d1-99a7-e69b70eb86b6", "node_type": "1", "metadata": {}, "hash": "852ddf3dc606dedcd9204f527592d9ded3f6f823a6b656a26f54acf6ba56ce6b", "class_name": "RelatedNodeInfo"}}, "text": "Control plates (only water amended PDA: (50.00), \n\n(49.00) and (49.67)) were observed highest growth of \n\nmycelium both at 7 days culture age and 14 days culture \n\nage. The highest concentration of botanical extracts was \n\nmore effective compared to low concentration in \n\nreducing the radial mycelia growth of the fungus as \n\nobserved in case of blast pathogen. With the \n\nconcentration increase of all tested botanical extracts, the \n\nmycelial growth of MoT reduced compared to control. \n\nDiscussion  \nIn Bangladesh about 80 % of people lean on directly on \n\nagriculture for their food and livelihood, with wheat \n\nbeing the second most important crop after rice. Wheat \n\nblast is caused by MoT is a new disease in Bangladesh \n\nwhich may cause up to 100% yield loss of wheat. In \n\nanother study significant losses in grain yield due to \n\nwheat blast were observed in all the survey sites of the \n\ntwo south-western districts of Bangladesh (Tanjina et al., \n\n2019) [25]. \n\nThe result evaluated that the lowest radial mycelial \n\ngrowth was recorded from Nigella sativa: 1:1 w/v @ 0.4% \n\n(3.00 mm) combination at 7 days culture age which is \n\nstatistically similar with Aloe vera: 1:1 w/v @ 0.4% \n\n(3.33mm) and Nigella sativa: 1:0.25 w/v @ 0.2% (4.33 mm) \n\ncombination. Control plates (only PDA) were always \n\nobserved highest growth of mycelium. At 14 days culture \n\nage, lowest mycelial growth was observed in Aloe vera: \n\n1:1 w/v @ 0.4% combination (6.00 mm). In 2nd lower \n\ngrowth recorder from Aloe vera: 1:0.25 w/v @0.2% (9.00 \n\nmm) and Nigella sativa: 1:1 w/v @0.4% (9.00) \n\ncombination, which were statistically identical each \n\nother.  Control plates (only PDA (50.00), (49.00) and \n\n(49.67)) were observed highest growth of mycelium both \n\nat 7 days culture age and 14 days culture age. The \n\nfindings are similar to Zohura et al. (2018) [26] who \n\nreported the effectiveness of 12 plant extracts namely \n\nneem leaf extract, bishkatali leaf extract, nishinda leaf \n\nextract, allamonda leaf extract, acasia leaf extract, tulsi \n\nleaf extract, mehendi leaf extract, datura leaf extract, \n\nbishkochu leaf, black cumin seed extract, garlic clove \n\nextract and mehogoni seed extracts against MoT in vitro. \n\nAmong these twelve plant extracts, four plant extracts \n\nviz. Tulsi leaf extract, Mehendi leaf extract, Datura leaf \n\nextract and Garlic clove extract impede the highest \n\npercentage (93.75%) of mycelial growth followed by \n\nBlack cumin seed extracts (90%) at 10 DAI where lowest \n\npercentage of mycelial growth inhibition (7.5%) were \n\nrecorded in Allamonda leaf extract over control.  \n\nThe highest concentration of botanicals extracts was more \n\neffective compare to low concentration in reducing the \n\nradial growth of the fungus as observed in case of blast \n\npathogen. The results are similar with the findings of \n\nTable 4.", "start_char_idx": 18412, "end_char_idx": 21254, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4a5c8b05-4940-46d1-99a7-e69b70eb86b6": {"__data__": {"id_": "4a5c8b05-4940-46d1-99a7-e69b70eb86b6", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "436ea7e0-d6d7-4fb6-b827-4c35bed4afb5", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "121c61ee94fc28c06c396c6e6f6826e852b3f93038238dbbbd0b5b76f2ee6f42", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fc1ef1c6-bacb-45d9-86aa-c4a22582fa0b", "node_type": "1", "metadata": {}, "hash": "e0a0f98147f195929c55840a6fc3595d73ffb5876f454e0fc1253e29316f678f", "class_name": "RelatedNodeInfo"}}, "text": "The results are similar with the findings of \n\nTable 4. Effects of ethanol extracts of botanicals on mycelia \ngrowth and colony characters of Magnaporthe oryzae triticum \n\nTreatments \n\nEthanol \n\nbotanicals \n\nratio (w/v) \n\nRadial mycelia \n\ngrowth (mm) \nColony character \n\n7 DAI 14 DAI \nColony \n\ncolor \nShape \n\nAllium \n\ncepa \n\n1:0.25 38.33b 58.33c \n\nGray ash Regular 1:0.50 36.00c 41.67hi \n\n1:1 31.33f 26.00l \n\nAllium \n\nsativum \n\n1:0.25 31.00f 60.33bc \n\nGray ash Regular 1:0.50 23.00i 56.33d \n\n1:1 11.00m 41.00i \n\nCurcuma \n\nlonga \n\n1:0.25 33.33e 59.00bc Gray with \n\nwhite \n\nmargin \n\nRegular 1:0.50 29.00g 49.00f \n\n1:1 18.00k 33.33j \n\nZingiber \n\nofficinale \n\n1:0.25 34.00de 46.33g \nGray with \n\nwhite \n\nmargin \n\nRegular 1:0.50 26.67h 40.33i \n\n1:1 19.67j 23.67m \n\nAzadiracht\n\na indica \n\n1:0.25 39.00b 61.00b \nGrey with \n\nwhite \nRegular 1:0.50 30.33fg 51.67e \n\n1:1 25.33h 43.33h \n\nNigella \n\nsativa \n\n1:0.25 5.00o 11.33n \n\nWhite Regular 1:0.50 4.33opq 9.67no \n\n1:1 3.00q 9.00o \n\nAllamanda \n\ncathartica \n\n1:0.25 35.00cd 51.00e \n\nWhite Regular 1:0.50 30.00fg 40.33i \n\n1:1 13.67l 29.67k \n\nAloe vera \n\n1:0.25 7.67n 11.67n \n\nWhite Regular 1:0.50 4.67op 9.00o \n\n1:1 3.33pq 6.00p \n\nControl \n\nOnly PDA 50.00a 69.33a \nGrey with \n\nwhite \nRegular Only PDA 49.00a 68.67a \n\nOnly PDA 49.67a 68.33a \n\nCV% 3.68 3.02  \n\nIn a column treatment means with the same letter are not significantly \n\ndifferent  DAI= Days after inoculation \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    83 \n\nZohura et al., (2018) [26] where no growth inhibition had \n\ntaken placed on control plate. Allamonda leaf extract \n\nperformed with the minimum percent growth inhibition \n\n(7.5%) compared to control while the maximum \n\ninhibition was observed in the plates containing Garlic \n\nclove, Tulsi leaf, Mehendi leaf (93.75%) and Black cumin \n\nseed extract (90%) respectively after 10 days of \n\ninoculation. This result is also in conformity with the \n\nfindings where they stated that H. anthelminthicus fruit \n\nextracts exhibited antifungal potential to growth \n\ninhibition at the 10,000-ppm concentration and 100% \n\ngrowth inhibition against Pyricularia oryzae, P. palmivora \n\nand R. solani were noted followed by S. rolfsii at 96.33% \n\nwhen compared with water control [27]. \n\nFor controlling the rice blast disease In-vitro and in-vivo \n\nexperiment were carried out by Hubert et al.", "start_char_idx": 21199, "end_char_idx": 23575, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fc1ef1c6-bacb-45d9-86aa-c4a22582fa0b": {"__data__": {"id_": "fc1ef1c6-bacb-45d9-86aa-c4a22582fa0b", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4a5c8b05-4940-46d1-99a7-e69b70eb86b6", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "c0cc717c4d16fe8c926c9e916ef7117393bd20d9c928cadb2ff909d230c12d0a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d1ab02df-b5f2-47e7-ab6e-19791974beb3", "node_type": "1", "metadata": {}, "hash": "d9a9e8edcd68baf9abb7217caa20c78e5d6d3464de6891f95694d96c446c8de2", "class_name": "RelatedNodeInfo"}}, "text": "(2015) [28] \n\nwhere the effects of Aloe vera, Allium sativum, \n\nAnnonamuricata, Azadirachta indica, Bidenspilosa, Camellia \n\nsinensis, Chrysanthemum coccineum, processed Coffee \n\narabica, Datura stramonium, Nicotiana tabacum and Zingiber \n\nofficinalis extracts for control of rice blast disease \n\n(Magnaporthe oryzae) both were assessed. At 10% and 25% \n\n(v/v) the highest (81.12%) and (89.40%) inhibitory effect \n\nwas observed in processed C. arabica against P. grisea, \n\nrespectively. To manage rice blast disease these plant \n\nextracts can be used. \n\nConclusions \nWheat blast caused by Magnaporthe oryzae triticum (MoT) \n\nrecognized as a devastating disease and caused up to \n\n100% yield loss first time in some wheat growing areas of \n\nBangladesh in the year 2016. As there are no appropriate \n\ncontrol measures have been developed till now so the \n\neffective management of this pathogen is the \n\nenvironment friendly management with botanical \n\nextracts until the establishment of resistance cultivars \n\nagainst this notorious pathogen in Bangladesh. \n\nThe highest radial mycelial growth inhibition Aloe vera \n\n(Aloe vera leaf) extracts and Nigella sativa (Black cumin \n\nseeds) extracts 1:1 w/v @ 0.4% (3.00mm and 3.33mm) \n\nconcentration at 7 days after inoculation, whereas the \n\nlowest inhibition Allium cepa (onion) extracts 1:0.25 w/v \n\n@ 0.1% (38.33mm) concentration of MoT under in-vitro \n\ncondition. However, this experiment with plant extracts \n\nurgent to be invented out to assess the field efficacy of \n\nthese botanical extracts with different concentrations and \n\nfrequencies in controlling blast of wheat.  \n\nAcknowledgements \nWe acknowledge Sher- e-Bangla Agricultural University \n\nResearch System (SAURES), Dhaka, Bangladesh for their \n\nsupport. \n\nConflict of interest \nThe authors declare no conflict of interest.  \n\nEthical issues \nThere are no ethical issues involved in this research work. \n\nFunding \nThis research work was supported by Sher- e-Bangla \n\nAgricultural University Research System (SAURES), No. \n\nSAU/SAURES/2019/1847(35), Dhaka, Bangladesh. \n\nAuthor contributions \nM. K. Rehena collected wheat blast samples from the \n\nfield, conducted the research and wrote the article; F. M. \n\nAminuzzaman designed and supervised the study and \n\nedited the manuscript; M. L. Ashrafi and U. A. Habiba \n\ncollected the blast samples from field and analyze the \n\ndata; M. S. M. Chowdhury, Z. Nazifa and M. Ahmed read \n\nthe manuscript contributed to the conceptualization, and \n\nmethodology of the study. \n\nReferences \n1. Lu, M., Cao, X., Pan, J., Gurajala, H. K., He, Z., Yang, X., and Khan, \n\nM. B. 2020. Genotypic variations in zinc accumulation and \nbioaccessibility among wheat (Triticum aestivum L.) genotypes under \ntwo different field conditions. Journal of Cereal Science, 93, 102953. \nhttps://doi.org/10.1016/j.jcs.2020.102953. \n\n2. Wiese, M. V. 1987. \u201cCompendium of wheat diseases\u201d.2nd Ed. \nAmerican Phytopathol. Soci. St. Paul. Minnesota. 112. \n\n3. BBS. (Bangladesh Bureau of Statistics). 2014. Statistical yearbook of \nBangladesh. Bureau of Statistics Division, Ministry of Planning, \nGovernment Republic of Bangladesh, Dhaka, Bangladesh. \n\n4. FAOSTAT. (Food and agriculture organization of the united nations \nstatistics division). 2017. Production/ crop rice paddy. \n\n5. FAOSTAT 2019.", "start_char_idx": 23576, "end_char_idx": 26893, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d1ab02df-b5f2-47e7-ab6e-19791974beb3": {"__data__": {"id_": "d1ab02df-b5f2-47e7-ab6e-19791974beb3", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fc1ef1c6-bacb-45d9-86aa-c4a22582fa0b", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "374f9a16685a69271a7854d474aabd71490c65bbfdce49f9db2b1fbcf15bd82a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "31513d1a-1fa5-4e7f-89b1-8ac76629fc09", "node_type": "1", "metadata": {}, "hash": "79fa737703f02eb89e79982d2ac07de91ae0c901be5c7412f7435d9af2c3fa4d", "class_name": "RelatedNodeInfo"}}, "text": "Production/ crop rice paddy. \n\n5. FAOSTAT 2019. Countries by commodity. Food and Agricultural \nOrganisation. \nhttp://www.fao.org/faostat/en/#rankings/countries_by_commod\nity. \n\n6. Malaker, P. K., Barma, N. C. D., Tiwari, T. P., Collis, W. J., Duveiller, \nE., Singh, P. K.,Joshi, A. K., Singh, R. P., Braun, H. J., Peterson, G. L., \nPedley, K. F., Farman, M. L. and Valent, B. 2016. First report of wheat \nblast caused by Magnaporthe oryzae pathotype triticum in Bangladesh. \nPlant Disease 100:2330\u20132330. https://doi.org/10.1094/PDIS-05-16-\n0666-PDN \n\n7. Islam, M. T., Croll, D., Gladieux, P., Soanes, D. M., Persoons, A., \nBhattacharjee, P., Hossain, M. S., Gupta, D. R., Rahman, M. M., \nMahboob, M. G., Cook, N., Salam, M. U., Surovy, M. Z., Sancho, V. \nB., Maciel, J. L. N., Nhani, A., Castroagudin, V. L., Reges, J. T. D., \nCeresini, P. C., Ravel, S., Kellner, R., Fournier, E., Tharreau, D., \nLebrun, M. H., Mcdonald, B. A., Stitt, T., Swan, D., Talbot, N. J., \nSaunders, D. G. O., Win, J. and Kamoun, S. 2016. Emergence of wheat \nblast in Bangladesh was caused by a South American lineage of \nMagnaporthe oryzae. BMC Biology14:84-11. \n\n8. BBS. (Bangladesh Bureau of Statistics). 2017. Statistical Pocket Book \nBangladesh. Bangladesh Bureau of Statistics, Dhaka, Bangladesh. \n\n9. Aman, A. 2016. 'Wheat blast' threatens yield. The Daily Star.Retrieved \nfrom https://www.thedailystar.net/backpage/wheat-blast-\nthreatens-yield-784372. \n\n10. USDA. 2019. Production, Supply and Distribution. Foreign \nAgricultural Service,United States Department of Agriculture. \nRetrived from https://apps.fas.usda.gov/psd \nonline/app/index.html#/app/downloads. \n\nhttps://doi.org/10.1094/PDIS-05-16-0666-PDN\nhttps://doi.org/10.1094/PDIS-05-16-0666-PDN\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 77-84     Rehena et al.  \n\n\u00a9NJB, BSN    84 \n\n11. West, J.S., Bravo, C., Oberit, R., Lemaire, D., Moshou, D., McCartney, \nH.A. 2003. The potential of optical canopy measurement for \ntargeted control of field crop diseases, Annual Review of \nPhytopathology, 41, 593\u2013\n614.http://doi.org/10.1146/annurev.phyto.41.121702.103726 \n\n12. Yoon, Mi-Young., Cha, B., Kim, Jin-Cheol. 2013. Recent Trends in \nStudies on Botanical Fungicides in Agriculture, Plant Pathol J., \n29(1), 1\u20139.doi: 10.5423/PPJ.RW.05.2012.0072 \n\n13. Rout, S., and Tewari, S.N. 2012.", "start_char_idx": 26846, "end_char_idx": 29169, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "31513d1a-1fa5-4e7f-89b1-8ac76629fc09": {"__data__": {"id_": "31513d1a-1fa5-4e7f-89b1-8ac76629fc09", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d1ab02df-b5f2-47e7-ab6e-19791974beb3", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "ea91d788fd703bc4b037fb9c628a9c442a42e5862f3206d6398386b7f94ef86e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e01da986-0152-4e19-ac88-3cbd0d6004af", "node_type": "1", "metadata": {}, "hash": "f066c11f3fbc6c3fdf6d9a00c2cdee483f26f579edbdcb00934fb751dc165ec1", "class_name": "RelatedNodeInfo"}}, "text": "Rout, S., and Tewari, S.N. 2012. Fungitoxic spectrum of Amalab-a \nagainst fungal pathogens in rice under in vitro, J Biopest, 5,161-167. \n\n14. Rumana I. 2004. Chromatographic separation of components in \ngarlic bulb and allamanda leaf extracts inhibitory to Phomopsis \nvegans. MS Thesis. Dept. of Plant Pathology, Bangladesh \nAgricultural University, Mymensingh. \n\n15. Khanzada, C. and Shah, B. 2012. Magnaporthe oryzae and rice blast \ndisease. In: Borkovich A, Ebbole D, eds. Cellular and Molecular \nBiology of Filamentous Fungi. Washington, DC, USA: ASM \nPress.593\u2013606.https://doi.org/10.1128/9781555816636.ch37 \n\n16. Iftikhar, T., Babar, L. K., Zahoor, S. and Khan, N. G. 2010. Best \nirrigation management practices in cotton. Pak. J. Bot. 42:3023\u20133028. \n\n17. Babar, M. and Khan, I. A. 1999. Genetic analysis of some agronomic \nand fibre characters in upland cotton (Gossypium hirsutum L.). \nPakistan Journal of Biological Sciences 2:1484-1487. \n\n18. Mondol, M. N., Sultana, A., Tumpa, F. H., Kashem, M. A. and \nKhokon, M. A. R. 2018. In-vitro suppression of wheat blast pathogen \nMagnaporthe oryzae pathotype triticum by elicitors. Bangladesh \nJournal of Plant Pathology 34 (1&2): 53-58. \n\n19. ISTA. (1996). International Rules for Seed Testing. Seed Science and \nTechnology 4:3\u201349. doi: 10.15258/istarules.2015.f. \n\n20. Somshetty, R., Bimla, R., Sirivella, N. And Vanama, S. 2020. In-Vitro \nevaluation of aqueous and ethanol extracts of botanicals against R. \nsolani causing sheath blight of rice. Int. J. Curr. Microbial App. Sci. \n11: 2772-2781. \n\n21. Nene, Y.L. and Thapliyal, P.N. 1979. Fungicides in Plant Disease \nControl. 2nd ed. Oxford and IBH pub.Co. New Delhi. \n\n22. Satish, S., Mohana, D. C., Ranhavendra, M. and Raveesha, K. A. \n2007. Antifungal activity of some plant extracts against important \nseed borne pathogens of Aspergillussp. Int J AgrilTechnol 3:109\u2013119. \n\n23. Dubey, R. C., Kumar, H. and Pandey, R. 2009. Fungitoxic effect of \nneem extracts on growth and sclerotial survival of Macrophomina \nphaseolina in-vitro. J. Am. Sci. 5:17\u201324. \n\n24. R Core Team. 2019. R: A language and environment for statistical \ncomputing. R Foundation for Statistical Computing, Vienna, \nAustria. URL: https://www.R- project.org/. \n\n25. Tanjina A, Aminuzzaman, FM, Islam MR, Joty AA, Laila L, \nRayhanul, M. I. 2019. Survey on wheat blast and morphological \nvariability of Magnaporthe oryzae triticum in two south-western \ndistricts of Bangladesh. Bangladesh Journal of Plant Pathology \n35(1&2):39-46.", "start_char_idx": 29137, "end_char_idx": 31644, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e01da986-0152-4e19-ac88-3cbd0d6004af": {"__data__": {"id_": "e01da986-0152-4e19-ac88-3cbd0d6004af", "embedding": null, "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-241", "node_type": "4", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "1e0077d2319a2efe46d34454e66bbeefe8340eb441de9d60f588707ca27c620c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "31513d1a-1fa5-4e7f-89b1-8ac76629fc09", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "3f0df6aa46ad7e58f391f6fd415cad0b24721cda8a16d28e93bd652bf47d1c1e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "33e0af6a-8abb-425a-8a5d-4fd65d41c855", "node_type": "1", "metadata": {}, "hash": "0b5b0acd5f1c04ce495acc6627d5b14ef86fcd21e467e2b5dbeb1f6b4bef6b65", "class_name": "RelatedNodeInfo"}}, "text": "Bangladesh Journal of Plant Pathology \n35(1&2):39-46. \n\n26. Zohura FT, Kashem MA, Hasan MAI, Hossain MA. 2018. Effects of \nplant extracts on controlling wheat blast disease caused by \nMagnaporthe oryzae Pathotype triticum in Bangladesh. Fundamental \nand Applied Agriculture 3(2): 422-433. doi: 10.5455/faa.293640. \n\n27. Jantasorn A, Moungsrimuangdee B, Dethoup T. 2016. In vitro \nantifungal activity evaluation of five plant extracts against five plant \npathogenic fungi causing rice and economic crop diseases. Journal \nof Biopesticides 9:1\u20137. \n\n28. Hubert J, Mabagala RB, Mamiro DP. 2015. Efficacy of selected plant \nextracts against Pyricularia grisea, causal agent of rice blast disease. \nAmerican Journal of Plant Science 06:602\u2013611. doi: \n10.4236/ajps.2015.65065. \n\n \n\nhttp://doi.org/10.1146/annurev.phyto.41.121702.103726\nhttps://dx.doi.org/10.5423%2FPPJ.RW.05.2012.0072\nhttps://doi.org/10.1128/9781555816636.ch37", "start_char_idx": 31591, "end_char_idx": 32511, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "33e0af6a-8abb-425a-8a5d-4fd65d41c855": {"__data__": {"id_": "33e0af6a-8abb-425a-8a5d-4fd65d41c855", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e01da986-0152-4e19-ac88-3cbd0d6004af", "node_type": "1", "metadata": {"identifier": "njb-241", "author": "Rehena, M. K.; Aminuzzaman, F. M.; Ashrafi, M. L.; Chowdhury, M.S.M.; Habiba, U. A.; Nazifa, Z.; Ahmed, M.", "title": "Efficacy of Ethanol Extract of Botanicals in Controlling Wheat Blast Fungus Magnaporthe oryzae triticum in vitro", "date": "2022-12-31", "file": "njb-241.pdf"}, "hash": "b9bad69c24c3b743c402d6234e939f5d96726ecda5df0cba44da81aa9c47234f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "12f61cb1-58b0-4483-875c-e1b07a2bfc46", "node_type": "1", "metadata": {}, "hash": "dd9afdab183be46c6f61e8b1c4523924a9c133b7de194f75f253e64c00574af4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0  (2): 85-90 Research article DOI: https://doi.org/10.54796/njb.v10i2.242 \n\n\u00a9NJB, BSN 85 \n\nMolecular Genetic Diversity in Bambara groundnut [Vigna subterranea \n(L.) Verdc] Assessed by Microsatellite Markers. \nNwakuche Chinenye Onwubiko1  , Michael Ifeanyi Uguru2 , Grace Ovute Chimdi3  \n1Department of Crop Science and Technology, Federal University of Technology, Owerri 1526, Nigeria \n2Department of Crop Science, University of Nigeria, Nsukka 410001, Nigeria \n3Department of Agricultural Technology, Federal Polytechnic Bauchi, Bauchi 0231, Nigeria \n\nReceived: 17 Mar 2022; Revised: 26 Nov 2022; Accepted: 03 Dec 2022; Published online: 31 Dec 2022 \n\nAbstract \nBambara groundnut is a valuable leguminous crop with many landraces. A study was carried out to establish genetic diversity \nand phylogenetic relationship, among 33 Bambara groundnut accessions based on simple sequence repeat (SSR) markers. The \nnine microsatellite markers amplified a total of 27 alleles with a mean of 6.00 alleles per locus. Marker P 36 had the highest \nnumber of polymorphic bands while makers P131 and P68 were monomorphic. Genetic distance among the accessions based \non Jaccard\u2019s similarity coefficient ranged from 0.84 to 1.00. Cluster analysis resolved the accessions into five major groups \nwith subgroups. Each group had a combination of distinct accessions from different geographical origin. A substantial level \nof intra-accession polymorphism was obtained among the evaluated collection of Bambara groundnut. The significant genetic \ndiversity observed can support the selection of appropriate parental genotypes for the improvement of Bambara groundnut \nthrough various breeding programmes.  \n\nKeywords: Accessions, Bambara groundnut, genetic diversity, microsatellite markers. \n\n Corresponding author, email: nwakuche.onwubiko@futo.edu.ng \n\nIntroduction \nVariation exists in crops; it can be interspecific or \n\nintraspecific. Naturally, variation occur due to mutation \n\nand it is maintained over the years by processes of \n\nevolution and natural selection. The reality of natural \n\nvariation in both wild and domesticated crops species is \n\nevident on existence of recognisable diverse forms of \n\ncrop species, which is the basis for selection and for crop \n\nimprovement. However not all recognisable variation in \n\ncrops is genetic. Variations triggered by the influence of \n\nthe environment occurs, in which differences observed in \n\nthe expression of some characters among crop species \n\nwere not intrinsic. On the other hand, variation in crops \n\nbecause of the action of gene(s) is genetic. Genes regulate \n\nstructure (size, shape, colour), physiological processes \n\nand functions, adaptability, phenology, and expression \n\nof characters (1).  \n\nConventionally assessment of genetic variability in crops \n\nis achieved through field screening in which \n\nmorphological descriptors are used to discriminate \n\nbetween and within Species. This approach is popular \n\namong workers because it is easy to carry out and is \n\nrelatively cost-effective (2-4). However, it has some \n\nlimitations because the expression of some characters \n\n(especially polygenic) is environment specific. Therefore, \n\nthe obtained result may not be reliable. Molecular \n\ncharacterisation on the other hand is the most reliable and \n\neffective technique to establish variations that exist in \n\ncrop species. It uses molecular descriptors or markers \n\nthat are not affected by the environment to discriminate \n\namong species at DNA level and can be used to screen \n\nthe entire population at any stage of crop development.", "start_char_idx": 48, "end_char_idx": 3681, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "12f61cb1-58b0-4483-875c-e1b07a2bfc46": {"__data__": {"id_": "12f61cb1-58b0-4483-875c-e1b07a2bfc46", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "33e0af6a-8abb-425a-8a5d-4fd65d41c855", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "b11963477b96e4f4ded26524af1921e82383f0cfa5b3ac4b63f5557b55a12cd4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c73b2d5a-f12b-4209-96ca-a408d401244f", "node_type": "1", "metadata": {}, "hash": "0120f070f03deb44b685c54225909246d70b0ceb32787742cc6650b7fceca1a6", "class_name": "RelatedNodeInfo"}}, "text": "The use of molecular markers to establish genetic \n\ndiversity has enabled breeders to identify the presence of \n\nallelic variations in the genes controlling different \n\nagronomic characters in crops (5), to differentiate \n\nbetween homozygote and heterozygote genotypes (6), \n\nand to manipulate important agronomic traits in crops \n\n(7).  \n\nBambara groundnut is a leguminous food security crop. \n\nIts seed is a complete food and many forms of this crop \n\nhas been reported by workers who made collections from \n\nmajor areas in west Africa eco-regions where this crop is \n\ngrown (25).  Unfortunately, the diversity reported by \n\nthese workers where based on field morphological \n\ncharacterization of accessions. (8-11), There are few \n\ndocumented reports on assessment of diversity on \n\nBambara groundnut based on molecular markers  (12 13, \n\n3, 14. 15), hence the need for this study whose primary \n\ntarget was to assess genetic diversity in Bambara \n\ngroundnut based on microsatellite markers with a view \n\nto establishing phylogenetic relationship among the \n\naccessions.  \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0003-3654-0861\nmailto:nwakuche.onwubiko@futo.edu.ng\nmailto:nwakuche.onwubiko@futo.edu.ng\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 85-90                 Onwubiko et al.  \n\n\u00a9NJB, BSN    86 \n\n \n\nMaterials and method \nThe plant materials were 33 accessions of Bambara \n\ngroundnut obtained from the germplasm collection \n\nmaintained at the gene bank of the International Institute \n\nof Tropical Agriculture (IITA), Ibadan (Table 1). \n\nDNA extraction  \nGenomic DNA was extracted using the modified \n\nminipreparation protocol described by Dellaporta et al \n\n(16).  Approximately 200 mg (0.2 gm) of lyophilized leaf \n\nsample was ground into fine powder with the aid of \n\nGenogrinder 2000.  \n\nTable 1: Passport data of the 33 Bambara groundnut \naccessions used for the study.", "start_char_idx": 3684, "end_char_idx": 5729, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c73b2d5a-f12b-4209-96ca-a408d401244f": {"__data__": {"id_": "c73b2d5a-f12b-4209-96ca-a408d401244f", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "12f61cb1-58b0-4483-875c-e1b07a2bfc46", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "bc1ada10debfe0e24d6eab8f40876ef3c2c4a83329f74ea832a454f600ef1656", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cbcd0992-c3ec-4143-9b14-5a3df3e0b344", "node_type": "1", "metadata": {}, "hash": "4360cb6cabb3c001e2a18a710e39ca6f496b0f473275017a57fbbeb13df1213b", "class_name": "RelatedNodeInfo"}}, "text": "s/n Accession name Country of origin \n\n  1 TVSu-1483 Ghana \n  2 TVSu-1503 Nigeria \n  3 TVSu-1504 Nigeria \n  4 TVSu-1509 Nigeria \n  5 TVSu-1510 Nigeria \n  6 TVSu-1512 Nigeria \n  7 TVSu-1513 Nigeria \n  8 TVSu-1552 Nigeria \n  9 TVSu-1554 Nigeria \n10 TVSu-1555 Nigeria \n11 TVSu-1559 Nigeria \n12 TVSu-1563 Nigeria \n13 TVSu-1584 Nigeria \n14 TVSu-1591 Togo \n15 TVSu-1604 Togo \n16 TVSu-1605 Togo \n17 TVSu-1610 Togo \n18 TVSu-1614 Togo \n19 TVSu-1620 Togo \n20 TVSu-1625 Togo \n21 TVSu-1627 Togo \n22 TVSu-1631 Togo \n23 TVSu-1638 Mali \n24 TVSu-1639 Mali \n25 TVSu-1688 Togo \n26 TVSu-1697 Togo \n27 TVSu-1702 Togo \n28 TVSu-1713 Zambia \n29 TVSu-1766 Malawi \n30 TVSu-1769 Malawi \n31 TVSu-1788 Malawi \n32 TVSu-1819 Cameroon \n33 TVSu-1917 Cameroon \n\nTo each tube 700 \u00b5L of hot (65oc) plant extraction \n\nbuffer(PEB) [which contains 637.5 mL of double distilled \n\nwater (ddH20), 100 mL of  1M Tris-HCl (pH 8.0), 100 mL \n\nof 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH \n\n8.0), 100 mL of 5M Nacl2 and 62.5mL of 20% sodium \n\ndodecylsulphate (SDS)] was added. One percent b-\n\nmercaptoethanol was added to the pre- warmed PEB just \n\nbefore use. The tubes were capped and inverted gently 6-\n\n7 times to mix the sample with buffer. The solution was \n\nincubated at 65\u00b0C in water bath for 20 minutes with \n\noccasional mixing to homogenize the samples. After 20 \n\nminutes, samples were removed from the water bath and \n\nuncapped. The tubes were allowed to cool at room \n\ntemperature for 2 minutes. After which 500 \u00b5L of 5M of \n\npotassium acetate (CH3COOK) was added to each tube \n\nand recapped. The tubes were mix inverted 6-7 times and \n\nincubated on ice for 20 minutes. After 20 minutes of \n\nincubation on ice, tubes were spun at 12,000 rpm for10 \n\nminutes at 4\u00b0C. The supernatant was transferred into new \n\n1.5 mL eppendorf tubes using wider bore pipette tips \n\n(1000 \u00b5L) and making sure debris were not taken along \n\nwith the supernatant. 700-\u00b5L chloroform isoamylalcohol \n\nwas added to the supernatant and spun at 10,000 rpm for \n\n10 minutes. The supernatant was transferred again to a \n\nnew correspondingly labeled tubes and 700-\u00b5L ice-cold \n\nisopropanol was added to each tube and mixed by gently \n\ninverting the tubes 6-10 times. The tubes were allowed to \n\nstand undisturbed in a rack and stored in a freezer (-20\u00b0c) \n\nfor at least 1 hour to precipitate the DNA. After 1-hour \n\nprecipitation in the tubes, the DNA were centrifuged at \n\n12,00 rpm for 10 minutes at 4\u00b0C. The supernatant in the \n\nfreezer, was carefully discarded with great care to \n\ndisallow the pellet from dislodging from the bottom of \n\nthe tube. The tubes were allowed to drain inverted on \n\nclean paper towels for 1 hour. The DNA pellets were \n\nwashed twice in 100\u00b5L, cold 70% ethanol for 20 minutes \n\nand air dried completely.", "start_char_idx": 5732, "end_char_idx": 8510, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cbcd0992-c3ec-4143-9b14-5a3df3e0b344": {"__data__": {"id_": "cbcd0992-c3ec-4143-9b14-5a3df3e0b344", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c73b2d5a-f12b-4209-96ca-a408d401244f", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "0a141bc763af6995911f11f93a99891f3a8321dec09fb7c5f550a68b650260d2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d130367f-798c-4ca5-9ba5-ab252c62875c", "node_type": "1", "metadata": {}, "hash": "15d77f060bf5a967e826aef07a5617bd9948cabb33ca572e7f68d77525f9ba62", "class_name": "RelatedNodeInfo"}}, "text": "After drying, 60\u00b5L of 1\u00d7TE \n\n[10mM Tris-HCL (pH 8.0), 1mM EDTA (Ph 8.0)] was \n\nadded to the pellets, followed by 2\u00b5L of 10 ng/ml RNAse \n\nto remove the RNA. The DNA was then diluted to 10 \n\nng/ul and then used for Polymerase Chain Reaction \n\n(PCR) \n\nPCR Amplification \nNine SSR markers (Inqaba Biotech, South Africa) for \n\nBambara groundnut was used to perform the PCR \n\nreactions and analysis for genetic diversity among the \n\nBambara groundnut accessions. The amplification was \n\nperformed in a 25 \u00b5L reaction volume containing 10X \n\nbuffer, 1.6 \u00b5L of 25 mM of Mgcl2, 2.0 of 5\u00b5/\u00b5L Tag, 8.0\u00b5L \n\nsterile distilled water and 4.0 \u00b5L sample DNA using a \n\nPTC-200 Thermal cycle. The PCR reaction was carried out \n\nwith the following protocol: initial denaturation at 94oC \n\nfor 5mins followed by 45 cycles of 30 secs at 94oC, then \n\n30mins at 65oC and continues in that order. The resulting \n\namplicons were loaded on 1.5% agarose. \n\nAgarose gel electrophoresis of the PCR \n\nproduct \n1.5% agarose was prepared, which was microwaved to \n\ndissolve the agarose and cooled down (560C). The gel  \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 85-90                 Onwubiko et al.  \n\n\u00a9NJB, BSN    87 \n\n \n\nwas poured into the gel tray that was prefixed with comb. \n\nThe gel tray was immersed into an electrophoresis tank \n\ncontaining 0.5 XTBE buffer. \n\nThe comb in the gel was removed to expose the wells \n\nformed. The amplified DNA (10 \u00b5L) was loaded into the \n\nwells of the gel with the aid of a pipette. A standard DNA \n\nmolecular size marker (1 kb DNA lambda) was also \n\nloaded as a check. The gel ran  for 2 hours at 150V and \n\n0.5mAmp. After electrophoresis, the gel was silver \n\nstained. Thereafter the gel was destained in distilled \n\nwater for 10 minutes. and the DNA bands in the gel was \n\nobserved under UV (Ultra violet) lamp and \n\nphotographed using a digital camera. \n\n Each accession was scored (1) for presence and (0) for \n\nabsence of polymorphic band for each primer.  The band \n\nscoring data was used to calculate genetic similarity \n\nbased on Jaccard\u2019s similarity coefficients (17) as follows: \n\n  GSij= a/(a+b+c),   \n\nWhere, \n\nGSij is the similarity between two accession i and j;  \n\na is the number of bands present in both I and j;  \n\nb is the number of bands present in I but absent in j; and  \n\n c is the number of bands present in j and absent in i.  \n\n In addition, the Jaccard\u2019s similarity coefficient was used \n\nto perform cluster analysis based on the Unweighted Pair \n\nGroup with Arithmetic mean (UPGMA) and in \n\nconstructing a dendrogram. The computer program \n\nNTSYS pc version 2.1 (18) was used for these analyses. \n\nFurthermore, the genetic diversity, allele frequency and \n\npolymorphic information content (PIC) were computed \n\nusing PowerMarker (Version 3.25). \n\nResults and discussion \n Polymorphism in several genes controls all phenotypic \n\nvariations within a species. In this respect, the assessment \n\nof genetic variability within crop species using molecular \n\nmarkers is of great importance to plant breeders (15, 19). \n\nThe molecular analysis of genetic diversity in the \n\nevaluated accessions of Bambara groundnut as \n\ndetermined by the SSR markers amplified a total of 27 \n\nalleles.", "start_char_idx": 8511, "end_char_idx": 11729, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d130367f-798c-4ca5-9ba5-ab252c62875c": {"__data__": {"id_": "d130367f-798c-4ca5-9ba5-ab252c62875c", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cbcd0992-c3ec-4143-9b14-5a3df3e0b344", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "a2ccd6637ad9017257e4b9d3dcfec5b3275106e51fcbb829f04e2d6a93292ce5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bda9c789-dbf6-4069-ac7d-76327fe77e30", "node_type": "1", "metadata": {}, "hash": "3be6328ad11c683df1d98ef01c382ed194f61af0a9047d610ff7ad6b6bdc2eb2", "class_name": "RelatedNodeInfo"}}, "text": "The PIC values, which is a measure of the allelic \n\ndiversity of SSRs, ranged from a minimum of 0.001 to a \n\nmaximum of 0.617 with a mean of 0.419. Seemingly the \n\nobtained PIC mean was relatively high when compared \n\nto the report from Basu et al (20), but however relatively \n\nlower than the report of Mohammed (21). Apparently the \n\nSSR makers used in this study were not Vigna subterranea \n\nspecific but Vigna unguiculata.  Concisely maker P36 had \n\nthe highest Marker Index (MI) which is a measure of \n\nefficiency to detect polymorphism. Invariably maker P36 \n\nwas more genetically efficient in distinguishing the \n\nphenotypical similarity that exist between the Bambara \n\ngroundnut accessions. On the other hand, markers P131 \n\nand P68 had monomorphic phenotype. These two \n\nmarkers recorded the least number of alleles per locus.  A \n\nsimilar result has been reported by other workers (14, 15, \n\n22, 19). \n\nGenerally, the 9 SSR markers (Table 2) showed the \n\navailability of a substantial level of polymorphism \n\namong the Bambara groundnut lines as revealed by both \n\ngenetic distance and cluster analysis. The accessions were \n\ngrouped into five groups based on Jaccard Neighbor-\n\njoining dendrogram. Each group had subgroups \n\ncomprised of distinct genotypes from different eco-\n\nregions. This implies that variations among individual \n\ngenotypes was mainly responsible for the observed \n\ngenetic variation among the Bambara groundnut lines \n\nand not variations established between specific accession \n\ngroups. Generally, reasonable intra-accessions \n\npolymorphism was observed in the cluster analysis of the \n\naccessions. In previous studies some workers reported \n\nextensive genetic diversity (12, 15), while others observed \n\nconsiderable genetic diversity (13, 3), yet some others  \n\nreported a low range of genetic diversity (23) in Bambara \n\ngroundnut. Further in this study, divergent genotype \n\nwas revealed by the cluster analysis using Unweighted \n\nPair-Group Method with Arithmetic Average (UPGMA). \n\nTVSU 1554 from Nigeria was the only accession found in \n\nGroup 5 among the five groups of the cluster analysis. \n\nInvariably this accession was dissimilar from other \n\naccessions evaluated. Divergent genotype(s) usually \n\nhave good breeding value which can be used for crop \n\nimprovement, in both direct selection and as parents for \n\nmaking crosses. \n\nTable 2. Description of the SSRs markers used in this study \n\nMarker Forward primer Reverse primer Gene bank ID \n\nP36 51-AAAATTGGAGAAAGGGGTTTTT-31 51-GATTTCGCCATATCCCCATC-31 GQ411715.1 \nP56 51-GCAATGGGTTCGTCGATACT-31 51-GCTCGATGCTTTTTGTTTCC-31 EU717407.1 \nP57 51-GGGAAACAAAAAGCATCGAG-31 51-CGCTACCCCAAAATACCAAA-31 EU717407.1 \nP61 51-GTCAGAGGCGAATTGAAAGC-31 51-AGGTCTTCCCGTTCCTTCAT-31 EU717373.1 \nP63 51-ATGAAGGAACGGGAAGACCT-31 51-CCTAAGGGCATATCGGTTGA-31 EU717373.1 \nP68 51-CAAGTCCCTCTATCCCCAAA-31 51-CAAGTCCCTCTATCCCCAAA-31 EU717348.1 \nP71 51-GTGTTGGGTTCAAAGCTGGT-31 51-CATCGGTCCACACAGTTGTC-31 EU717266.1 \n\nP131 51-CAAAGCCATTGCTGAAGACA-31 51-GGATGCTACACCGTTCGATT-31 HB823749.1 \nP184 51-GCCAGAGACTCTCACGTTCC-31 51-TGCATGGTCCCTGTTGTAGA-31 HB465729.1", "start_char_idx": 11730, "end_char_idx": 14852, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bda9c789-dbf6-4069-ac7d-76327fe77e30": {"__data__": {"id_": "bda9c789-dbf6-4069-ac7d-76327fe77e30", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d130367f-798c-4ca5-9ba5-ab252c62875c", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "e42aaeb620659c33e114dbab0934d0d6e41bfca1dc486c499f23d47a692bde5a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "eea3a7ee-9fbc-4993-8306-f9817970511a", "node_type": "1", "metadata": {}, "hash": "a19c3f3ca4a493b349e4b20674b0fc6b970a7f3c50b76a2ba8de9af47db92356", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 85-90                 Onwubiko et al.  \n\n\u00a9NJB, BSN    88 \n\n \n\nFigure.1:  Molecular dendrogram analysis of 33 accession of \nBambara groundnut with unweighted pair group method with \narithmetic method (UPGMA).  \n\nThe genetic distance from the molecular dendrogram \n\nanalysis based on Jaccard\u2019s similarity coefficient ranged \n\nfrom 0.84 to 1.00 (Figure 1).  \n\nThis implies that at 100% level of similarity, all the \n\naccessions were distinct from each other, while at 84% \n\nlevel of similarity all the accessions clustered to form a \n\nsingle accession. Invariably it means that each accession \n\nhad at least one neighbour with more than 84% similarity. \n\nHowever, at 94% level of similarity, the accessions were \n\ngrouped into five clusters. \n\nThe pattern of clustering of the Bambara groundnut \n\naccessions in groups with the Unweighted Pair Group \n\nwith Arithmetic mean (UPGMA) and Jaccard\u2019s \n\nNeighbour-joining dendrogram was similar (Figure 2). \n\nAccessions were clustered in the same group based on \n\ngenetic similarity and not on sources of collection or eco-\n\nregion of origin. There were five heterogenic groups in \n\nthe two-cluster analysis. The reason why accessions from \n\nthe same geographical area could not form a distinct \n\ncluster was because they were genetically dissimilar. \n\nEach cluster, therefore, contained accessions with similar \n\ngenetic characters. For example, group one of the \n\nUnweighted Pair Group with Arithmetic mean \n\n(UPGMA) grouping was the smallest group. It had two \n\naccessions, and both were collected from different \n\ngeographical areas as can be seen in their identification \n\nnumbers; TVSU 1483 was from Ghana, while TVSU 1631 \n\nfrom Togo.  \n\nHowever, both were clustered together in group one, \n\nimplying that the two accessions were duplicates or \n\nclosely similar, and not two different accessions from two \n\ndifferent localities as indicated in their identification \n\nnumbers and places of collections. Another outstanding \n\nexample was observed in group four. The accessions \n\nclustered in this group were TVSU 1584, TVSU 1591 and \n\nTVSU 1604. Accession TVSU 1584 was collected from \n\nNigeria, while TVSU 1591 and TSVU 1604 were from \n\nTogo. A detailed analysis of this result surprisingly \n\nshowed that these three accessions, that were collected \n\nfrom different geographical areas, were the most \n\ngenetically similar lines among the evaluated Bambara \n\ngroundnut collections. Similarly, in group 1 of Jaccard \n\nNeighbour-joining (JNJ) dendrogram, a comparable \n\nassociation was substantiated between TVSU 1697 and \n\nTVSU 1627 (from Togo) and TVSU 1503 (from Nigeria).  \n\nThese complex linkages suggest the possibility that these \n\naccessions were related. They either had similar genes or \n\nwhere from a common origin but were given different \n\nidentification numbers. Apparently, these results showed \n\nthe potentials of the SSR markers in detecting differences \n\nand establishing the extent of genetic relatedness in \n\nexistence among the evaluated genetic materials. It also \n\nemphasizes the superiority of SSR markers in classifying \n\ncollections of Bambara groundnut more precisely, as \n\nagainst the use of morphological markers in germplasm \n\ncharacterization (19).", "start_char_idx": 14857, "end_char_idx": 18120, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "eea3a7ee-9fbc-4993-8306-f9817970511a": {"__data__": {"id_": "eea3a7ee-9fbc-4993-8306-f9817970511a", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bda9c789-dbf6-4069-ac7d-76327fe77e30", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "5662a19d32b76aaa10513297918aa36ced61964f3dd2aba39d2ea963c094cfac", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "985d85c0-f232-4506-a881-58dd1eb9f491", "node_type": "1", "metadata": {}, "hash": "bd4364220eed9f8094e68356439010205699c10ac77f4ebb9cde27e1cd4b4f37", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 85-90                 Onwubiko et al.  \n\n\u00a9NJB, BSN    89 \n\n \n\nFigure 2: Jaccard Neighbour-joining dendrogram illustrating \ngenetic diversity and relationships among 33 Bambara \n\ngroundnut accessions. \n\nClustering of accessions of Bambara groundnut collected \n\nfrom different geographical areas in the same group may \n\nhave arisen due to duplication of genotypes caused by \n\nhigh rate of seed exchange between farmers from diverse \n\nethnic and agro-geographical areas. In fact, there was a \n\nreport from a previous study on a similar trend of \n\nassociation between Bambara groundnut accessions \n\ncollected from different geographical areas, and in that \n\nstudy, it was concluded that these accessions were either \n\nrelated, or the genotypes were the same (22). \n\nA detailed examination of the result from the \n\ndendrogram showed that accessions from Nigeria were \n\nmore dispersed among the clusters of accessions than \n\naccessions from other African countries. These accessions \n\nwere found in four out of the five genetic groups.  This \n\nobservation may have some implication on the origin of \n\nBambara groundnut, in that it supports the report of \n\nearlier studies on the origin of this crop;  Nigeria may \n\nhave been a regional centre of diversity of Bambara \n\ngroundnut (24), which other studies confirmed by the \n\nexistence of genuinely wild state of the crop in this area \n\n(25, 26).  \n\nConclusion \nMolecular analysis of genetic diversity of 33 accessions of \n\nBambara groundnut based on SSR maker was reported. \n\nGenetic distances result and the cluster analysis revealed \n\nhigh level of polymorphism, indicating the existence of a \n\nwide range of genetic diversity among the accessions. \n\nThis result can facilitate selection of appropriate \n\ngenotypes for the development of improved lines of \n\nBambara groundnut through various breeding \n\nprograms. This study has contributed to broadening the \n\ngenetic base of Bambara groundnut. The usefulness of \n\nthis report in the effective utilisation, management, and \n\nconservation of Bambara groundnut germplasm is \n\nundoubtable.  \n\nAuthor\u2019s contribution \nMIU conceptualized the research proposal and \n\nsupervised the research activities. NCO performed the \n\nlab works, scoring, data analysis and interpretation. NCO \n\nand GOC wrote the first draft of the paper.  All authors \n\nread and approved the final manuscript. \n\nCompeting Interests \n No competing interests were disclosed.  \n\nFunding \nThis research work was not funded by any organization. \n\nAcknowledgment  \nThe authors thank the staff of the International Institute \n\nof Tropical Agriculture (IITA), Ibadan for providing us \n\nwith seeds of the accessions of Bambara groundnut used \n\nfor this study. \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 85-90                 Onwubiko et al.  \n\n\u00a9NJB, BSN    90 \n\n \n\nReferences  \n1. Ma Q, Wenheng, Z, Xiang Q. Evolution and developmental genetics \n\nof floral display\u2014A review of progress. 2017.Volume 55 Issue 6, \nPages 487-515.  \n\n2. Afolayan GO, Aladele, SE, Danquah EU, Bley E. Marker assisted \nselection: A potent tool for sorghum improvement.  38th annual GSN \nconference 2014 October, Edo, Benin State, Nigeria. Pp 193-196. \n\n3. Ntundu WH, Bach IC, Christiansen JL, Andersen SB. Analysis of \ngenetic diversity in Bambara groundnut (Vigna subterranea (L.) \nVerdc.) landraces using amplified fragment length polymorphism \n(AFLP) markers. African Journal Biotechnology 2004.3(4):220\u2013225 \n\n4. Afuape SO. Morphological diversity of Sweet potato (Ipomoea \nbatatas (L.) Lam) landraces as expressed in contrasting agro-\necologics in Nigeria. Nigerian Journal of Genetics. 2014 28:55-77.  \n\n5. Ejeta G,  Knoll JE,.", "start_char_idx": 18126, "end_char_idx": 21831, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "985d85c0-f232-4506-a881-58dd1eb9f491": {"__data__": {"id_": "985d85c0-f232-4506-a881-58dd1eb9f491", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "eea3a7ee-9fbc-4993-8306-f9817970511a", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "c970cd126b7bc9c6971495c9741160633cd4e95fe9f5236d7ac5953299dcbbc2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "90dfcab3-b72a-4dd6-ad8f-0e9b0d1247ae", "node_type": "1", "metadata": {}, "hash": "a2602fcc971dea94494ee06e9ef51ff0b8ae72e76e97b0e2166c6110a2feef8c", "class_name": "RelatedNodeInfo"}}, "text": "5. Ejeta G,  Knoll JE,. Marker-assisted selection in sorghum. 2007 \npp.187-205 In: R.  Vashney and R. Tuberosa (eds) Genomic Assisted \nCrop Improvement: Vol 2, Genomic  Applications in Crops. \nSpringer-Verlag, Berlin. \n\n6. Collard, B.C.Y., Grams, R. A., Bovill, W.D., Percy, C.D., Jolley, R., \nLehmensiek A., Wildermuth, G., and Sutherland, M.W. \nDevelopment of molecular markers for crown rot resistance in \nwheat: mapping of QTL for seedling resistance in a \u20182-49\u2019 x \u2018Janz\u2019 \npopulation. Plant Breed 2005. 124:532\u2013537 \n\n7. Salgotra RS, Stewart CN. Functional Markers for Precision Plant \nBreeding. International Journal of Molecular Sciences, 2020, 21, \n4792. \n\n8. Drabo I, Sereme P, Dabire C. Burkina Faso: In: Heller J, Begemann, \nF, and Mushonga J. (Eds.), pp 11-18. Bambara Groundnut (Vigna \nsubterranea L. Verdc.). Promoting the Conservation and Use of \nunderutilised and Neglected Crops.  Proceeding of Workshop on \nConservation and Improvement of Bambara Groundnuts (Vigna \nsubterranea L. Verdc.), 1995.14-16 November, Harare, Zimbabwe \n\n9. Massawe FJ, Azam-Ali SM, Roberts JA. The use of molecular \nmarkers to explore phenotypic variation between and within \nlandraces of Bambara groundnut. Journal of Experimental Biology \n(Supplement). 2000. 51:  71. \n\n10. Ntundu WH. Genetic diversity of Bambara groundnut (Vigna \nsubterranea (L.) Verdc) in Tanzania, 2002. PhD Thesis, the Royal \nVeterinary and Agricultural University, Copenhagen, Denmark. \n\n11. Ofori K, Kumaqa FK, Tonyiqah A. Morphological characterization \nand agronomic evaluation of Bambara groundnut (Vigna subterranea \n[L.] Verdc.) Germplasm in Ghana. International Plant Genetic \nResources Newsletter/FAO Bioversity, 2006.145: 23-2 \n\n12. Massawe, FJ, Robert JA, Azam-Ali SN, Davey MR. Genetic diversity \nin Bambara groundnut (Vigna subterranea (L.) Verdc.) landraces \nassessed by random amplified polymorphic DNA (RAPD) markers. \nGenetic Resources and Crop Evolution. 2003.50:737\u2013741  \n\n13. Amadou HI,  Bebeli PJ,  Kaltsikes PJ. Genetic diversity in Bambara \ngroundnut (Vigna subterreneae) germplasm revealed by RAPD \nmarkers. Genome.2001 44:995-999. \n\n14. Somta P, Chankaew S, Rungnoi O, Sioles G. Genetic diversity of the \nBambara groundnut (Vigna subterranea (L.) Verdc.), as assessed by \nSSR markers. Genome. 2011.54:11. 898-910. \n\n15. Alaka CO, Okanlola GO, Akanbi AG, Olalekan OJ. Genetic \nvariability in Bambara groundnut (Vigna subterranea (L.) Verdc) \nusing molecular markers. 38th annual GSN conference 2014 October \nEdo Benin State Nigeria. Pp 241 \u2013 247.   \n\n16. Dellaporta, S.L., wood, V.P and Hicks, J.B. (1983) A plant DNA mini \npreparation. Vision 11. Plant Mol. Boil. Rept 1:19-21. \n\n17.", "start_char_idx": 21808, "end_char_idx": 24482, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "90dfcab3-b72a-4dd6-ad8f-0e9b0d1247ae": {"__data__": {"id_": "90dfcab3-b72a-4dd6-ad8f-0e9b0d1247ae", "embedding": null, "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-242", "node_type": "4", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "32f67a274a21ec788e18f2ece31ea97cab5b19beadc61dddc1610da6dfb780b0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "985d85c0-f232-4506-a881-58dd1eb9f491", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "831f305c8f0d852a57412258c0db9d3347d0725153cd09144b0da7a29e965791", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "62c51fd3-d7f0-4442-9bae-549618640bec", "node_type": "1", "metadata": {}, "hash": "d3c0140fffa4f19090cf359345d6163e8e05dd4c91770804faa1fa73a1253402", "class_name": "RelatedNodeInfo"}}, "text": "Boil. Rept 1:19-21. \n\n17. Jaccard P. Nouvelles researchers sur la distribution florale. Bull. Soc. \nVaud. Sci. 1908.44:22-270. \n\n18. Rohlf FJ. NTSYSpc: Numerical taxonomy and multivariate analysis \nsystem. 2000. Exeter Software, New York. \n\n19. Siise A. Massawe, FJ.  Microsatellites based marker molecular \nanalysis of Ghanaian Bambara groundnut (Vigna subterranea (L.) \nVerdc.) landraces alongside morphological characterization. \nGenetic Resources and Crop Evolution. 2013.60:777\u2013787 \n\n20. Basu S, Roberts J, Azam-Ali S, Mayes S. Development of \nmicrosatellite markers for Bambara groundnut (Vigna subterranea \n[L.] Verdc.) \u2013 an underutilized African legume crop species. \nMolecular Ecology Note. 2007a.7: 1326-1328. \n\n21. Mohammed SM (2014) Pre-breeding of Bambara Groundnut (Vigna \nsubterranea [L.] Verdc.), PhD thesis, University of KwaZulu-Natal, \nPietermaritzburg Campus, South Africa \n\n22. Ntundu WH, Shillah, SA, Marandu WYF, Christiansen JL, \nMorphological diversity of Bambara groundnut (Vigna subterranea \n(L.) Verdc.) landraces in Tanzania. Genetic Resources and Crop \nEvolution. 2006..53:367\u2013378 \n\n23. Pasquet RS, Scwedes S, Gepts P. Isozyme diversity in Bambara \ngroundnut Crop Science. 1999.39: 1226\u20131236. \n\n24. Tanimu B, Aliyu L. Country Report on Bambara groundnut \nproduction in Nigeria. In J. Heller, F. Begemann, and J. Mushonga \n(Eds.), Bambara Groundnut Vigna Subterranea (L.) Verdc: \nProceedings of the Workshop on Conservation and Improvement of \nBambara Groundnut (Vigna Subterranea (L.) Verdc.): 1995. 14-16 \nNovember, Harare, Zimbabwe Bioversity International.pp.89. \n\n25. Hepper FN. 1970. Bambara groundnut (Voandzeia subterranea). \nReview article. Field Crop Abstract. 23 (1):1-6. \n\n26. Goli AE, Begemann F, Ng, NQ. Characterization of IITA\u2019s Bambara \ngroundnut collection. In: Proceedings of the workshop, \nconservation and improvement of Bambara groundnut (Vigna \nsubterranea (L.) Verdc.). 1995. 14\u201316 November, Harare Zimbabwe", "start_char_idx": 24457, "end_char_idx": 26417, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "62c51fd3-d7f0-4442-9bae-549618640bec": {"__data__": {"id_": "62c51fd3-d7f0-4442-9bae-549618640bec", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "90dfcab3-b72a-4dd6-ad8f-0e9b0d1247ae", "node_type": "1", "metadata": {"identifier": "njb-242", "author": "Chinenye Onwubiko, Nwakuche; Ifeanyi Uguru, Michael; Ovute Chimdi, Grace", "title": "Molecular Genetic Diversity in Bambara groundnut [Vigna subterranea (L.) Verdc] Assessed by Microsatellite Markers", "date": "2022-12-31", "file": "njb-242.pdf"}, "hash": "6b827b05f2054e00b2e3b72cc6f98fff50cbdd5692e1f3cc0eef875f0843b1b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4b6a4935-c012-407d-9748-130352d204c0", "node_type": "1", "metadata": {}, "hash": "5835bd5d6c025d863e33d4e9d0f9aa8e47735c002aab95d536be2a28903a5ec3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0  (2): 57-76   Review article  DOI: https://doi.org/10.54796/njb.v10i2.243 \n\n \n\n\u00a9NJB, BSN  57 \n\nDetoxification and Removal of Hexavalent Chromium in Aquatic \nSystems: Applications of Bioremediation \nA.M.K.C.B. Aththanayake1 , I.V.N. Rathnayake1 , M.P. Deeyamulla2  \n1Department of Microbiology, Faculty of Science, University of Kelaniya, Kelaniya, GQ 11600, Sri Lanka. \n2Department of Chemistry, Faculty of Science, University of Kelaniya, Kelaniya, GQ 11600, Sri Lanka. \n\nReceived: 01 Oct 2022; Revised: 01 Nov 2022; Accepted: 09 Nov 2022; Published online: 31 Dec 2022 \n\nAbstract. \nChromium is a transition metal with a wide range of applications in leather tanning, textile, electroplating, stainless steel \nproduction, inorganic chemical production and wood preservation industries due to yellow colouration, corrosion resistance, \nhigher melting-point and crystalline structure with raging of oxidation states from 0 to +6. Trivalent and hexavalent \nchromium are the most abundant forms of chromium discharged into the aquatic environment by industries. It has been \nreported that hexavalent chromium is highly toxic than trivalent chromium due to the higher solubility, mobility and \ntendency to accumulate in higher trophic levels, which, therefore, become bioavailable and causes carcinogenic, mutagenic \nand teratogenic effects on most microorganisms and animals, growth inhibition, morphological and physiological changes \nand yield reductions in plants. Therefore, it is essential to detoxify the above hazardous pollutants up to permissible limits, \nwhich local and international authorities have legislated concerning its threat towards biotic components. Hexavalent \nchromium detoxification is possible to achieve using three methods i.e. physical, chemical and biological methods. These \nremediation processes can eliminate highly toxic hexavalent chromium or transform it into a less toxic form of trivalent \nchromium, completely or partially by adsorption and reduction. Biological remediation is considered a cost-effective and eco-\nfriendly method compared to physical and chemical remediation. Further, many biological agents have been identified as \nagents that can tolerate the hexavalent chromium toxicity up to certain higher levels depending on the internal and external \nenvironmental factors, indicating different metal tolerance mechanisms that are assumed to be applied in metal remediation \naspects. According to the testimonies of novel bioremediation studies, some hexavalent chromium tolerant organisms such \nas plants, bacteria, unicellular and multicellular fungi and algae are promising eco-friendly alternatives in detoxification and \nhexavalent chromium removal perspective. This article reviews the bioremediation approaches available for hexavalent \nchromium detoxification and removal and highlights the strengths and weaknesses of current bioremediation methods.  \n\nKeywords: Hexavalent chromium, toxicity, biological remediation, chemical remediation, physical remediation, biofilms \n\n Corresponding author, email: vayanga@kln.ac.lk \n\nIntroduction \nChromium is a highly valued industrial raw material \n\nwith a wide range of industrial applications such as \n\npigment production for paints, inks and plastics, anti-\n\ncorrosion coating production, stainless steel production, \n\nwood preservation and leather tannins which are \n\ndischarged both trivalent chromium (Cr(III)) and \n\nhexavalent chromium (Cr(VI)) in higher quantities [1,2]. \n\nAccording to toxicological studies, Cr(VI) is 100 times \n\nmore toxic than Cr(III) due to solubility, mobility and \n\npermeability in biota [3\u20137]. From the view of toxicology, \n\nprolonged exposure to Cr(VI) can lead to carcinogenic, \n\nmutagenic and teratogenic effects on animals which has \n\nbeen clinically proved, and morphological and \n\nphysiological effects on plants, algae and other \n\nmicroorganisms [8\u201312].", "start_char_idx": 48, "end_char_idx": 3977, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4b6a4935-c012-407d-9748-130352d204c0": {"__data__": {"id_": "4b6a4935-c012-407d-9748-130352d204c0", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "62c51fd3-d7f0-4442-9bae-549618640bec", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "b2daeb622364d82b21277611cd0792074de1add790f71fa7897ac31c6b89360b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b23a5469-80ff-4a94-bcc4-6b690ea5cd06", "node_type": "1", "metadata": {}, "hash": "dc87c81ef7138fc5fcc38fad26f15976ebc976bd79923f7966b6b07112d7608e", "class_name": "RelatedNodeInfo"}}, "text": "The working community in \n\nchromium based industries, including chrome mining, \n\nhas the highest potential for chromium poisoning [8,13\u2013\n\n16]. Therefore, international authorities for the \n\noccupational community, such as Occupational Safety \n\nand Health Administration (OSHA), has set the \n\nmaximum limit for Cr(VI) exposures as similar to the \n\nconventional public health concerning local and \n\ninternational authorities; World Health Organization \n\n(WHO), United States Environmental Protection Agency \n\n(US EPA). The minimization and detoxification of Cr(VI) \n\nin industrial effluents can be achieved by Physical, \n\nChemical, and biological methods. Among the above \n\nmethods, biological remediation is considered the most \n\ncost-effective and environmentally friendly method. [17\u2013\n\n20].  \n\nChemical nature and uses of Chromium. \nChromium is the 21st most abundant element in earth\u2019s \n\ncrust, which belongs to d-block in the periodic table with \n\na molar mass of 51.9961 g mol-1 with a wide range of \n\nindustrial applications based on chemical and physical \n\nproperties such as inert nature, hardness, strength, high-\n\ntemperature resistance and corrosion resistance etc. [21]. \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0001-7743-0074\nhttps://orcid.org/0000-0003-3476-7018\nmailto:vayanga@kln.ac.lk\nhttps://orcid.org/0000-0002-3085-4280\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  58 \n\nCr(III) and Cr(VI) are the most stable and domain \n\noxidation forms of chromium that exists in nature among \n\nthe other oxidation forms of metallic chromium (Cr(0)), \n\ndivalent chromium (Cr(II)), tetravalent chromium \n\n(Cr(IV)) and pentavalent chromium (Cr(V)).  \n\nThe majority of chromium is used for metallurgical (67%) \n\nand refractories (18%), while the rest of 15 % are used for \n\nchromium induced chemical production, which is used \n\nfor wood preservation, leather tanning, metal finishing, \n\npigment production and textile industry as a raw \n\nmaterial [2] (Table 1). \n\nTable 1 \u2013 Industrial applications of Cr(0), Cr(III) and Cr(VI). \n\nOxidation \n\nstate \nIndustrial application Reference  \n\nCr (0) Stainless steel production \n\nAlloy production  \n\nMetal manufacturing  \n\n[2] \n\nCr (III) Metal and alloy production \n\nTextile and leather tanning \n\nCopy machine toners  \n\nBrick lining \n\nChrome plating  \n\nCatalysts production \n\nPaint production \n\n[2,22] \n\nCr (VI) Chrome plating \n\nLeather tanning \n\nTextile industry \n\nCopy machine toners \n\nDye/paint pigment \n\nproduction \n\nWood preservation \n\nHigh temperature battery \n\nproduction \n\nMetal finishing \n\nCatalyst production \n\nStainless steel production \n\nPlastic production \n\n[2,22,23] \n\nToxicity of Chromium.  \nAmong the most stable oxidation states of chromium in \n\nnature, Cr(III) is considered an essential micronutrient of \n\nhigher organisms with less toxic effects due to lower \n\nsolubility and impermeability [6,24]. Further, it has been \n\nreported that Cr(III) can assist in regulating the glucose \n\nlevel of the human body [25]. In contrast, Cr(VI) has been \n\ncategorized as a carcinogenic agent by the USEPA and \n\nInternational Agency for Research on Cancer (IARC) due \n\nto high water solubility and mobility [26].  \n\nToxicity of Cr(VI) to humans. \nProlonged Cr(VI) exposes through breathing, ingesting, \n\nand skin contacts can cause nasal irritations, nasal \n\nperforations, skin irritations, skin ulcerations, skin \n\nallergies, lung cancers, stomach upsets, convulsions, \n\nkidney and liver damages [2].", "start_char_idx": 3978, "end_char_idx": 7637, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b23a5469-80ff-4a94-bcc4-6b690ea5cd06": {"__data__": {"id_": "b23a5469-80ff-4a94-bcc4-6b690ea5cd06", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4b6a4935-c012-407d-9748-130352d204c0", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "9166821a30b358060dec4d279a1afbbe842f4e5dd845fc1436f0ae3b1b6d9a0b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1734a97f-d2b1-4e5b-b92e-aa7edcd923be", "node_type": "1", "metadata": {}, "hash": "69d567ce01dfdce5e4bccb5062dc34a7992d6d2f6b749e59e1006a72796bc830", "class_name": "RelatedNodeInfo"}}, "text": "The working community in chromium-based industries \n\n(leather tanning, electroplating, mining and pigment \n\nproduction, etc.) has a high tendency to be affected by \n\nCr(VI) toxicity. Cr(VI) can produce highly reactive \n\nhydroxyl radicals in blood vessels during the reduction \n\ninto Cr(III), which can cause blood cell damages with \n\norgan degradations and cellular activity interruption by \n\nmetal-DNA bindings [27]. Further it believes that  Cr(VI) \n\nis responsible for causing teratogenic effects in human as \n\nit has proven with animal model trials [28]. \n\nToxicity of Cr(VI) to plants.  \nChromium being a non-essential element it does not have \n\na specific mechanism of uptake into plants. It is believed \n\nthat, the plants use a passive process to uptake Cr(III) and \n\nan active process for uptake Cr(VI) with carriers \n\ncompeting with iron, sulphur and phosphorus [29]. Part \n\nof the Cr(VI) is taken up into plants after reducing into Cr \n\n(III) on the root surface, and the rest of the  Cr(VI) is taken \n\nup by plants by dissolving in water and without reducing \n\n[29,30]. In the toxicological point of view, Cr(VI) affect \n\nplants both morphologically and physiologically. It has \n\nbeen found that, high concentrations of Cr(VI) affect the \n\nseed germination negatively due to the depressive effects \n\non enzyme activity and sugar transport to embryo axes \n\n[31,32], It also reduces the root growth due to inhibition \n\nof water absorption [33], and shoot growth due to \n\nchromium transportation in aerial parts [31]. \n\nToxicological Studies done else ware using Oryza sativa, \n\nAcacia holosericea, Leucaena leucocephala and Albizia lebbek \n\nand Phaseolus vulgaris reported that leaf area and biomass \n\ncan be adversely affected by Cr(VI) [31,34]. \n\nPlant physiological studies revealed that Cr(VI) can lead \n\nto yield reduction by decreasing chlorophyll a, \n\nchlorophyll b and carotenoid pigments and affecting \n\nwater and mineral transportation due to high oxidative \n\npotential [31,35]. \n\nToxicity of Cr(VI) to microorganisms.  \nMicroorganisms are commonly exposed to many \n\npollutants, including toxic metals, as they are widely \n\ndispersed in the environment, causing many toxic effects. \n\nCr(VI) can become toxic to most bacterial strains causing \n\ncell enlargements, cell elongations and cell division \n\ninhibitions [23]. Cr(VI) can rapidly enter into the bacterial \n\ncytoplasm and reduces to lower oxidation states which \n\nare free radicals such as Cr(V), which leads to genotoxic \n\neffects by causing oxidative damages to DNA. Moreover, \n\nit has been found 400 \u2013 800 \ud835\udf07g of Cr(VI) can directly \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  59 \n\ninteract with bacterial DNA causing frameshift \n\nmutations and base-pair replacements [36]. \n\nThe Cr(VI) tolerance limits of bacteria have not clearly \n\nbeen defined as it can depend on several factors, \n\nincluding the type of the strain and physio-chemical \n\nconditions of the habitat, nature of the waste etc. \n\nProviding evidence to the above assumption a study of  \n\nreported that 10 \u2013 12 mg/L of Cr(VI) was adequate to \n\ninhibit most soil bacteria [22], while some strains in \n\nactivated sludge can tolerate up to 80 mg/L of Cr(VI) \n\n[37]. Further, they have reported that Cr (VI) was able to \n\nstimulate bacterial growth up to 25 mg/L of Cr(VI). \n\nCompered to bacteria, fungi are less sensitive to Cr(VI) \n\ndue to the decreased uptake and production of \n\nantioxidants [38,39,39\u201341].", "start_char_idx": 7640, "end_char_idx": 11133, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1734a97f-d2b1-4e5b-b92e-aa7edcd923be": {"__data__": {"id_": "1734a97f-d2b1-4e5b-b92e-aa7edcd923be", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b23a5469-80ff-4a94-bcc4-6b690ea5cd06", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "3023099e9603a4cce2869496b7c79b17804da5c72205259848b147315a166b8f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1f631274-ff5e-4b26-97cb-057202d0c4c6", "node_type": "1", "metadata": {}, "hash": "7d2f962c2778935e8cb5a609f3cfe209f41b2ac87fc13749c042e91b0e8a245c", "class_name": "RelatedNodeInfo"}}, "text": "However, some studies \n\ndescribe that, Cr(VI) can  cause genotoxic and mutagenic \n\neffects on several strains of fungi, including \n\nSaccharomyces cerevisiae, Sclerotium rolfsii and Pycnoporus \nsanguineus leading to complex physiological changes and \n\nfunctional changes such as inhibition of oxygen uptake, \n\ninduction of petite mutations and inducing \n\nmitochondrial functional damages [24,42\u201344]. Further \n\nstudies on fungi have shown that effects of Chromium \n\ntoxicity vary on the nature of carbon substrate [45].  \n\nCr(VI) can affect PS II reaction centers of algae, which \n\nleads to inhibition of photosynthesis and cause \n\nsignificant morphological changes in some genera, \n\nincluding Chlorella, Scenedesmas, Ulva, Isochrysis, \n\nMicrasterias, and Chlamydomonas [36,46\u201351]. \n\nDisposal and remediation process of \nChromium wastes. \nThe chromium-based industries i.e., electroplating, \n\ntanning, water cooling, textile, wood preservation, alloy \n\nmanufacturing, dye and pigment production discharge \n\nlarge quantities of contaminated chromium containing \n\nwaste to soil, air and water annually. Considerable \n\nproportions of used chromium as a raw material and / or \n\na reagent for industries including tannery (40%), chrome \n\nplating (35%), academic, research and industry \n\nlaboratories (100%) discharge Cr(III) and Cr(VI) as \n\neffluents [52].  \n\nThese chromium contaminated effluents should be \n\nremediated before discharging into the environment, due \n\nto toxicity of chromium to the environment and public \n\nhealth. Therefore, rules and regulations have been \n\nlegislated and implemented by national and international \n\nauthorized bodies such as WHO, US EPA, and national \n\nenvironmental acts of host countries for industrial \n\nwastewater and drinking water. \n\nAccording to the US EPA and WHO standards maximum \n\npermissible level of Cr(VI) in drinking water and \n\nindustrial wastewater have been legislated to 0.05 mg/L \n\nand 0.10 mg/L, respectively. Considering the health \n\nhazard to the occupational community in chromium-\n\nbased industries, Occupational Safety and Health \n\nAdministration (OSHA) has set the maximum limit for \n\nCr(VI) compounds for 8-hour work shifts and 40-hour \n\nworkweeks as 0.052 mg/L [23,53]. \n\nBased on these regulations, Cr(VI) contaminated wastes \n\nshould be remediated before being discharged into the \n\nenvironment. Remediation of chromium containing \n\nwaste can be carried out using three (03) methods i.e. \n\nchemical, physical and biological which are summarized \n\nin the Table 2. \n\nChemical methods of Cr(VI) remediation. \nChemical reduction and photocatalysis are the most \n\ncommon chemical remediation methods that have been \n\napplied in chemical remediation processes. The chemical \n\nreduction uses reducing agents such as sulfur dioxide \n\n(SO2), calcium polysulfide (CaS5), ferrous sulfate (FeSO4), \n\nsodium metabisulfite (NaHSO3), sodium sulfite \n\n(Na2SO3), barium sulfite (BaSO3), hydrazine hydrate \n\n(N2H4), hydrogen peroxide (H2O2) and, calcium \n\ncarbonate (Na2CO3) [19,54\u201356]. Redox reactions of above \n\nmentioned reducing agents are kinetically slow at low \n\nCr(VI) concentrations [57]. Therefore, it may require \n\ndifferent methods to remediate residual Cr(VI), which \n\nare even higher than the discharge limits. Further, it has \n\nbeen found that this reduction process is also influenced \n\nby physical and chemical characteristics of the \n\ndischarging sites (pH, conductivity, soil type and texture, \n\npresence of transition metals) [58,59].  \n\nSemiconductor based photocatalysis is a developing \n\ntechnology for toxic metal remediation such as Cr(VI), \n\nHg(II), As(V), Cu(II), and Pb(II) [62]. This technology is \n\nmore advantageous as there are no requirements for \n\nsecondary disposal methods. Titania based \n\nphotocatalysts such as TiO2 and La2Ti2O7 are extensively \n\nused for photocatalytic reduction of Cr(VI) in specific \n\nvalues [19,61].", "start_char_idx": 11134, "end_char_idx": 15035, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1f631274-ff5e-4b26-97cb-057202d0c4c6": {"__data__": {"id_": "1f631274-ff5e-4b26-97cb-057202d0c4c6", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1734a97f-d2b1-4e5b-b92e-aa7edcd923be", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "6b68e15350480df2773746c01fa202c8493778004bf98e2ddad15cc317f33151", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "af0bec92-6fce-48d7-9d5b-b8df8f0e7616", "node_type": "1", "metadata": {}, "hash": "ba020552dc4b804aeb439750b60e8928f6eae82f7cdf32fefee20e7eee14399a", "class_name": "RelatedNodeInfo"}}, "text": "But these Titania based photocatalysts \n\ncannot be applied practically to mass-scale commercial \n\nreactor systems due to high cost and operational \n\ndisturbances due to sunlight irradiation and highly acidic \n\nconditions [62]. \n\nPhysical methods of Cr(VI) removal. \nPhysical remediation is achieved by techniques such as \n\nadsorption, electrolysis, ion exchange, membrane \n\nfiltration and capping [19,63,64]. Adsorption is widely \n\nused for chromium removal in wastewater, consisting of \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  60 \n\nsignificant advantages such as low cost, profitability, \n\navailability, high efficiency, and minimum effort \n\noperation than other physio-chemical methods. A range \n\nof synthetic and natural adsorbents, including activated \n\ncarbon, zeolite, chitosan, treated wastes, biological \n\nmaterials of coconut shell, wood husk, orange peel, \n\nhazelnut shell, sawdust, are used for Cr(VI) removal with \n\na wide range of removal percentages under different pH \n\nvalues which are mostly laid on the extreme acidic range \n\n[65\u201369]. As some of the above adsorbents are freely \n\navailable in nature, so that, adsorption is considered as \n\none of the cost-effective methods of physical remediation. \n\nMembrane filtration technology is implemented with \n\nreverse osmosis, which is considered as one of the best \n\navailable technology for removing all forms of chromium \n\n[70\u201372]. Literature shows that different membrane \n\ntechnology modifications to enhance Cr(VI) removal \n\neffectiveness, including micellar enhanced ultrafiltration, \n\npolymer inclusion membranes, ion exchange membranes \n\nand nanofiltration [73,74]. However, membrane \n\ntechnology is considered as a costly method with \n\ngenerating a large volume of concentrated liquid toxic \n\nwastes [72]. \n\nThe acidic and basic ion exchange resins have also \n\nreported as effective Cr(VI) removal methods from \n\nchromium contaminated wastewater.  A study of [75] \n\nhave developed a complete Cr (VI) removal process from \n\nreal wastewater by using a strongly basic synthetic \n\nDowex 2-X4 resin without affecting pH. Further studies \n\nof [57] indicate 99.5% of Cr(VI) removal from \u201csynthetic \n\nwastewater\u201d using solvent impregnated resins which are \n\nacidic. \n\nBiological methods of Cr(VI) removal. \nRemediation of chromium contaminated sources using \n\nbiological agents including bacteria, fungi, algae, and \n\nplants play an important role in remediation approaches. \n\nBacteria and fungi have shown efficient remediation \n\nagents than other agents. It has shown that organisms \n\nthat can survive in a contaminated site may have the \n\nability to remediate the contaminated site by themselves \n\nup to a certain level by transforming toxic pollutants into \n\nnontoxic forms [19,76\u201381]. This detoxification is achieved \n\nthrough biosorption, bioaccumulation and \n\nbiotransformation. \n\nBioremediation is affected by several physio-chemical \n\nfactors including, energy source (electron donors), \n\nelectron acceptors, nutrients, pH, temperature and \n\ninhibitory substrates or metabolites [82]. Bioremediation \n\nof chromium is implemented in both in situ and ex situ \n\ndepending on the nature and requirements of the \n\ncontaminated site [83,84]. \n\nBiological methods are considered as more advantageous \n\nfrom the economic and environmental point of view as \n\nthey are cost-effective due to low installation and \n\noperational cost, eco-friendly with generating much less \n\nsecondary pollutants, convenient and straightforward \n\noperation compared to physiochemical methods [63,85\u2013\n\n87]. \n\nBioremediation of Cr(VI) by bacteria. \nBacterial bioremediation of Cr(VI) is explained in terms \n\nof chromium tolerance mechanisms such as biosorption \n\nand biotransformation/ bioreduction in both Gram-\n\npositive and Gram-negative strains [19]. During the \n\nbioreduction, highly toxic Cr(VI) is reduced into lesser \n\ntoxic Cr (III) inside the bacterial cytoplasm, cell wall, or \n\nin both. The bacterial strains that can reduce Cr (VI) are \n\nusually named Chromium Reducing Bacteria (CRB).", "start_char_idx": 15036, "end_char_idx": 19125, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "af0bec92-6fce-48d7-9d5b-b8df8f0e7616": {"__data__": {"id_": "af0bec92-6fce-48d7-9d5b-b8df8f0e7616", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1f631274-ff5e-4b26-97cb-057202d0c4c6", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "a75f6ddae9c6b40c7a4163ce2782aa3e038a9c1d5ed95a37707ac91fc1f05245", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fbb8ba36-266d-454d-8785-3f98a82b80d0", "node_type": "1", "metadata": {}, "hash": "6a99f631ae6554505d28c3b38fc0edac4c9922ce77fa16065a36a8319b5ea015", "class_name": "RelatedNodeInfo"}}, "text": "It is \n\nbelieved that Gram-positive CRB have a significant high \n\ntolerance to high Cr(VI) concentrations than gram-\n\nnegative CRB [88].  \n\nAccording to previous studies, bacterial genera such as \n\nPseudomonas, Bacillus, Enterobacter, Deinococcus, \n\nShewanella, Agrobacterium, Escherichia, Thermus, \n\nMicrobacterium, Desulfovibrio, Deinococcus, Brucella, \n\nand Staphylococcus have the potential to reduce Cr(VI) \n\n\u201cdirectly\u201d with enzymes and \u201cindirectly\u201d with metabolic \n\nend products [88\u201392]. It has also been reported that \n\nchromium tolerance and reduction are independent \n\nproperties of bacteria, which means not all Cr(VI) \n\nresistant bacteria can reduce Cr(VI) into Cr(III) [88,93].  \n\nBacterial Cr(VI) reduction is achieved under aerobic, \n\nanaerobic and both conditions [94]. Aerobic reduction is \n\nassociated with soluble proteins and NADH as electron \n\ndonors to enhance the reduction process, while anaerobic \n\nCr(VI) reduction is associated with cell membrane bound \n\nreductase (flavin reductase, cytochromase, \n\nhydrogenases) and soluble reductase or both [95,96]. \n\nBacterial bioreduction rate of Cr(VI) is influenced by \n\ninitial cell density/ concentration, initial chromium \n\nconcentration, initial pH, temperature, electron donors, \n\noxyanions, salt concentration, presence of other heavy \n\nmetals, metabolic inhibitors and oxidation-reduction \n\npotential of culture [96,97]. Further, the bacterial strains \n\nin the same species have different Cr(VI) tolerance and \n\nremoval potentials depending on the level of the \n\ncontaminants in the environment. This phenomena was \n\nevidenced in a comparative study carried out between \n\nuncontaminated and Cr(VI) polluted environments [98].  \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  61 \n\nTable 2. Chemical, physical and biological method of Cr(VI) removal \n\nMethod Technique Mechanism \n\nType of \ncontaminated \n\nsource \n(Tested) \n\nCr, Cr \n(VI) \n\nremoval \npercentag\n\ne \n\nTime pH \nTemp. \n\n(\uf0b0C) \nReference \n\nChemical \n\nReduction \nCr(VI) reduction and \n\nadsorption by ED-\nRGO. \n\nSynthetic \nwastewater \n\n100% 24 hrs. 2.0 N/A [120] \n\nReduction \nCr(VI) reduction by \nCalcium polysulfide \n\n(CaSx). \n\nContaminated \nground water \n\n90% 4 days \n8 -\n\n12.5 \nN/A [121] \n\nReduction \nand \n\nbiosorption \n\nCr(VI) reduction and \nbiosorption by \n\nchemically treated \nbrown seaweed \n\n(Ecklonia sp.). \n\nSynthetic \nwastewater \n\n100% 12 hrs. 2.0 25 [122] \n\nReduction \nCr(VI) removal by \n\nchemically and \nelectrochemically. \n\nSynthetic \nwastewater \n\n99.99% 10 min. \n8.5 \u2013 \n10.0 \n\nN/A [123] \n\nReduction \n\nCr(VI) reduction by \nSodium \n\ncorboxymethyl \nstabilized nanoscale \n\nzero valent iron. \n\nSynthetic \nwaste soil \n\nsample \n80% 72 hrs. \n\n4.73 \u2013 \n7.36 \n\nN/A [124] \n\nReduction \nand \n\ncoagulation \n\nCr(VI) removal by \nFerrous sulfate \n\n(FeSO4). \n\nSpiked ground \nwater \n\n95% 46 hrs. > 7.5 N/A [125] \n\nPhysical \n\nAdsorption \nChromium Removal \n\nby fly ash. \nIndustrial \n\nwaste \n97.86% 12 hrs. N/A 25 [126] \n\nAdsorption \nCr(VI) removal by \nRagi husk powder. \n\nSynthetic \nwastewater \n\n81.34% 2 hrs.", "start_char_idx": 19126, "end_char_idx": 22197, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fbb8ba36-266d-454d-8785-3f98a82b80d0": {"__data__": {"id_": "fbb8ba36-266d-454d-8785-3f98a82b80d0", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "af0bec92-6fce-48d7-9d5b-b8df8f0e7616", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "a8610d6957d002fafbc7c6bfb4e296f0a2325286757c32c4892c0d18ba0fe743", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "18b96755-f961-47d4-9ce7-e305c4cdd03f", "node_type": "1", "metadata": {}, "hash": "b7654c0e2911d9aba6fde92643083bca4fc396bd0abf3f28a8b41bb2f28a6f30", "class_name": "RelatedNodeInfo"}}, "text": "Synthetic \nwastewater \n\n81.34% 2 hrs. 1.75 N/A [127] \n\nAdsorption \nCr(VI) removal by \n\ngreen algae and \nactivated carbon. \n\nWaste water 99.52% 2 hrs. 1.0 25 [110] \n\nAdsorption \nCr(VI) removal by \n\ntreated waste \nnewspaper (TWNP). \n\nSynthetic \nwastewater \n\n64% 1 hrs. 3.0 25 [65] \n\nAdsorption \nCr(VI) removal by \n\ngreen coconut shell. \nSynthetic \n\nwastewater \n95% 30 min. 6.5 28 [66] \n\nAdsorption \n\nCr(VI) removal by \nagriculture wastes. \n\nMaize corncob. \nCane bagasse. \n\nJatrophica oil cake. \n\nSynthetic \nwastewater \n\n \n \n\n62% \n92% \n97% \n\n \n \n \n\n1 hrs. \n\n \n \n \n\n2.0 \n\n \n \n \n\n30 \n\n[128] \n\nAdsorption \nCr(VI) removal by \n\nMangifera indica \nleaves. \n\nSynthetic \nwastewater \n\n91% 2 hrs. 2.0 30 [129] \n\nRetention/ \nfiltration \n\nCr(VI) removal by \nAromatic polymide \nthin film membrane. \n\nSynthetic \nwastewater \n\n77% N/A 8.0 25 [73] \n\nAdsorption \nCr(VI) removal by \n\nanion exchange \nresins. \n\nSynthetic \nwastewater \n\n99.4% 30 min. 3.0 \u2013 5.0 \n25  \n60 \n\n[130] \n\nAdsorption \nCr(VI) removal by \nhydrophobic resin. \n\nSynthetic \nwastewater \n\n99.5% \n92% \n\n24 hrs. 3.0 25 [131] \n\nAdsorption \nCr(VI) removal by \nboiled rice husk. \n\nSynthetic \nwastewater \n\n71% 3 hrs. 2.0 27 [132] \n\nAdsorption \nCr(VI) removal by \n\nformaldehyde treated \nrice husk. \n\nSynthetic \nwastewater \n\n76.5% 3 hrs. 2.0 27 [132]", "start_char_idx": 22160, "end_char_idx": 23443, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "18b96755-f961-47d4-9ce7-e305c4cdd03f": {"__data__": {"id_": "18b96755-f961-47d4-9ce7-e305c4cdd03f", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fbb8ba36-266d-454d-8785-3f98a82b80d0", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "effc89567126763b18b1339bf8a4a9079901a75bde1808dc61392a4aa0940223", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "381d6cb5-618c-44c8-b392-ec458395cf5c", "node_type": "1", "metadata": {}, "hash": "ed67fcc92c44b3a69fe2ab25411c69d660bcb0142cbe386f9b1f776ed1c17b46", "class_name": "RelatedNodeInfo"}}, "text": "2.0 27 [132] \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  62 \n\nPhysical \n\nAdsorption \n\nCr(VI) removal by \nmodified \n\nmontmorillonite clay \nnanocomposite. \n\nSynthetic \nwastewater \n\n99.9% 24 hrs. \n2.0 - \n6.6 \n\n25 [133] \n\nAdsorption \nCr(VI) removal by Fe-\n\n2O3/ graphene \nadsorbents. \n\nSynthetic \nwastewater \n\n70.33% N/A 3 - 4 25 [134] \n\nAdsorption \n\nCr(VI) removal by \nsynthesized \n\nhydroxyapatite \nmicrofibrillated \n\ncellulose \n(CHA/MFC) \n\nSynthetic \nwastewater \n\n94% 5 min. 7  - 5 25 [135] \n\nAdsorption \nCr(VI) removal by \n\nMagnetite \nnanoparticles \n\nSynthetic \nwastewater \n\n66% 2 hrs. 3.0 25 [136] \n\nAdsorption \nCr(VI) removal by \n\nmixed waste tea and \ncoffee ground \n\nSynthetic \nwastewater \n\n95% 3 hrs. 2.0 50 - 65 [137] \n\nAdsorption \n\nCr(VI) removal by \nnatural adsorbents. \n\nWool \nOlive cake \nSawdust \n\nPine needles \nAlmond \n\nCoal \nCactus \n\nSynthetic \nwastewater \n\n \n \n\n69.3% \n47.1% \n53.5% \n42.9% \n23.5% \n23.6% \n19.8% \n\n2 hrs. 2.0 30 \n\n[138] \n \n \n \n \n \n \n \n \n\nAdsorption \nPolypyrrole \u2013\n\nmontmorillonite clay \ncomposite \n\nSynthetic \nwastewater \n\n100% 24 hrs. 2.0 25 [139] \n\nAdsorption \nNanocomposite of \nZnO with cotton \n\nstalks biochar \n\nSynthetic \nwastewater \n\n96.19% 1 hr. 2-4 25 [140] \n\nFiltration \nGreen emulsion \n\nliquid membrane \nSynthetic \n\nwastewater \n97-99% 0.5 hrs. 0.45 30 [71] \n\nFiltration \nGreen synthesized \nCuO nanoparticles \n\nSynthetic \nwastewater \n\n88.08% 2 hrs. 6.9 25 [70] \n\nBiological \n\nReduction \nCr (VI) bioreduction \nby effluent bacteria \nStaphylococcus cohnii \n\nSynthetic \nwastewater \n\n90% 96 hrs. 7.2 37 [141] \n\nReduction \nCr (VI) bioreduction \n\nby Pseudomonas \numsongensis \n\nSynthetic \nwastewater \n\n93.9% 72 hrs. 7.0 30 [142] \n\nReduction \nAdsorption \n\nCr (VI) bioreduction \nand biosorption by \n\nBacillus sp. \n\nSynthetic \nwastewater \n\n97.04% 96 hrs. 7.0 37 [143] \n\nReduction \nCr (VI) bioreduction \n\nby Aeromonas \nhydrophila \n\nSynthetic \nwastewater \n\n88% 72 hrs. 7.2 30 [144] \n\nReduction \nCr(VI) reduction by \nBacillus thuringiensis \n\nSynthetic \nwastewater \n\n86.42% 96 hrs. 7.0 35 [91] \n\nReduction \nCr(VI) reduction by \nStaphylococcus capitis \n\nSynthetic \nwastewater \n\n97.34% 96 hrs. 7.0 35 [91] \n\nReduction \nCr(VI) reduction by \n\nBacillus cereus \nSynthetic \n\nwastewater \n98.5% 72 hrs. 7.1 26 [98] \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.", "start_char_idx": 23431, "end_char_idx": 25776, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "381d6cb5-618c-44c8-b392-ec458395cf5c": {"__data__": {"id_": "381d6cb5-618c-44c8-b392-ec458395cf5c", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "18b96755-f961-47d4-9ce7-e305c4cdd03f", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "9c821d47fbf48b07d2343358bbc317b8b825e043d2e4a95a559f6fb55f2c9127", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70ae5417-7cda-4dc1-ad96-b63ee0b8982a", "node_type": "1", "metadata": {}, "hash": "ee805a9a82e21e73b14be7783ac6d254ee873e0a5c096a605de154f45d7f12d9", "class_name": "RelatedNodeInfo"}}, "text": "\u00a9NJB, BSN  63 \n\nReduction \nSorption \n\nCr(VI) reduction and \nsorption by \n\nEnterobacter sp. \n\nSynthetic \nwastewater \n\n99.1% 25 hrs. 6.0 45 [145] \n\nReduction \nCr(VI) reduction by \nMorganella morganii \n\nSynthetic \nwastewater \n\n \nRaw tannery \n\neffluent \n\n92% \n \n \n\n90% \n\n48 hrs. \n \n \n\n48 hrs. \n\n7.0 \n \n \n\n7.0 \n \n\n37 \n \n \n\n37 \n\n[146] \n\nReduction \nSorption \n\nCr(VI) reduction and \nsorption by \n\nStenotrophomonas \nrhizophila \n\nSynthetic \nwastewater \n\n100% 28 hrs. 7.5 30 [147] \n\nReduction \nCr(VI) reduction by \n\nCellulosimicrobium sp. \nSynthetic \n\nwastewater \n100% 48 hrs. 7.0 30 [148] \n\nReduction \nCr(VI) reduction by \n\nGeobacter \nsulfurreducens \n\nSynthetic \nwastewater \n\n99% 2hrs. N/A 30 [149] \n\nReduction \nCr(VI) reduction by \n\nPseudomonas \naeruginosa \n\nSynthetic \nwastewater \n\n93% 96 hrs. 7-8 30 [150] \n\nSorption \nCr(VI) biosorption by \nShewanella putrefaciens \n\nSynthetic \nwastewater \n\n85.68% 17 hrs. 8.0 38.44 [151] \n\nReduction \nCr(VI) bioreduction \nby mixed bacterial \n\nconsortium. \n\nSynthetic \nwastewater \n\n100% \n120 \nhrs. \n\n8.0 30 [152] \n\nReduction \nAdsorption \n\nCr(VI) bioreduction \nand biodorption by \n\nCorynebacterium \npaurometabolum, \n\nSynthetic \nwastewater \n\n55% 2 hrs. 3.0 30 [153] \n\nReduction \n \n\nCr(VI) bioreduction \nby Cellulosimicrobium \n\nfunkei \n\nSynthetic \nwastewater \n\n80.43% \n120 \nhrs. \n\n7.0 35 [154] \n\nBiological \n \n \n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nReduction \n \n\nCr(VI) bioreduction \nby Pseudomonas \n\nstutzeri \n\nSynthetic \nwastewater \n\n97% 24 hrs. 7.0 37 [155] \n\nReduction \nCr(VI) bioreduction \n\nby Acinetobacter \nbaumannii \n\nSynthetic \nwastewater \n\n99.58% 24 hrs. 8.0 37 [155] \n\nReduction \nCr(VI) bioreduction \nby Ochrobactrum sp. \n\nSynthetic \nwastewater \n\n96.5% N/A 7.0 30 [156] \n\nAdsorption \nCr(VI) biosorption by \n\nTrichoderma sp. \nSynthetic \n\nwastewater \n97.39% 2 hrs. 5.5 25 [103] \n\nReduction \nAdsorption \n\nCr(VI) biosorption \nand bioreduction by \nPaecilomyces lilacinus \n\nSynthetic \nwastewater \n\n100% \n120 \nhrs. \n\n5.5 25 [157] \n\nAdsorption \nCr(VI) biosorption by \n\nPhanerochaete \nchrysosporium \n\nSynthetic \nwastewater \n\n99.7% 72 hrs. 7.0 40 [158] \n\nAdsorption \nCr(VI) biosorption by \n\nPleurotus ostreatus \nSynthetic \n\nwastewater \n80% 12 hrs. \n\n2.0 \u2013 \n11.0 \n\n65 [159] \n\nAdsorption \n\nCr(VI) adsorption by \nCationic surfactant-\n\nmodified, \nKazachstania \nyasuniensis \n\nKodamaea transpacifica \nSaturnispora quitensis \n\nSaccharomyces \ncerevisiae \n\nSynthetic \nwastewater \n\n \n \n\n80.70% \n85.80% \n\n \n85.40% \n75.80% \n\n4 hrs.", "start_char_idx": 25781, "end_char_idx": 28253, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70ae5417-7cda-4dc1-ad96-b63ee0b8982a": {"__data__": {"id_": "70ae5417-7cda-4dc1-ad96-b63ee0b8982a", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "381d6cb5-618c-44c8-b392-ec458395cf5c", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "4a4ea196a89d718e624b5d3259f55ad65821463a62d30cccc523896cdeecb380", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8b9eccb3-53e3-4c70-8496-c768d6a42d55", "node_type": "1", "metadata": {}, "hash": "a02a26569bb210e32d46fdca56256d01cebc586dd5b0d1dd3dab2b76d7ff6eb8", "class_name": "RelatedNodeInfo"}}, "text": "4.5 25 [105] \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al. \n \n\n\u00a9NJB, BSN  64 \n\n \n\n \n\n \n\n \n\n \n\nBiological \n\nAdsorption \n\nCr(VI) adsorption by \na hydroxyl-\n\nfunctionalized \nmagnetic Aspergillus \nniger nanocomposite \n\nSynthetic \nwastewater \n\n64.91% 4 hrs. 5.0 50 [160] \n\nAdsorption \nCr (VI) adsorption by \nChlorella sorokiniana. \n\nSynthetic \nwaste water \n\n99.68% 72 hrs. 7.0 40 [161] \n\nReduction \nAdsorption \n\nCr (VI) removal by \ngreen algal strain \nCladophora albida \n\nSynthetic \nwaste water \n\nindustrial \nwaste water \n\n100% \n120 \nhrs. \n\n0.5 25 [162] \n\n \nAdsorption \n\nCr(VI) removal by \nmarine algae \n\nTurbinaria ornata \n\nSynthetic \nwastewater \n\n95.25% 3.5 hrs. 4.7 33.6 [163] \n\nN/A \u2013 Not applicable \n\nTable 3. Microorganisms and substrates used in biofilm formation for bioremediation of Cr(VI) \n\nOrganism Adhesion substrate \nCr (VI) removal \npercentage (%) \n\npH \nTemp. \n\n(\u2103) \nTime Reference \n\nArthrobacter viscosus \nGranular activated \n\ncarbon \n99.9% 5 \u2013 5.5 28 30 days [190] \n\nPseudomonas sp. \nBacillus sp. \nAzotobacter sp. \nAcremonium sp. \n\nGlass wool 90% 5.6-6.1 30 10 days [172] \n\nStreptomyces strain CG252 Glass bead 100% N/A 30 \n48 \u2013 72 \n\nhrs. \n[191] \n\nArthrobacter sp. \nGravel packed bed \n\nreactors \n100% N/A 30 26 hrs. [192] \n\nMorganella morganii STB5 \nPolystyrene \nPolysulfone \n\n99.47% \n90.78% \n\n7.0 30 72 hrs. [193] \n\nArthrobacter sp. SUK 1205 Glass beads 100% 7.0 37 96 hrs. [194] \n\nHalomonas sp. Pumic particle stones 94.5% 6.5 28 48 hrs. [195] \n\nBacillus subtilis \nEscherichia coil \nAcinetobacter junii \n\nAlginate bead 97.84% 7.0 25 7 hrs. [196] \n\nWickerhamomyces anomalus Wood husk 92.5% 3.72 30 N/A [171] \n\nAcinetobacter haemolyticus Wood husk 97% 7.0 25 72 hrs. [69] \n\nStreptococcus salivarius Stainless steel AISI 316L 42% N/A 37 72 hrs. [197] \nPseudomonas fluorescens LB \n300 \n\nGlass beads 100% 6.8-7.0 30 8 days [198] \n\nCellulosimicrobium sp. \n\nPVC \nRubber tubing \n\nSand \nSmall stone \n\n99.5% \n90.0% \n96% \n\n88.4% \n\nN/A 25 11 days [199] \n\nEscherichia coli Kaolin 100% 4.6-5.1 37 10 days [200] \n\nNostoc sp. Polystyrene 86.49% 7.0 25 7 days [201] \n\nShewanella xiamenensis Zeolite 100% 3.0 22-25 35 days [202] \n\nCunninghamella elegans \nStainless steel \n\ncompression springs \n98.6% 7.0-3.0 28 40 hrs. [203] \n\nArthrobacter sp.", "start_char_idx": 28254, "end_char_idx": 30521, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8b9eccb3-53e3-4c70-8496-c768d6a42d55": {"__data__": {"id_": "8b9eccb3-53e3-4c70-8496-c768d6a42d55", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70ae5417-7cda-4dc1-ad96-b63ee0b8982a", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "0f5411db44ae017525502b8f3c79d512d5036132cf8fe4896fb3dd6537dc58e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "853b544b-00db-4bf3-a926-6dbea6ddd51e", "node_type": "1", "metadata": {}, "hash": "6588b02b92a94c03669637c45a360e4a5dc9856b3752a909773b495a7595758a", "class_name": "RelatedNodeInfo"}}, "text": "[203] \n\nArthrobacter sp. SUK 1201 Glass beads 100% 7.0 37 3 days [204] \n\nLysinibacillus sphaericus \nRTA-01 \n\nGlass slide 82.8% 5.2 37 72 hrs. [205] \n\nOchrobactrum \npseudintermedium ADV31 \n\nPolyurethane foam 82% 7.0 45 5 days [206] \n\nN/A \u2013 Not applicable \n\n \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  65 \n\nBioremediation of Cr(VI) by fungi. \nSimilar to the bacterial remediation of toxic metals, some \n\nfungal strains have been investigated for bioremediation \n\nwith the same metal removal techniques used with \n\nbacteria, i.e. biosorption, bioaccumulation and \n\nbiotransformation/ bioreduction. \n\nFungi Aspergillus sp. was reported to remove chromium \n\nthrough bioreduction from contaminated effluents [99]. \n\nThis study also indicates 65% of chromium removal from \n\ntannery effluent and 85% of Cr(VI) removal from the \n\nsynthetic medium at pH 6 within 07 days. The similar \n\nbioreduction of Cr(VI) has been also reported by [19,100\u2013\n\n102] with  Hypocrea tawa, Trichoderma inhamatum and \n\nisolated Yeast strains. However, the Cr(VI) reduction \n\ncapability of fungal strains can be changed with the initial \n\nCr(VI) concentration and initial biomass of the strain.  \n\nFusarium oxysporum and Trichoderma sp. have shown to \n\nadsorb Cr(VI) on to their cell surface by forming chemical \n\nbonds with cell surface proteins with analytically verified \n\nevidence using FT-IR spectrum [19,103]. Comparison of \n\nCr(VI) removal in synthetic and raw wastes  was found \n\nto be 77% and 85% of Cr(VI) removal respectively, with \n\n200-1000 mg/L of initial Cr(VI) concentrations using \n\nimmobilized Baker\u2019s yeast strain (Saccharomyces \n\ncerevisiae) in Biomass/Polymer Matrices Beads (BPMM) \n\nthrough biosorption [104]. A study comparing Cr(VI) \n\nbiosorption by native Ecuadorian yeast species, reported \n\nthat Kazachstania yasuniensis, Kodamaea transpacifica, and \n\nSaturnispora quitensis have the ability to remove Cr(VI). \n\nFurthermore, they have reported that efficient Cr(VI) \n\nremoval can be achieved by inducing belzalkonium \n\nchloride (BZK) to cell surface as a chemical modification \n\nto the applying bio agent  [105]. \n\nBioremediation of Cr(VI) by algae. \nIt is evident that both freshwater and marine algal species \n\nsuch as  Cladophora sp., Selenastrum sp., Spirogyra sp., \n\nCeramium sp, Chlorella sp. and Ulva sp. can be used to \n\nremediate chromium contaminated wastewater by \n\napplying as cultures or incorporating with other physio-\n\nchemical methods following biosorption and \n\nbioreduction [19,106\u2013109]. Unlike other organisms, algae \n\nhave been used in both living and non-living forms for \n\nCr(VI) remediation. Introducing an efficient Cr(VI) \n\nremoval method [110] reports that dried Ulva lactuca \n\nincorporated into activated carbon can be used to \n\nremediate highly acidic and halophilic wastewater.  \n\nChlorella sp. has been used in most Cr(VI) algal \n\nremediation bioreactors as it is widely dispersed in the \n\naquatic environment with higher Cr(VI) tolerance \n\n[109,111\u2013114]. Constructing a hybrid remediation system \n\n[115] has introduced efficient and reusable alumina \n\nhollow fibers immobilized with TiO2 and Chlorella \n\nvulgaris cells. Additionally, this hybrid system has been \n\nachieved greater than 90% of Cr(VI) removal after five \n\nsequential reuses.", "start_char_idx": 30497, "end_char_idx": 33810, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "853b544b-00db-4bf3-a926-6dbea6ddd51e": {"__data__": {"id_": "853b544b-00db-4bf3-a926-6dbea6ddd51e", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8b9eccb3-53e3-4c70-8496-c768d6a42d55", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "9da4d16454cc089aed4747e0336a70f7bb82ca7f5c6b6b9e65873000294a746f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "dcc3a362-5588-4433-bbcb-5434de6d2530", "node_type": "1", "metadata": {}, "hash": "4bb051ff93ab27114b5a14d628fbcdd63e1393a2abd4185fe7744fbc85f0756e", "class_name": "RelatedNodeInfo"}}, "text": "However, similar to bacterial bioremediation, algal \n\nCr(VI) bioremediation is influenced by physio-chemical \n\nparameters including pH, temperature, initial biomass, \n\ninitial Cr(VI) concentration, light intensity, contact time \n\nof cells and wastes of the treatment process \n\nbioremediation [19,116].  \n\nBioremediation of Cr(VI) by plants. \nLimited studies have reported that the Cr(VI) \n\ndetoxification and removal potential of plants compared \n\nto the other biological agents. Green plants detoxify \n\nmany pollutants using various mechanisms followed by \n\nuptake, known as phytoremediation [117]. Plants can \n\neither store heavy metals in roots or partially translocate \n\nto shoot through the xylem after getting diffused into the \n\nroot system. Further, it has been reported that upward \n\ntranslocation of heavy metals is retarded by the cation \n\nexchange process in plant tissues and leads to \n\nconsiderable heavy metal accumulation in roots \n\ncompared to axial parts of plants.  In the point of view of \n\nCr(VI) and other chromium forms, this phenomenon has \n\nbeen reported else ware using Phragmitus australis, \n\nAilantus altissima and Salix viminalis [118]. This may be \n\ndue to the encapsulation in vacuoles of root cells based \n\non natural counteraction of plants against chromium \n\ntoxicity [119]. According to observations over 360 days, a \n\nstudy suggests that Salix viminalis used for large scale \n\nphytoremediation application for removal of Cr(VI) and \n\nother chromium forms from contaminated sources as \n\nS.viminalis were removed 70% of total chromium and \n\n90% of Cr(VI) removal with indicating higher \n\ntranslocation capacity [118]. \n\nIn vitro study of Nopalea cochenillifera found that it has the \n\npotential to accumulate a wide range of Cr(VI) (600 \u2013 \n\n26,000 mg/ Kg) from the growth medium. As N. \n\ncochenillifera is a non-consuming plant for diets, the risk \n\nof bioaccumulation can be avoided in the ecosystem. \n\nFurthermore, the above study has also reported plant \n\nspecies with different chromium accumulation potentials \n\nincluding Gynura pseudochina, Brassica napus, Prospis \n\njuliflora, Leersia hexandra, Urtica dioica, Salix matsudana, \n\nBrassica napus, Helianthus annuus, Lycopersicon \n\nlycopersicum and Saponaria officinalis perhaps considered \n\nfor the phytoremediation [117]. \n\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  66 \n\nBiofilms \nAn aggregated community of prokaryotic and eukaryotic \n\nmicroorganisms adhering to substance/matrix surface \n\nand submerged/embedded in self-produced \n\nextracellular polymeric substances (EPS) is termed a \n\n\"biofilm\" [164,165]. These aggregates are omnipresent in \n\nthe biosphere, including soil, water, plant and animal \n\ntissues, abiotic substances like pipelines, ship hulls and \n\nfilters. These biofilms can be developed in solid\u2013water, \n\nwater\u2013air and solid\u2013air interfaces with composing EPS, \n\nmultivalent cations, biogenic particles, colloidal and \n\ndissolved compounds [166]. Biofilms are comprised of \n\nboth single and multiple microbial species. Among them, \n\nmultiple species biofilms are the most dispersed biofilm \n\ntype in the environment [167]. \n\nBiofilm formation is a subsequent process that consists of \n\n03 main steps; surface attachment, biofilm maturation \n\nand dispersal [165]. In surface attachment, microbial cells \n\nundergo reversible attachment at cell poles by involving \n\ncell appendages (flagella, pilli and fimbriae) followed by \n\nirreversible attachment. After the reversible attachment \n\nstage, microbial cells can be adapted to biofilm lifestyle \n\nor left the matrix. During the irreversible attachment, \n\nstage cells adhere to the matrix by EPS and surface \n\nproteins (Sad B and Lap A). \n\nBiofilm maturation starts after the irreversible \n\nattachment with developing microcolonies.", "start_char_idx": 33814, "end_char_idx": 37645, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "dcc3a362-5588-4433-bbcb-5434de6d2530": {"__data__": {"id_": "dcc3a362-5588-4433-bbcb-5434de6d2530", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "853b544b-00db-4bf3-a926-6dbea6ddd51e", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "1dc68061494cc409c4a9f98eef63d97bc16daf2887e0901ff2ab568b7d3b4932", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6bb83aa0-54ab-4bc6-a772-813893256cd3", "node_type": "1", "metadata": {}, "hash": "0da7c3f1bbc7355c9ce4f09a23c10f284f9225a2031cd1f8f717a5b302fa657f", "class_name": "RelatedNodeInfo"}}, "text": "Biofilm maturation starts after the irreversible \n\nattachment with developing microcolonies. At this stage, \n\nprevious microbial cells are assembled and proliferated \n\nalong with producing EPS. Further studies explain that \n\nbiofilm structure, including thickness and cell density, is \n\ndynamically changed according to environmental \n\nconditions such as temperature, presence of oxygen, pH \n\nand amounts of nutrients [164,165]. \n\nThe immobilized microbial cells are transferred back to \n\nplanktonic growth as the final stage of the biofilm \n\nlifecycle and as an initial step of a naval forming biofilm. \n\nThis dispersal can be happened \u201cactively\u201d by cell motility \n\nand EPS degradation or \u201cpassively\u201d by external physical \n\nforces. \n\nEnvironmental applications of biofilms \nEven though free-living microorganisms are capable of \n\nbioremediating polluted environments, the remediation \n\nprocess can be disrupted due to high concentrated toxic \n\ncompounds, availability of nutrients and environmental \n\nstress. Applying sessile or floating biofilms for \n\nremediation is highly advantageous as biofilm \n\ncommunities have higher tolerance towards \n\nenvironmental stress, including lack of nutrient \n\navailability, high concentrated chemical exposures, pH \n\nand temperature fluctuations, lack of moisture content \n\nthan free-living microorganisms [168].  \n\nWhen selecting biofilms for remediation purposes, \n\nseveral factors must be considered: the capability to \n\ntolerate environmental stress, exchange of genetic \n\nmaterials, growth rates, metabolic diversity, and \n\nsymbiotic relationships. Based on above factors bacterial, \n\nalgal and fungal biofilms are used in bioreactors to \n\nremediate contaminated sources by a wide range of \n\npollutants including, organic pollutants (polyaromatic \n\nhydrocarbons, chlorinated aromatic compounds, \n\naromatic amine compounds, polyethylene and \n\npolythene), heavy metals (Cu, Zn, Cd, Ni, As, Fe, Hg, \n\nMn), inorganic pollutants (nitrate ions and synthetic \n\ndyes) contaminates which can adversely affect the  eco-\n\nsystems [168\u2013170].  \n\nCr(VI) remediation by biofilms \nCr(VI) bioremediation is achieved using bacterial, algal \n\nand fungal single species and multi-species biofilms \n\ngrowing on either natural or artificial substrates through \n\nbioremediation and biosorption techniques [171\u2013174].  \n\nBiofilms are more effective for Cr(VI) remediation than \n\nplanktonic cells. The study investigating Streptomyces sp. \n\nstrain CG252 and Pseudomonas aeruginosa A2Chr, \n\nrespectively, reported evidence for the above phenomena \n\n[102,175]. Another study using three (03) different \n\nbiosorbents including lyophilized Escherichia coli AUS 7 \n\ncells, granulated activated carbon (GAC) and biofilm of \n\nEscherichia coli AUS 7 on GAC  exhibited that biofilms are \n\nable to achieve a higher adsorption of Cr(VI) than GAC \n\nand lyophilized cells in according to Langmuir and \n\nFreundlich isotherm models from aqueous solutions \n\nunder acidic conditions [176]. However, contradictory \n\nresults were also reported else ware, that the planktonic \n\ncells have more significant potential for Cr(VI) reduction \n\nthan biofilms based on their study of Bacillus subtilis \n\nATCC-6633. Furthermore above study revealed that, \n\nbiofilm debris are susceptible to immobilized reduced \n\nCr(III) ions completely [177]. \n\nCompared to bacteria, reports on Cr(VI) remediation by \n\nfungal biofilms are scarce in the literature.  Immobilized \n\ncells of Aspergillus niger, Coriolus versicolor, Saccharomyces \n\ncerevisiae, and Lentinus sajorcaju have been used in \n\nsorption and reduction techniques  [99,178\u2013180]. \n\nMoreover, immobilized algal cells on different matrixes \n\nhave been used for Cr(VI) remediation by sorption of \n\nmetal ions to the cell wall components [181\u2013185]. \n\nAlgal-bacterial biofilms/consortia have also been used \n\nfor Cr(VI) bioremediation. Further, it is reported that \n\nthese consortia have symbiotic effects on each other by \n\n\n\nNepal J Biotechnol.", "start_char_idx": 37553, "end_char_idx": 41535, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6bb83aa0-54ab-4bc6-a772-813893256cd3": {"__data__": {"id_": "6bb83aa0-54ab-4bc6-a772-813893256cd3", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "dcc3a362-5588-4433-bbcb-5434de6d2530", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "fd39caba45c47809f9d9605b14c24d655cef3645f36516b76351fac6e257d7b6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "eb4031b6-488c-4384-adeb-6e811eaf6707", "node_type": "1", "metadata": {}, "hash": "4c9e20f310ffc890b2f19359ee3e0edfb0141c4e04867d4b431c08a7cb7e7be8", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  67 \n\nsupplying each other\u2019s nutritional needs such as O2 for \n\naerobic bacteria by algae and CO2 for algae by bacteria \n\nduring the remediation process. Therefore, algal-\n\nbacterial consortia are termed as a self-sustaining system \n\n[186\u2013188]. The algal-bacterial system has the potential to \n\nremove higher Cr(VI) contents such as 100 mg/L, 75 \n\nmg/L and 50 mg/L providing a carbon source to the \n\nmixed consortium of chromium reducing bacterial (CRB) \n\ncultures of Escherichia coli, Bacillus thermoamylovorans and \n\nCitrobacter sedakii from the algal strain of Chlamdomonas \n\nreinhartii. Furthermore, the above study suggests that the \n\nalgal-biofilm consortia as a cost-effective method that \n\nprevents cost of carbon sources as it fulfils by algae even \n\nthough algal-bacterial consortia takes a longer time \n\nduration for the Cr(VI) removal [189].   Table 3 illustrates \n\nsome of the   microorganisms   and substrates used in \n\nbioremediation of Cr(VI). \n\nLimitations and remedial actions of the \ncurrent bioremediation methods in Cr(VI) \nremoval \nThe notable limitations of biological methods available \n\nfor Cr(VI) removal have been identified including, \n\nvarying Cr(VI) tolerance and removal levels \n\nenvironmental conditions and nutritional requirements \n\nof biological components used, toxic substances present \n\nin wastewater which can interfere with the biological \n\ncomponents, disposal of the accumulated Cr in the \n\nbiological component, and practical difficulties in \n\nextrapolating bench/pilot-scale to full-scale field \n\napplication [207\u2013209]. Customized solutions need to be \n\nsought by assessing the remediation requirements at \n\nindividual level, because the environmental conditions \n\nand the nutritional requirements of the biological \n\ncomponent vary depending on the contaminated site. \n\nMoreover, to overcome the Cr disposal after \n\nbioremediation, it is possible to percolate the \n\nbiotransformed Cr(III) through reduction at low pH \n\nconditions and tend to be reoxidised to Cr(VI) in the \n\npresence of manganese oxide and chlorine in treated \n\neffluent or discharging environment [210,211]. In order to \n\nprevent the discharge of higher amounts of chromium \n\nand to enhance the sorption capacity of biomasses, the \n\nmetal desorption process should be followed. This \n\ndesorption can be done by acid digestions [212\u2013215] and \n\nalkaline treatments [216\u2013220] as a hybrid Cr(VI) \n\nremediation process, which has many benefits such as \n\nreduction of generation of secondary pollutants and \n\nrecovery of valuable metals. These recovered Cr(VI) and \n\nCr(III) can be applied for tannery and chromium-based \n\nchemical production as raw materials [221]. \n\nConclusion \nThe wide industrial and research application of \n\nChromium followed by emitting considerable amounts \n\nof Cr(VI), coupled with the fact that it leads to serious \n\nproblems to all components of the ecosystem. Therefore, \n\nit has been legislated to remediate Cr(VI) contaminated \n\neffluents by national and international authorities before \n\nit being discharged to the environment. This remediation \n\nis carried out by chemical, physical and biological \n\nmethods. Biological remediation is considered as the \n\nmost environment-friendly and cost-effective method \n\nrather than chemical and physical remediation. \n\nHowever, considering the limitations of the current \n\nbioremediation processes, hybrid remediation processes \n\ncombining the bioremediation with other chemical and \n\nphysical methods are being used for the effective \n\nremediation of Cr(VI) in aquatic systems. \n\nAuthors Contribution \nThe authors confirm contribution to the paper as follows: \n\nstudy conception and design: A.M.K.C.B. Aththanayake, \n\nI.V.N. Rathnayake, and M.P. Deeyamulla; draft \n\nmanuscript preparation: A.M.K.C.M. Aththanayake; \n\nReview, and editing the final draft: A.M.K.C.B. \n\nAththanayake, I.V.N. Rathnayake; All authors read and \n\napproved the final manuscript.", "start_char_idx": 41516, "end_char_idx": 45534, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "eb4031b6-488c-4384-adeb-6e811eaf6707": {"__data__": {"id_": "eb4031b6-488c-4384-adeb-6e811eaf6707", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6bb83aa0-54ab-4bc6-a772-813893256cd3", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "d25c4affb47ae6a9083255bade6a0fccd871386146bfc1ec206de299560e9ad8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "515c6703-4e35-499b-9cbe-1d28b0536d41", "node_type": "1", "metadata": {}, "hash": "3e23822dd4950a6eed0e60171337a98f3ef1e30af8e8bf3787345781bec25826", "class_name": "RelatedNodeInfo"}}, "text": "Rathnayake; All authors read and \n\napproved the final manuscript. \n\nCompeting interests \nThe authors declare that they have no any competing \n\ninterest.  \n\nAcknowledgement \nThis research was supported by the National Research \n\nCouncil, Sri Lanka Investor Driven Research Grant No. \n\n18-083. \n\nReferences  \n1.  Park D, Yun Y-S, Park JM. Use of dead fungal biomass for the \n\ndetoxification of hexavalent chromium: screening and kinetics. \nProcess Biochem [Internet]. 2005 Jun [cited 2020 Apr 9];40(7):2559\u2013\n65. Available from: https://linkinghub.elsevier.com/ \nretrieve/pii/S0032959205000038 \n\n2.  Saha R, Nandi R, Saha B. Sources and toxicity of hexavalent \nchromium. J Coord Chem [Internet]. 2011 May 20 [cited 2020 Apr \n9];64(10):1782\u2013806. Available from: https://www.tandfonline. \ncom/doi/full/10.1080/00958972.2011.583646 \n\n3.  Ashraf A, Bibi I, Niazi NK, Ok YS, Murtaza G, Shahid M, et al. \nChromium(VI) sorption efficiency of acid-activated banana peel \nover organo-montmorillonite in aqueous solutions. Int J \nPhytoremediation [Internet]. 2017 Jul 3 [cited 2021 Sep \n18];19(7):605\u201313. Available from: https://www.tandfonline.com \n/doi/full/10.1080/15226514.2016.1256372 \n\n4.  Choppala G, Bolan N, Lamb D, Kunhikrishnan A. Comparative \nSorption and Mobility of Cr(III) and Cr(VI) Species in a Range of \nSoils: Implications to Bioavailability. Water Air Soil Pollut \n[Internet]. 2013 Dec [cited 2021 Jul 11];224(12):1699. Available \nfrom: http://link.springer.com/10.1007/s11270-013-1699-6 \n\n5.  Lacalle RG, Garbisu C, Becerril JM. Effects of the application of an \norganic amendment and nanoscale zero-valent iron particles on \n\nhttp://link.springer.com/10.1007/s11270-013-1699-6\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  68 \n\nsoil Cr(VI) remediation. Environ Sci Pollut Res [Internet]. 2020 Sep \n[cited 2021 Sep 18];27(25):31726\u201336. Available from: \nhttps://link.springer.com/10.1007/s11356-020-09449-x \n\n6.  Megharaj M, Avudainayagam S, Naidu R. Toxicity of Hexavalent \nChromium and Its Reduction by Bacteria Isolated from Soil \nContaminated with Tannery Waste. Curr Microbiol [Internet]. \n2003 Jul 1 [cited 2019 Feb 27];47(1):51\u20134. Available from: \nhttp://link.springer.com/10.1007/s00284-002-3889-0 \n\n7.  Xia S, Song Z, Jeyakumar P, Shaheen SM, Rinklebe J, Ok YS, et al. \nA critical review on bioremediation technologies for Cr(VI)-\ncontaminated soils and wastewater. Crit Rev Environ Sci Technol \n[Internet]. 2019 Jun 18 [cited 2020 May 5];49(12):1027\u201378. Available \nfrom: https://www.tandfonline.com/doi/full/10.1080/ \n10643389.2018.1564526 \n\n8.  Anttila S, Boffetta P, editors. Occupational Cancers [Internet]. \nCham: Springer International Publishing; 2020 [cited 2021 Sep 20].", "start_char_idx": 45469, "end_char_idx": 48205, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "515c6703-4e35-499b-9cbe-1d28b0536d41": {"__data__": {"id_": "515c6703-4e35-499b-9cbe-1d28b0536d41", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "eb4031b6-488c-4384-adeb-6e811eaf6707", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "e4a47b4542ddfcbb6af4d816b2e462aa76807bc46ff29ede0d74116fe3735c51", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "72faeafa-1889-4869-941e-a5e907b10ca9", "node_type": "1", "metadata": {}, "hash": "cd0e8792b7aeec9adb20ba7484d34cc335a531faef87e7a8cffae08284b4bee7", "class_name": "RelatedNodeInfo"}}, "text": "Available from: http://link.springer.com/10.1007/978-3-030-\n30766-0 \n\n9.  Chen QY, Murphy A, Sun H, Costa M. Molecular and epigenetic \nmechanisms of Cr(VI)-induced carcinogenesis. Toxicol Appl \nPharmacol [Internet]. 2019 Aug [cited 2021 Jul 12];377:114636. \nAvailable from:  https://linkinghub.elsevier.com/ \nretrieve/pii/S0041008X19302443 \n\n10.  Chervona Y, Costa M. Hexavalent Chromium and Cancer. In: \nKretsinger RH, Uversky VN, Permyakov EA, editors. \nEncyclopedia of Metalloproteins [Internet]. New York, NY: \nSpringer New York; 2013 [cited 2021 Sep 20]. p. 969\u201375. Available \nfrom: http://link.springer.com/10.1007/978-1-4614-1533-6_10 \n\n11.  Prado FE, Hilal M, Chocobar-Ponce S, Pagano E, Rosa M, Prado C. \nChromium and the Plant. In: Plant Metal Interaction [Internet]. \nElsevier; 2016 [cited 2021 Sep 20]. p. 149\u201377. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/B978012803158200\n0060 \n\n12.  Wani PA, Wani JA, Wahid S. Recent advances in the mechanism \nof detoxification of genotoxic and cytotoxic Cr (VI) by microbes. J \nEnviron Chem Eng [Internet]. 2018 Aug [cited 2021 Sep \n20];6(4):3798\u2013807. Available from: https://linkinghub \n.elsevier.com/retrieve/pii/S2213343718302884 \n\n13.  Oginawati K, Susetyo SH, Rosalyn FA, Kurniawan SB, Abdullah \nSRS. Risk analysis of inhaled hexavalent chromium (Cr6+) \nexposure on blacksmiths from industrial area. Environ Sci Pollut \nRes [Internet]. 2021 Mar [cited 2021 Sep 20];28(11):14000\u20138. \nAvailable from: http://link.springer.com/10.1007/s11356-020-\n11590-6 \n\n14.  Wild P, Bourgkard E, Paris C. Lung Cancer and Exposure to \nMetals: The Epidemiological Evidence. In: Verma M, editor. \nCancer Epidemiology [Internet]. Totowa, NJ: Humana Press; 2009 \n[cited 2021 Sep 20]. p. 139\u201367. (Walker JM, editor. Methods in \nMolecular Biology; vol. 472). Available from: \nhttp://link.springer.com/10.1007/978-1-60327-492-0_6 \n\n15.  Yoshinaga M, Ninomiya H, Al Hossain MMA, Sudo M, Akhand \nAA, Ahsan N, et al. A comprehensive study including monitoring, \nassessment of health effects and development of a remediation \nmethod for chromium pollution. Chemosphere [Internet]. 2018 Jun \n[cited 2021 Sep 20];201:667\u201375. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004565351830431\n4 \n\n16.  Zhang X-H, Zhang X, Wang X-C, Jin L-F, Yang Z-P, Jiang C-X, et \nal. Chronic occupational exposure to hexavalent chromium causes \nDNA damage in electroplating workers. BMC Public Health \n[Internet]. 2011 Dec [cited 2021 Sep 20];11(1):224. Available from: \nhttps://bmcpublichealth.biomedcentral.com/articles/10.1186/1\n471-2458-11-224 \n\n17.  Haq I, Kalamdhad AS, editors. Emerging Treatment Technologies \nfor Waste Management [Internet].", "start_char_idx": 48207, "end_char_idx": 50897, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "72faeafa-1889-4869-941e-a5e907b10ca9": {"__data__": {"id_": "72faeafa-1889-4869-941e-a5e907b10ca9", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "515c6703-4e35-499b-9cbe-1d28b0536d41", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "023f1ba99e7ca44cd912ffb9be6a00744720260134e9df3ce601b5c7c3be9a8e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2a4ba3dd-c6e9-4a7d-a36a-714130ede260", "node_type": "1", "metadata": {}, "hash": "1b5796869ad4488dccf768f2da9fccef6e83a2f5a0452f81600bd7df68ca2a89", "class_name": "RelatedNodeInfo"}}, "text": "Emerging Treatment Technologies \nfor Waste Management [Internet]. Singapore: Springer Singapore; \n2021 [cited 2021 Sep 20]. Available from: \nhttps://link.springer.com/10.1007/978-981-16-2015-7 \n\n18.  He C, Gu L, Xu Z, He H, Fu G, Han F, et al. Cleaning chromium \npollution in aquatic environments by bioremediation, \nphotocatalytic remediation, electrochemical remediation and \ncoupled remediation systems. Environ Chem Lett [Internet]. 2020 \nMay [cited 2021 Sep 20];18(3):561\u201376. Available from: \nhttp://link.springer.com/10.1007/s10311-019-00960-3 \n\n19.  Jobby R, Jha P, Yadav AK, Desai N. Biosorption and \nbiotransformation of hexavalent chromium [Cr(VI)]: A \ncomprehensive review. Chemosphere [Internet]. 2018 Sep [cited \n2020 Apr 9];207:255\u201366. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004565351830898\n1 \n\n20.  Karimi-Maleh H, Ayati A, Ghanbari S, Orooji Y, Tanhaei B, Karimi \nF, et al. Recent advances in removal techniques of Cr(VI) toxic ion \nfrom aqueous solution: A comprehensive review. J Mol Liq \n[Internet]. 2021 May [cited 2021 Sep 20];329:115062. Available \nfrom:  https://linkinghub.elsevier.com/retrieve/pii/S016 \n7732220373049 \n\n21.  Barnhart J. Occurrences, Uses, and Properties of Chromium. Regul \nToxicol Pharmacol [Internet]. 1997 Aug [cited 2020 Apr \n9];26(1):S3\u20137. Available from:  https://linkinghub.elsevier.com/ \nretrieve/pii/S0273230097911326 \n\n22.  Losi ME, Amrhein C, Frankenberger WT. Environmental \nBiochemistry of Chromium. In: Ware GW, editor. Reviews of \nEnvironmental Contamination and Toxicology [Internet]. New \nYork, NY: Springer New York; 1994 [cited 2020 Apr 10]. p. 91\u2013121. \n(Reviews of Environmental Contamination and Toxicology; vol. \n136). Available from: http://link.springer.com/10.1007/978-1-\n4612-2656-7_3 \n\n23.  Mishra S, Bharagava RN. Toxic and genotoxic effects of hexavalent \nchromium in environment and its bioremediation strategies. J \nEnviron Sci Health Part C [Internet]. 2016 Jan 2 [cited 2021 Jul \n10];34(1):1\u201332. Available from: \nhttp://www.tandfonline.com/doi/full/10.1080/10590501.2015.1\n096883 \n\n24.  Cervantes C, Campos-Garc\u00eda J, Devars S, Guti\u00e9rrez-Corona F, \nLoza-Tavera H, Torres-Guzm\u00e1n JC, et al. Interactions of chromium \nwith microorganisms and plants. FEMS Microbiol Rev [Internet]. \n2001 May [cited 2020 Apr 9];25(3):335\u201347. Available from: \nhttps://academic.oup.com/femsre/article-\nlookup/doi/10.1111/j.1574-6976.2001.tb00581.x \n\n25.  Costa M, Klein CB. Toxicity and Carcinogenicity of Chromium \nCompounds in Humans. Crit Rev Toxicol [Internet]. 2006 Jan \n[cited 2020 Apr 9];36(2):155\u201363. Available from: \nhttp://www.tandfonline.com/doi/full/10.1080/10408440500534\n032 \n\n26.  McLean JE, McNeill LS, Edwards MA, Parks JL.", "start_char_idx": 50832, "end_char_idx": 53547, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2a4ba3dd-c6e9-4a7d-a36a-714130ede260": {"__data__": {"id_": "2a4ba3dd-c6e9-4a7d-a36a-714130ede260", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "72faeafa-1889-4869-941e-a5e907b10ca9", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "80db587c06603fcb33336eb88e18d5e33c3648ce0c61e8470234eeb3c654150b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f66f9ded-54c4-4a5c-a999-674572b80597", "node_type": "1", "metadata": {}, "hash": "04be52f34da0b11792eba0ae195b0765c4924c6826da71db75073933230cdcc4", "class_name": "RelatedNodeInfo"}}, "text": "McLean JE, McNeill LS, Edwards MA, Parks JL. Hexavalent \nchromium review, part 1: Health effects, regulations, and analysis. \nJ - Am Water Works Assoc [Internet]. 2012 Jun [cited 2020 Apr \n9];104(6):E348\u201357. Available from: \nhttp://doi.wiley.com/10.5942/jawwa.2012.104.0091 \n\n27.  Kim HS, Kim YJ, Seo YR. An Overview of Carcinogenic Heavy \nMetal: Molecular Toxicity Mechanism and Prevention. J Cancer \nPrev [Internet]. 2015 Dec 30 [cited 2020 Apr 9];20(4):232\u201340. \nAvailable from: http://www.jcpjournal.org/journal/view.html? \ndoi=10.15430/JCP.2015.20.4.232 \n\n28.  Baruthio F. Toxic effects of chromium and its compounds. Biol \nTrace Elem Res [Internet]. 1992 Jan [cited 2020 Apr 9];32(1\u20133):145\u2013\n53. Available from: http://link.springer.com/10.1007/ \nBF02784599 \n\n29.  Oliveira H. Chromium as an Environmental Pollutant: Insights on \nInduced Plant Toxicity. J Bot [Internet]. 2012 May 20 [cited 2020 \nApr 9];2012:1\u20138. Available from: https://www.hindawi.com/ \narchive/2012/375843/ \n\n30.  Zayed A, Lytle CM, Qian J-H, Terry N. Chromium accumulation, \ntranslocation and chemical speciation in vegetable crops. Planta \n[Internet]. 1998 Aug 6 [cited 2020 Apr 9];206(2):293\u20139. Available \nfrom: http://link.springer.com/10.1007/s004250050403 \n\n31.  Shanker A, Cervantes C, Lozatavera H, Avudainayagam S. \nChromium toxicity in plants. Environ Int [Internet]. 2005 Jul [cited \n2020 Apr 9];31(5):739\u201353. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S016041200500023\n1 \n\n32.  Zeid IM. Responses of Phaseolus Vulgaris Chromium and Cobalt \nTreatments. Biol Plant [Internet]. 2001 [cited 2020 Apr 9];44(1):111\u2013\n5. Available from: http://link.springer.com/10.1023/ \nA:1017934708402 \n\n33.  Barcel\u00f3 J, Poschenrieder C, Guns\u00e9 B. Water Relations of Chromium \nVI Treated Bush Bean Plants ( Phaseolus vulgaris L. cv.", "start_char_idx": 53503, "end_char_idx": 55316, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f66f9ded-54c4-4a5c-a999-674572b80597": {"__data__": {"id_": "f66f9ded-54c4-4a5c-a999-674572b80597", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2a4ba3dd-c6e9-4a7d-a36a-714130ede260", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "818c3bea38388ce94c408f8c0eca3aee207c5383468f5d364c4a49ddabf8d3ec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3fe1befd-6ea0-4e3d-9da3-e6172fc488cc", "node_type": "1", "metadata": {}, "hash": "5012cd0bd8b9496c53846c2f8dea4965bddcbc36c803e511bd6b14c61d6be911", "class_name": "RelatedNodeInfo"}}, "text": "Contender) \n\nhttps://link.springer.com/10.1007/s11356-020-09449-x\nhttp://link.springer.com/10.1007/s00284-002-3889-0\nhttp://link.springer.com/10.1007/978-3-030-30766-0\nhttp://link.springer.com/10.1007/978-3-030-30766-0\nhttp://link.springer.com/10.1007/978-1-4614-1533-6_10\nhttps://linkinghub.elsevier.com/retrieve/pii/B9780128031582000060\nhttps://linkinghub.elsevier.com/retrieve/pii/B9780128031582000060\nhttp://link.springer.com/10.1007/s11356-020-11590-6\nhttp://link.springer.com/10.1007/s11356-020-11590-6\nhttp://link.springer.com/10.1007/978-1-60327-492-0_6\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653518304314\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653518304314\nhttps://bmcpublichealth.biomedcentral.com/articles/10.1186/1471-2458-11-224\nhttps://bmcpublichealth.biomedcentral.com/articles/10.1186/1471-2458-11-224\nhttps://link.springer.com/10.1007/978-981-16-2015-7\nhttp://link.springer.com/10.1007/s10311-019-00960-3\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653518308981\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653518308981\nhttp://link.springer.com/10.1007/978-1-4612-2656-7_3\nhttp://link.springer.com/10.1007/978-1-4612-2656-7_3\nhttp://www.tandfonline.com/doi/full/10.1080/10590501.2015.1096883\nhttp://www.tandfonline.com/doi/full/10.1080/10590501.2015.1096883\nhttps://academic.oup.com/femsre/article-lookup/doi/10.1111/j.1574-6976.2001.tb00581.x\nhttps://academic.oup.com/femsre/article-lookup/doi/10.1111/j.1574-6976.2001.tb00581.x\nhttp://www.tandfonline.com/doi/full/10.1080/10408440500534032\nhttp://www.tandfonline.com/doi/full/10.1080/10408440500534032\nhttp://doi.wiley.com/10.5942/jawwa.2012.104.0091\nhttp://link.springer.com/10.1007/s004250050403\nhttps://linkinghub.elsevier.com/retrieve/pii/S0160412005000231\nhttps://linkinghub.elsevier.com/retrieve/pii/S0160412005000231\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  69 \n\nunder both Normal and Water Stress Conditions. J Exp Bot \n[Internet]. 1986 [cited 2020 Apr 9];37(2):178\u201387. Available from: \nhttps://academic.oup.com/jxb/article-\nlookup/doi/10.1093/jxb/37.2.178 \n\n34.  Karunyal S, Renuga G, Kailash P. Effects of tannery effluent on \nseed germination, leaf area, biomass and mineral content of some \nplants. Bioresour Technol [Internet]. 1994 Jan [cited 2020 Apr \n9];47(3):215\u20138. Available from: https://linkinghub.elsevier.com/ \nretrieve/pii/096085249490183X \n\n35.", "start_char_idx": 55317, "end_char_idx": 57733, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3fe1befd-6ea0-4e3d-9da3-e6172fc488cc": {"__data__": {"id_": "3fe1befd-6ea0-4e3d-9da3-e6172fc488cc", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f66f9ded-54c4-4a5c-a999-674572b80597", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "8d53fec247f5ff46ad205ebdba6e2efe65a91dc4fd9d4e23ff1f092669f409ef", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ebec6eb6-daf3-4e05-8495-59c72093c2d9", "node_type": "1", "metadata": {}, "hash": "1fd4a6c3ef3d83aee0a0eee53516cc8a4d9fe6a4f99b324a9135724830c4fa56", "class_name": "RelatedNodeInfo"}}, "text": "Nichols PB, Couch JD, Al-Hamdani SH. Selected physiological \nresponses of Salvinia minima to different chromium \nconcentrations. Aquat Bot [Internet]. 2000 Dec [cited 2020 Apr \n9];68(4):313\u20139. Available from: https://linkinghub.elsevier.com/ \nretrieve/pii/S0304377000001285 \n\n36.  Laxmi V, Kaushik G. Toxicity of Hexavalent Chromium in \nEnvironment, Health Threats, and Its Bioremediation and \nDetoxification from Tannery Wastewater for Environmental \nSafety. In: Saxena G, Bharagava RN, editors. Bioremediation of \nIndustrial Waste for Environmental Safety [Internet]. Singapore: \nSpringer Singapore; 2020 [cited 2020 Apr 11]. p. 223\u201343. Available \nfrom: http://link.springer.com/10.1007/978-981-13-1891-7_11 \n\n37.  Sepideh S, S B, C S, B K. A Study of Toxic Dosage of Combined \nSelenium and Hexavalent Chromium on Activated Sludge \nBacteria. Int J Water Wastewater Treat [Internet]. 2019 [cited 2020 \nApr 11];5(1). Available from: https://www.sciforschenonline.org \n/journals /water-and-waste/IJWWT161.php \n\n38.  Ahemad M. Bacterial mechanisms for Cr(VI) resistance and \nreduction: an overview and recent advances. Folia Microbiol \n(Praha) [Internet]. 2014 Jul [cited 2021 Jul 12];59(4):321\u201332. \nAvailable from: http://link.springer.com/10.1007/s12223-014-\n0304-8 \n\n39.  Kumar V, Dwivedi SK. Hexavalent chromium stress response, \nreduction capability and bioremediation potential of Trichoderma \nsp. isolated from electroplating wastewater. Ecotoxicol Environ \nSaf [Internet]. 2019 Dec [cited 2021 Sep 7];185:109734. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/ \nS0147651319310656 \n\n40.  Poljsak B, P\u00f3csi I, Raspor P, Pesti M. Interference of chromium \nwith biological systems in yeasts and fungi: a review: Effects of \nchromium on yeast and fungi. J Basic Microbiol [Internet]. 2010 \nFeb [cited 2020 Apr 10];50(1):21\u201336. Available from: \nhttp://doi.wiley.com/10.1002/jobm.200900170 \n\n41.  Vajpai S, Taylor PE, Adholeya A, Leigh Ackland M. Chromium \ntolerance and accumulation in Aspergillus flavus isolated from \ntannery effluent. J Basic Microbiol [Internet]. 2020 Jan [cited 2021 \nSep 7];60(1):58\u201371. Available from: \nhttps://onlinelibrary.wiley.com/doi/10.1002/jobm.201900389 \n\n42.  Feng M, Yin H, Peng H, Liu Z, Lu G, Dang Z. Hexavalent \nchromium induced oxidative stress and apoptosis in Pycnoporus \nsanguineus. Environ Pollut Barking Essex 1987. 2017 May \n18;228:128\u201339.  \n\n43.  Kharab P, Singh I. Genotoxic effects of potassium dichromate, \nsodium arsenite, cobalt chloride and lead nitrate in diploid yeast. \nMutat Res Toxicol [Internet]. 1985 Mar [cited 2020 Apr \n10];155(3):117\u201320. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/0165121885901284 \n\n44.  Shahid M, Shamshad S, Rafiq M, Khalid S, Bibi I, Niazi NK, et al.", "start_char_idx": 57735, "end_char_idx": 60499, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ebec6eb6-daf3-4e05-8495-59c72093c2d9": {"__data__": {"id_": "ebec6eb6-daf3-4e05-8495-59c72093c2d9", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3fe1befd-6ea0-4e3d-9da3-e6172fc488cc", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "6a93db03486bfbc3bb151f5366179cf3a377e9cacdf9bb12949f4218afb078ce", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "af706b74-fbfb-45a0-bb04-4d686c746687", "node_type": "1", "metadata": {}, "hash": "33d04cec59f16a7e6ea685fbcf66fd7a68cc7049660526151c8e392aa6d97724", "class_name": "RelatedNodeInfo"}}, "text": "Chromium speciation, bioavailability, uptake, toxicity and \ndetoxification in soil-plant system: A review. Chemosphere \n[Internet]. 2017 Jul [cited 2020 Apr 24];178:513\u201333. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004565351730437\nX \n\n45.  Henderson G. A comparison of the effects of chromate, molybdate \nand cadmium oxide on respiration in the yeastSaccharomyces \ncerevisiae. Biol Met [Internet]. 1989 [cited 2020 Apr 12];2(2):83\u20138. \nAvailable from: http://link.springer.com/10.1007/BF01129205 \n\n46.  Cozza D, Torelli A, Veltri A, Ferrari M, Marieschi M, Cozza R. \nUltrastructural features, chromium content and in situ \nimmunodetection of 5-methyl-cytosine following Cr (VI) \ntreatment in two strains of Scenedesmus acutus M. (Chlorophyceae) \nwith different chromium sensitivity. Eur J Phycol [Internet]. 2016 \nJul 2 [cited 2021 Sep 10];51(3):294\u2013306. Available from: \n\nhttps://www.tandfonline.com/doi/full/10.1080/09670262.2016.\n1157902 \n\n47.  H\u00f6rcsik ZT, Kov\u00e1cs L, L\u00e1posi R, M\u00e9sz\u00e1ros I, Lakatos G, Garab G. \nEffect of chromium on photosystem 2 in the unicellular green alga, \nChlorella pyrenoidosa. Photosynthetica [Internet]. 2007 Mar [cited \n2020 May 25];45(1):65\u20139. Available from: \nhttp://link.springer.com/10.1007/s11099-007-0010-8 \n\n48.  Jin M, Xiao X, Qin L, Geng W, Gao Y, Li L, et al. Physiological and \nmorphological responses and tolerance mechanisms of Isochrysis \ngalbana to Cr(VI) stress. Bioresour Technol [Internet]. 2020 Apr \n[cited 2021 Sep 10];302:122860. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096085242030129\n2 \n\n49.  Toranzo R, Ferraro G, Beligni MV, Perez GL, Castiglioni D, \nPasquevich D, et al. Natural and acquired mechanisms of tolerance \nto chromium in a Scenedesmus dimorphus strain. Algal Res \n[Internet]. 2020 Dec [cited 2021 Sep 10];52:102100. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S221192642030968\n1 \n\n50.  \u00dcnal D, I\u015fik NO, Sukatar A. Effects of Chromium VI stress on \ngreen alga Ulva lactuca (L.). 2008;6.  \n\n51.  Volland S, L\u00fctz C, Michalke B, L\u00fctz-Meindl U. Intracellular \nchromium localization and cell physiological response in the \nunicellular alga Micrasterias. Aquat Toxicol [Internet]. 2012 Mar \n[cited 2021 Sep 10];109:59\u201369. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S0166445X1100329\n8 \n\n52.  Saha B, Orvig C. Biosorbents for hexavalent chromium elimination \nfrom industrial and municipal effluents. Coord Chem Rev \n[Internet]. 2010 Dec [cited 2020 Apr 12];254(23\u201324):2959\u201372. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0010854510001591 \n\n53.  Altun T, Parlay\u0131c\u0131 \u015e, Pehlivan E. Hexavalent chromium removal \nusing agricultural waste \u201crye husk.\u201d Desalination Water Treat \n[Internet].", "start_char_idx": 60501, "end_char_idx": 63241, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "af706b74-fbfb-45a0-bb04-4d686c746687": {"__data__": {"id_": "af706b74-fbfb-45a0-bb04-4d686c746687", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ebec6eb6-daf3-4e05-8495-59c72093c2d9", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "65ec32f4e6f179bcc86cab60d224e98ac3c3fed0f9d0a73e4da60c8d309d8429", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b4d93010-3779-45f5-b2b0-9a3551817c87", "node_type": "1", "metadata": {}, "hash": "4a2094dc9960a78dfe6e48bc5f397a9ed8cbca47eafe751d772993d7435a6a57", "class_name": "RelatedNodeInfo"}}, "text": "2016 Aug 14 [cited 2020 Apr 13];57(38):17748\u201356. \nAvailable from: http://www.tandfonline.com/doi/full/10.1080/ \n19443994.2015.1085914 \n\n54.  Dong C, Ji J, Shen B, Xing M, Zhang J. Enhancement of H 2 O 2 \nDecomposition by the Co-catalytic Effect of WS 2 on the Fenton \nReaction for the Synchronous Reduction of Cr(VI) and \nRemediation of Phenol. Environ Sci Technol [Internet]. 2018 Oct 2 \n[cited 2021 Sep 10];52(19):11297\u2013308. Available from: \nhttps://pubs.acs.org/doi/10.1021/acs.est.8b02403 \n\n55.  Jacobs J, L. Hardison R, Rouse.V. J. In-situ remediation of heavy \nmetals using sulfur-based treatment technologies. 2001;4.  \n\n56.  Ma Y, Li F, Jiang Y, Yang W, Lv L, Xue H, et al. Remediation of \nCr(VI)-Contaminated Soil Using the Acidified Hydrazine \nHydrate. Bull Environ Contam Toxicol [Internet]. 2016 Sep [cited \n2021 Sep 10];97(3):392\u20134. Available from: \nhttp://link.springer.com/10.1007/s00128-016-1862-z \n\n57.  Kabay N, Arda M, Saha B, Streat M. Removal of Cr(VI) by solvent \nimpregnated resins (SIR) containing aliquat 336. React Funct \nPolym [Internet]. 2003 Jan [cited 2020 Apr 15];54(1\u20133):103\u201315. \nAvailable from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S138151480200186\n4 \n\n58.  Jin W, Du H, Zheng S, Zhang Y. Electrochemical processes for the \nenvironmental remediation of toxic Cr(VI): A review. \nElectrochimica Acta [Internet]. 2016 Feb [cited 2021 Sep \n11];191:1044\u201355. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S001346861630133\n5 \n\n59.  Li D, Ji G, Hu J, Hu S, Yuan X. Remediation strategy and \nelectrochemistry flushing & reduction technology for real Cr(VI)-\ncontaminated soils. Chem Eng J [Internet]. 2018 Feb [cited 2021 Sep \n11];334:1281\u20138. Available from: https://linkinghub.elsevier. \ncom/retrieve/pii/S1385894717319861 \n\n60.  Belver C, Bedia J, G\u00f3mez-Avil\u00e9s A, Pe\u00f1as-Garz\u00f3n M, Rodriguez JJ. \nSemiconductor Photocatalysis for Water Purification. In: \nNanoscale Materials in Water Purification [Internet]. Elsevier; 2019 \n[cited 2021 Aug 2]. p. 581\u2013651.", "start_char_idx": 63242, "end_char_idx": 65247, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b4d93010-3779-45f5-b2b0-9a3551817c87": {"__data__": {"id_": "b4d93010-3779-45f5-b2b0-9a3551817c87", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "af706b74-fbfb-45a0-bb04-4d686c746687", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "8f23921dd98de7130dbc4202c2d05ada16ce89c12b97c3a22b4210c62af7d9ea", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2433d9f4-037b-4ea6-9361-d606515c013c", "node_type": "1", "metadata": {}, "hash": "570b9a7d3585e8fd1f3d7b961003da91b02d77d7433bf4e5865693a49a787657", "class_name": "RelatedNodeInfo"}}, "text": "p. 581\u2013651. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/B978012813926400\n0288 \n\nhttps://academic.oup.com/jxb/article-lookup/doi/10.1093/jxb/37.2.178\nhttps://academic.oup.com/jxb/article-lookup/doi/10.1093/jxb/37.2.178\nhttp://link.springer.com/10.1007/978-981-13-1891-7_11\nhttp://link.springer.com/10.1007/s12223-014-0304-8\nhttp://link.springer.com/10.1007/s12223-014-0304-8\nhttp://doi.wiley.com/10.1002/jobm.200900170\nhttps://onlinelibrary.wiley.com/doi/10.1002/jobm.201900389\nhttps://linkinghub.elsevier.com/retrieve/pii/0165121885901284\nhttps://linkinghub.elsevier.com/retrieve/pii/S004565351730437X\nhttps://linkinghub.elsevier.com/retrieve/pii/S004565351730437X\nhttp://link.springer.com/10.1007/BF01129205\nhttps://www.tandfonline.com/doi/full/10.1080/09670262.2016.1157902\nhttps://www.tandfonline.com/doi/full/10.1080/09670262.2016.1157902\nhttp://link.springer.com/10.1007/s11099-007-0010-8\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852420301292\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852420301292\nhttps://linkinghub.elsevier.com/retrieve/pii/S2211926420309681\nhttps://linkinghub.elsevier.com/retrieve/pii/S2211926420309681\nhttps://linkinghub.elsevier.com/retrieve/pii/S0166445X11003298\nhttps://linkinghub.elsevier.com/retrieve/pii/S0166445X11003298\nhttps://pubs.acs.org/doi/10.1021/acs.est.8b02403\nhttps://pubs.acs.org/doi/10.1021/acs.est.8b02403\nhttp://link.springer.com/10.1007/s00128-016-1862-z\nhttps://linkinghub.elsevier.com/retrieve/pii/S1381514802001864\nhttps://linkinghub.elsevier.com/retrieve/pii/S1381514802001864\nhttps://linkinghub.elsevier.com/retrieve/pii/S0013468616301335\nhttps://linkinghub.elsevier.com/retrieve/pii/S0013468616301335\nhttps://linkinghub.elsevier.com/retrieve/pii/B9780128139264000288\nhttps://linkinghub.elsevier.com/retrieve/pii/B9780128139264000288\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  70 \n\n61.  Izzudin NM, Jalil AA, Aziz FFA, Azami MS, Ali MW, Hassan NS, \net al. Simultaneous remediation of hexavalent chromium and \norganic pollutants in wastewater using period 4 transition metal \noxide-based photocatalysts: a review. Environ Chem Lett \n[Internet]. 2021 Jul 21 [cited 2021 Sep 11]; Available from: \nhttps://link.springer.com/10.1007/s10311-021-01272-1 \n\n62.  Idris A, Hassan N, Mohd Ismail NS, Misran E, Yusof NM, \nNgomsik A-F, et al. Photocatalytic magnetic separable beads for \nchromium (VI) reduction. Water Res [Internet]. 2010 Mar [cited \n2020 Apr 15];44(6):1683\u20138.", "start_char_idx": 65236, "end_char_idx": 67734, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2433d9f4-037b-4ea6-9361-d606515c013c": {"__data__": {"id_": "2433d9f4-037b-4ea6-9361-d606515c013c", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b4d93010-3779-45f5-b2b0-9a3551817c87", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "ce5c2fb408ceba2afeefdbad72cadf69f719325c3954de5f890235602b6ac16b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5f8de965-b639-43cb-88e8-b54140706eb3", "node_type": "1", "metadata": {}, "hash": "182eca5fd1f0c669f8b988a0d2ef9478f730cdd76487ad18f920e286e17a4f99", "class_name": "RelatedNodeInfo"}}, "text": "Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004313540900768\n4 \n\n63.  Xia S, Song Z, Jeyakumar P, Shaheen SM, Rinklebe J, Ok YS, et al. \nA critical review on bioremediation technologies for Cr(VI)-\ncontaminated soils and wastewater. Crit Rev Environ Sci Technol \n[Internet]. 2019 Jun 18 [cited 2021 Jul 12];49(12):1027\u201378. Available \nfrom: \nhttps://www.tandfonline.com/doi/full/10.1080/10643389.2018.\n1564526 \n\n64.  Zhang D, Xu Y, Li X, Wang L, He X, Ma Y, et al. The \nImmobilization Effect of Natural Mineral Materials on Cr(VI) \nRemediation in Water and Soil. Int J Environ Res Public Health \n[Internet]. 2020 Apr 20 [cited 2021 Sep 11];17(8):2832. Available \nfrom: https://www.mdpi.com/1660-4601/17/8/2832 \n\n65.  Dehghani MH, Sanaei D, Ali I, Bhatnagar A. Removal of \nchromium(VI) from aqueous solution using treated waste \nnewspaper as a low-cost adsorbent: Kinetic modeling and \nisotherm studies. J Mol Liq [Internet]. 2016 Mar [cited 2020 Apr \n12];215:671\u20139. Available from: https://linkinghub.elsevier. \ncom/retrieve/pii/S0167732215313039 \n\n66.  Kumar S, Meikap BC. Removal of Chromium(VI) from waste \nwater by using adsorbent prepared from green coconut shell. \nDesalination Water Treat [Internet]. 2014 May 12 [cited 2020 Apr \n9];52(16\u201318):3122\u201332. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.8\n01796 \n\n67.  Owlad M, Aroua MK, Daud WAW, Baroutian S. Removal of \nHexavalent Chromium-Contaminated Water and Wastewater: A \nReview. Water Air Soil Pollut [Internet]. 2009 Jun [cited 2020 Apr \n9];200(1\u20134):59\u201377. Available from: http://link.springer.com/ \n10.1007/s11270-008-9893-7 \n\n68.  Ramirez Losada VA, Bonilla EP, Carvajal Pinilla LA, Serrezuela \nRR. Removal of chromium in wastewater from tanneries applying \nbioremediation with algae, orange peels and citrus pectin. \nContemp Eng Sci [Internet]. 2018 [cited 2020 Apr 9];11(9):433\u201349. \nAvailable from: http://www.m-hikari.com/ces/ces2018/ces9-12-\n2018/8235.html \n\n69.  Zakaria ZA, Zakaria Z, Surif S, Ahmad WA. Biological \ndetoxification of Cr(VI) using wood-husk immobilized \nAcinetobacter haemolyticus. J Hazard Mater [Internet]. 2007 Sep \n[cited 2020 Jun 4];148(1\u20132):164\u201371. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940700243\n9 \n\n70.  Choudhury P, Mondal P, Majumdar S, Saha S, Sahoo GC. \nPreparation of ceramic ultrafiltration membrane using green \nsynthesized CuO nanoparticles for chromium (VI) removal and \noptimization by response surface methodology. J Clean Prod \n[Internet]. 2018 Dec [cited 2021 Sep 11];203:511\u201320. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S095965261832650\n7 \n\n71.  Kumar A, Thakur A, Panesar PS.", "start_char_idx": 67735, "end_char_idx": 70416, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5f8de965-b639-43cb-88e8-b54140706eb3": {"__data__": {"id_": "5f8de965-b639-43cb-88e8-b54140706eb3", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2433d9f4-037b-4ea6-9361-d606515c013c", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "e0ccf876b5280b7a5fb7e0b13b69b9e61b21060f93286e3ea920edf3f0613fba", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f718e374-f8a5-4669-beb5-8744359e107e", "node_type": "1", "metadata": {}, "hash": "c84b6e33cdb2f10113ef2bff77ada0a73f14772946f8173aa4c83445c5d6d015", "class_name": "RelatedNodeInfo"}}, "text": "Kumar A, Thakur A, Panesar PS. Extraction of hexavalent \nchromium by environmentally benign green emulsion liquid \nmembrane using tridodecyamine as an extractant. J Ind Eng Chem \n[Internet]. 2019 Feb [cited 2021 Sep 11];70:394\u2013401. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S1226086X1830821\n9 \n\n72.  Sharma SK, Petrusevski B, Amy G. Chromium removal from \nwater: a review. J Water Supply Res Technol-Aqua [Internet]. 2008 \nDec [cited 2020 Apr 15];57(8):541\u201353. Available from: \nhttps://iwaponline.com/aqua/article/57/8/541/31147/Chromi\num-removal-from-water-a-review \n\n73.  Hafiane A, Lemordant D, Dhahbi M. Removal of hexavalent \nchromium by nanofiltration. Desalination [Internet]. 2000 Nov \n\n[cited 2020 Apr 16];130(3):305\u201312. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S001191640000094\n1 \n\n74.  Peng H, Guo J. Removal of chromium from wastewater by \nmembrane filtration, chemical precipitation, ion exchange, \nadsorption electrocoagulation, electrochemical reduction, \nelectrodialysis, electrodeionization, photocatalysis and \nnanotechnology: a review. Environ Chem Lett [Internet]. 2020 Nov \n[cited 2021 Sep 11];18(6):2055\u201368. Available from: \nhttps://link.springer.com/10.1007/s10311-020-01058-x \n\n75.  Sapari N, Idris A, Hamid NHAb. Total removal of heavy metal \nfrom mixed plating rinse wastewater. Desalination [Internet]. 1996 \nAug [cited 2020 Apr 15];106(1\u20133):419\u201322. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S001191649600139\n7 \n\n76.  Dangi AK, Sharma B, Hill RT, Shukla P. Bioremediation through \nmicrobes: systems biology and metabolic engineering approach. \nCrit Rev Biotechnol [Internet]. 2019 Jan 2 [cited 2021 Sep \n26];39(1):79\u201398. Available from: \nhttps://www.tandfonline.com/doi/full/10.1080/07388551.2018.\n1500997 \n\n77.  Verma S, Kuila A. Bioremediation of heavy metals by microbial \nprocess. Environ Technol Innov [Internet]. 2019 May [cited 2021 \nSep 26];14:100369. Available from: https://linkinghub \n.elsevier.com/retrieve/pii/S2352186418305911 \n\n78.  Rabani MS, Sharma R, Singh R, Gupta MK. Characterization and \nIdentification of Naphthalene Degrading Bacteria Isolated from \nPetroleum Contaminated Sites and Their Possible Use in \nBioremediation. Polycycl Aromat Compd [Internet]. 2020 May 6 \n[cited 2021 Sep 26];1\u201312. Available from: \nhttps://www.tandfonline.com/doi/full/10.1080/10406638.2020.\n1759663 \n\n79.  Onta\u00f1on OM, Fernandez M, Agostini E, Gonz\u00e1lez PS. \nIdentification of the main mechanisms involved in the tolerance \nand bioremediation of Cr(VI) by Bacillus sp. SFC 500-1E. Environ \nSci Pollut Res [Internet]. 2018 Jun [cited 2021 Jun 5];25(16):16111\u2013\n20. Available from: http://link.springer.com/10.1007/s11356-\n018-1764-1 \n\n80.  Diaconu M, Pavel LV, Hlihor R-M, Rosca M, Fertu DI, Lenz M, et \nal.", "start_char_idx": 70386, "end_char_idx": 73181, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f718e374-f8a5-4669-beb5-8744359e107e": {"__data__": {"id_": "f718e374-f8a5-4669-beb5-8744359e107e", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5f8de965-b639-43cb-88e8-b54140706eb3", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "1634b1d7231ced280486b685d278665d43f0684d56bcaae319bf0358064c665c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "89e7a201-87b7-4864-861c-84d78dc5064f", "node_type": "1", "metadata": {}, "hash": "ecec74ee0570949c05e79db33c0b541e451f0e5c3e7ce336f8f9300d07b009b6", "class_name": "RelatedNodeInfo"}}, "text": "Characterization of heavy metal toxicity in some plants and \nmicroorganisms\u2014A preliminary approach for environmental \nbioremediation. New Biotechnol [Internet]. 2020 May [cited 2021 \nSep 26];56:130\u20139. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S187167841930060\n3 \n\n81.  Chen H, Wang Q. Microalgae-based nitrogen bioremediation. \nAlgal Res [Internet]. 2020 Mar [cited 2021 Sep 26];46:101775. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS2211926419311233 \n\n82.  Boopathy R. Factors limiting bioremediation technologies. \nBioresour Technol [Internet]. 2000 Aug [cited 2020 Apr \n19];74(1):63\u20137. Available from: https://linkinghub. \nelsevier.com/retrieve/pii/S0960852499001443 \n\n83.  Brar SK, Verma M, Surampalli RY, Misra K, Tyagi RD, Meunier N, \net al. Bioremediation of Hazardous Wastes\u2014A Review. Pract \nPeriod Hazard Toxic Radioact Waste Manag [Internet]. 2006 Apr \n[cited 2020 May 6];10(2):59\u201372. Available from: \nhttp://ascelibrary.org/doi/10.1061/%28ASCE%291090-\n025X%282006%2910%3A2%2859%29 \n\n84.  Salunkhe PB, Dhakephalkar PK, Paknikar KM. Bioremediation of \nhexavalent chromium in soil microcosms. Biotechnol Lett \n[Internet]. 1998 [cited 2020 May 5];20(8):749\u201351. Available from: \nhttp://link.springer.com/10.1023/A:1005338820430 \n\n85.  Molokwane PE, Nkhalambayausi-Chirwa EM. Microbial culture \ndynamics and chromium (VI) removal in packed-column \nmicrocosm reactors. Water Sci Technol [Internet]. 2009 Jul 1 [cited \n2020 May 5];60(2):381\u20138. Available from: \nhttps://iwaponline.com/wst/article/60/2/381/17771/Microbia\nl-culture-dynamics-and-chromium-VI-removal \n\n86.  Pavithra KG, Kumar PS, Jaikumar V, Vardhan KH, SundarRajan \nP. Microalgae for biofuel production and removal of heavy metals: \na review. Environ Chem Lett [Internet].", "start_char_idx": 73182, "end_char_idx": 74962, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "89e7a201-87b7-4864-861c-84d78dc5064f": {"__data__": {"id_": "89e7a201-87b7-4864-861c-84d78dc5064f", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f718e374-f8a5-4669-beb5-8744359e107e", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "ac2298e0b59c815dab186af5e92c98c1a7bd1ea25e5adc00bc015d88ec21c0be", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "69c32e3b-4d52-489b-9b65-490812b5e0a4", "node_type": "1", "metadata": {}, "hash": "e700bfb7f966d1b33f44b64c410eb35745fff05f448259d3983a2b28519f0d7b", "class_name": "RelatedNodeInfo"}}, "text": "Environ Chem Lett [Internet]. 2020 Nov [cited 2021 Sep \n\nhttps://link.springer.com/10.1007/s10311-021-01272-1\nhttps://linkinghub.elsevier.com/retrieve/pii/S0043135409007684\nhttps://linkinghub.elsevier.com/retrieve/pii/S0043135409007684\nhttps://www.tandfonline.com/doi/full/10.1080/10643389.2018.1564526\nhttps://www.tandfonline.com/doi/full/10.1080/10643389.2018.1564526\nhttps://www.mdpi.com/1660-4601/17/8/2832\nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.801796\nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.801796\nhttp://www.m-hikari.com/ces/ces2018/ces9-12-2018/8235.html\nhttp://www.m-hikari.com/ces/ces2018/ces9-12-2018/8235.html\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407002439\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407002439\nhttps://linkinghub.elsevier.com/retrieve/pii/S0959652618326507\nhttps://linkinghub.elsevier.com/retrieve/pii/S0959652618326507\nhttps://linkinghub.elsevier.com/retrieve/pii/S1226086X18308219\nhttps://linkinghub.elsevier.com/retrieve/pii/S1226086X18308219\nhttps://iwaponline.com/aqua/article/57/8/541/31147/Chromium-removal-from-water-a-review\nhttps://iwaponline.com/aqua/article/57/8/541/31147/Chromium-removal-from-water-a-review\nhttps://linkinghub.elsevier.com/retrieve/pii/S0011916400000941\nhttps://linkinghub.elsevier.com/retrieve/pii/S0011916400000941\nhttps://link.springer.com/10.1007/s10311-020-01058-x\nhttps://linkinghub.elsevier.com/retrieve/pii/S0011916496001397\nhttps://linkinghub.elsevier.com/retrieve/pii/S0011916496001397\nhttps://www.tandfonline.com/doi/full/10.1080/07388551.2018.1500997\nhttps://www.tandfonline.com/doi/full/10.1080/07388551.2018.1500997\nhttps://www.tandfonline.com/doi/full/10.1080/10406638.2020.1759663\nhttps://www.tandfonline.com/doi/full/10.1080/10406638.2020.1759663\nhttp://link.springer.com/10.1007/s11356-018-1764-1\nhttp://link.springer.com/10.1007/s11356-018-1764-1\nhttps://linkinghub.elsevier.com/retrieve/pii/S1871678419300603\nhttps://linkinghub.elsevier.com/retrieve/pii/S1871678419300603\nhttp://ascelibrary.org/doi/10.1061/%28ASCE%291090-025X%282006%2910%3A2%2859%29\nhttp://ascelibrary.org/doi/10.1061/%28ASCE%291090-025X%282006%2910%3A2%2859%29\nhttp://link.springer.com/10.1023/A:1005338820430\nhttps://iwaponline.com/wst/article/60/2/381/17771/Microbial-culture-dynamics-and-chromium-VI-removal\nhttps://iwaponline.com/wst/article/60/2/381/17771/Microbial-culture-dynamics-and-chromium-VI-removal\n\n\nNepal J Biotechnol.", "start_char_idx": 74933, "end_char_idx": 77375, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "69c32e3b-4d52-489b-9b65-490812b5e0a4": {"__data__": {"id_": "69c32e3b-4d52-489b-9b65-490812b5e0a4", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "89e7a201-87b7-4864-861c-84d78dc5064f", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "6d09f9c91c76fa2539f529778f36b775f8a87053126dc5a4865979f6eb9722b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8df451a6-24d8-4bcb-8c54-16c788669e4a", "node_type": "1", "metadata": {}, "hash": "02df478e3b5374027a87781583d299e6a6605f1500a6bd262213e64eb5a6d966", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  71 \n\n11];18(6):1905\u201323. Available from: \nhttps://link.springer.com/10.1007/s10311-020-01046-1 \n\n87.  Rahman Z, Singh VP. Bioremediation of toxic heavy metals \n(THMs) contaminated sites: concepts, applications and challenges. \nEnviron Sci Pollut Res [Internet]. 2020 Aug [cited 2021 Sep \n11];27(22):27563\u201381. Available from: \nhttps://link.springer.com/10.1007/s11356-020-08903-0 \n\n88.  Thatoi H, Das S, Mishra J, Rath BP, Das N. Bacterial chromate \nreductase, a potential enzyme for bioremediation of hexavalent \nchromium: A review. J Environ Manage [Internet]. 2014 Dec [cited \n2020 Apr 9];146:383\u201399. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030147971400354\n5 \n\n89.  Mistry K, Desai C, Lal S, Patel K, Patel B. Hexavalent Chromium \nReduction by Staphylococcus Sp. Isolated From Cr (Vi) \nContaminated Land Fill. 2010;17.  \n\n90.  Ohtake H, Cervantes C, Silver S. Decreased chromate uptake in \nPseudomonas fluorescens carrying a chromate resistance plasmid. \nJ Bacteriol [Internet]. 1987 [cited 2020 Apr 28];169(8):3853\u20136. \nAvailable from: https://JB.asm.org/content/169/8/3853 \n\n91.  Suresh G, Balasubramanian B, Ravichandran N, Ramesh B, \nKamyab H, Velmurugan P, et al. Bioremediation of hexavalent \nchromium-contaminated wastewater by Bacillus thuringiensis \nand Staphylococcus capitis isolated from tannery sediment. \nBiomass Convers Biorefinery [Internet]. 2021 Apr [cited 2021 Sep \n12];11(2):383\u201391. Available from: \nhttp://link.springer.com/10.1007/s13399-020-01259-y \n\n92.  Vatsouria A, Vainshtein M, Kuschk P, Wiessner A, D K, Kaestner \nM. Anaerobic co-reduction of chromate and nitrate by bacterial \ncultures of Staphylococcus epidermidis L-02. J Ind Microbiol \nBiotechnol [Internet]. 2005 Sep [cited 2021 Jun 9];32(9):409\u201314. \nAvailable from: \nhttps://academic.oup.com/jimb/article/32/9/409-414/5992807 \n\n93.  Bopp LH, Ehrlich HL. Chromate resistance and reduction in \nPseudomonas fluorescens strain LB300. Arch Microbiol [Internet]. \n1988 Sep [cited 2020 Apr 9];150(5):426\u201331. Available from: \nhttp://link.springer.com/10.1007/BF00422281 \n\n94.  Campos J, Martinez-Pacheco M, Cervantes C. Hexavalent-\nchromium reduction by a chromate-resistantBacillus sp. strain. \nAntonie Van Leeuwenhoek [Internet]. 1995 Oct [cited 2020 Apr \n9];68(3):203\u20138. Available from: \nhttp://link.springer.com/10.1007/BF00871816 \n\n95.  Opperman DJ, van Heerden E. Aerobic Cr(VI) reduction by \nThermus scotoductus strain SA-01: Cr(VI) reduction by Thermus \nscotoductus. J Appl Microbiol [Internet].", "start_char_idx": 77356, "end_char_idx": 79944, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8df451a6-24d8-4bcb-8c54-16c788669e4a": {"__data__": {"id_": "8df451a6-24d8-4bcb-8c54-16c788669e4a", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "69c32e3b-4d52-489b-9b65-490812b5e0a4", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "c70a35e75d8fa4c5f2f4e08be9b2883d929234fbbe701d78b48661ddcb9b3682", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b02a4016-0657-468d-8686-0b145e6a5d93", "node_type": "1", "metadata": {}, "hash": "f69671ef1ccaf0e542d1450ee72fbe88f4fc4a018880ea649b9ff52e68ad46c2", "class_name": "RelatedNodeInfo"}}, "text": "J Appl Microbiol [Internet]. 2007 Nov [cited 2020 Apr \n21];103(5):1907\u201313. Available from: \nhttp://doi.wiley.com/10.1111/j.1365-2672.2007.03429.x \n\n96.  Wang Y. Factors affecting hexavalent chromium reduction in pure \ncultures of bacteria. Water Res [Internet]. 1995 Nov [cited 2020 Apr \n28];29(11):2467\u201374. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/004313549500093Z \n\n97.  Narayani M, Shetty KV. Chromium-Resistant Bacteria and Their \nEnvironmental Condition for Hexavalent Chromium Removal: A \nReview. Crit Rev Environ Sci Technol [Internet]. 2013 Jan [cited \n2020 Apr 17];43(9):955\u20131009. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/10643389.2011.6\n27022 \n\n98.  Tamind\u017eija D, Chromikova Z, Spai\u0107 A, Barak I, Bernier-Latmani R, \nRadnovi\u0107 D. Chromate tolerance and removal of bacterial strains \nisolated from uncontaminated and chromium-polluted \nenvironments. World J Microbiol Biotechnol [Internet]. 2019 Apr \n[cited 2021 Sep 12];35(4):56. Available from: \nhttp://link.springer.com/10.1007/s11274-019-2638-5 \n\n99.  Srivastava S, Thakur IS. Isolation and process parameter \noptimization of Aspergillus sp. for removal of chromium from \ntannery effluent. Bioresour Technol [Internet]. 2006 Jul [cited 2020 \nApr 22];97(10):1167\u201373. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096085240500272\n5 \n\n100.  Martorell MM, Fern\u00e1ndez PM, Fari\u00f1a JI, Figueroa LIC. Cr(VI) \nreduction by cell-free extracts of Pichia jadinii and Pichia anomala \nisolated from textile-dye factory effluents. Int Biodeterior \nBiodegrad [Internet]. 2012 Jul [cited 2020 Apr 16];71:80\u20135. \n\nAvailable from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051200093\n5 \n\n101.  Morales-Barrera L, Guill\u00e9n-Jim\u00e9nez F de M, Ortiz-Moreno A, \nVillegas-Garrido TL, Sandoval-Cabrera A, Hern\u00e1ndez-Rodr\u00edguez \nCH, et al. Isolation, identification and characterization of a \nHypocrea tawa strain with high Cr(VI) reduction potential. \nBiochem Eng J [Internet]. 2008 Jun [cited 2020 Apr 16];40(2):284\u2013\n92. Available from: https://linkinghub.elsevier \n.com/retrieve/pii/S1369703X0800003X \n\n102.  Morales-Barrera L, Cristiani-Urbina E. Hexavalent Chromium \nRemoval by a Trichoderma inhamatum Fungal Strain Isolated \nfrom Tannery Effluent. Water Air Soil Pollut [Internet]. 2007 Nov \n29 [cited 2020 Apr 16];187(1\u20134):327\u201336. Available from: \nhttp://link.springer.com/10.1007/s11270-007-9520-z \n\n103.  Vankar PS, Bajpai D. Phyto-remediation of chrome-VI of tannery \neffluent by Trichoderma species. Desalination [Internet]. 2008 Mar \n[cited 2020 Apr 16];222(1\u20133):255\u201362. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S001191640700776\nX \n\n104.  Mahmoud MS, Mohamed SA.", "start_char_idx": 79916, "end_char_idx": 82604, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b02a4016-0657-468d-8686-0b145e6a5d93": {"__data__": {"id_": "b02a4016-0657-468d-8686-0b145e6a5d93", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8df451a6-24d8-4bcb-8c54-16c788669e4a", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "9dd15d478d385852e9b0ba59f9c7f1770dc708fd84fe49e9b30b293dba3a4deb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "116a3d4a-fa98-4008-8949-59b4835931a5", "node_type": "1", "metadata": {}, "hash": "f51373a9f1f9f365ce8cbc7f3a2208d169445a82a23463778ecc1ce2981d6d58", "class_name": "RelatedNodeInfo"}}, "text": "Mahmoud MS, Mohamed SA. Calcium alginate as an eco-friendly \nsupporting material for Baker\u2019s yeast strain in chromium \nbioremediation. HBRC J [Internet]. 2017 Dec [cited 2020 Apr 17]; \n13(3):245\u201354. Available from: \nhttps://www.tandfonline.com/doi/full/10.1016/j.hbrcj.2015.06.\n003 \n\n105.  Campa\u00f1a-P\u00e9rez JF, Portero Barahona P, Mart\u00edn-Ramos P, Carvajal \nBarriga EJ. Ecuadorian yeast species as microbial particles for \nCr(VI) biosorption. Environ Sci Pollut Res [Internet]. 2019 Sep \n[cited 2021 Sep 16];26(27):28162\u201372. Available from: \nhttp://link.springer.com/10.1007/s11356-019-06035-8 \n\n106.  Adam S, P SK, P S, S DK, P P. Bioremediation of Tannery \nWastewater Using Immobilized Marine Microalga Tetraselmis sp.: \nExperimental Studies and Pseudo-Second Order Kinetics. J Mar \nBiol Oceanogr [Internet]. 2015 [cited 2020 Apr 9];04(01). Available \nfrom: https://www.scitechnol.com/peer-\nreview/bioremediation-of-tannery-wastewater-using-\nimmobilized-marine-microalga-tetraselmis-sp-experimental-\nstudies-and-pseudosecond-order-Uoxt.php?article_id=3505 \n\n107.  Gupta VK, Shrivastava AK, Jain N. Biosorption of Chromium(VI) \nFrom Aqueous solutions by green algae spirogyra species. Water \nRes [Internet]. 2001 Dec [cited 2020 Apr 17];35(17):4079\u201385. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0043135401001385 \n\n108.  Lee D-C, Park C-J, Yang J-E, Jeong Y-H, Rhee H-I. Screening of \nhexavalent chromium biosorbent from marine algae. Appl \nMicrobiol Biotechnol [Internet]. 2000 Sep 15 [cited 2020 Apr \n17];54(3):445\u20138. Available from: http://link.springer.com \n/10.1007/s002530000387 \n\n109.  Yewalkar SN, Dhumal KondiramN, Sainis JK. Chromium(VI)-\nreducing Chlorella spp. isolated from disposal sites of paper-pulp \nand electroplating industry. J Appl Phycol [Internet]. 2007 Aug 28 \n[cited 2020 May 28];19(5):459\u201365. Available from: \nhttp://link.springer.com/10.1007/s10811-007-9153-z \n\n110.  El-Sikaily A, Nemr AE, Khaled A, Abdelwehab O. Removal of \ntoxic chromium from wastewater using green alga Ulva lactuca \nand its activated carbon. J Hazard Mater [Internet]. 2007 Sep [cited \n2020 Apr 17];148(1\u20132):216\u201328. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940700250\n6 \n\n111.  Aksu Z, A\u00e7ikel \u00dc, Kutsal T. Investigation of Simultaneous \nBiosorption of Copper(II) and Chromium(VI) on Dried Chlorella \nVulgaris from Binary Metal Mixtures: Application of \nMulticomponent Adsorption Isotherms. Sep Sci Technol \n[Internet]. 1999 Feb 22 [cited 2020 May 28];34(3):501\u201324. Available \nfrom: http://www.tandfonline.com/doi/abs/10.1081/SS-\n100100663 \n\n112.", "start_char_idx": 82581, "end_char_idx": 85172, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "116a3d4a-fa98-4008-8949-59b4835931a5": {"__data__": {"id_": "116a3d4a-fa98-4008-8949-59b4835931a5", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b02a4016-0657-468d-8686-0b145e6a5d93", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "3c05c23f3c68619d37f4545c8a8c4c9c981b3c27df3c38f13e9597b9c6873b4b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "739cb038-98c2-4f0b-bcd4-f8cc68258aa1", "node_type": "1", "metadata": {}, "hash": "44c58d88f8ff39149948c43725fff7e29a672dece022230acee24987c5b61325", "class_name": "RelatedNodeInfo"}}, "text": "Athira K, Sathish A, Nithya K, Guhananthan A. Corn cob \nimmobilised Chlorella sorokiniana for the sequestration of \nchromium ions from aqueous solution. Mater Today Proc \n[Internet]. 2020 Apr [cited 2020 May 28];S2214785320319313. \nAvailable from: https://linkinghub.elsevier.com \n/retrieve/pii/S2214785320319313 \n\nhttps://link.springer.com/10.1007/s10311-020-01046-1\nhttps://link.springer.com/10.1007/s11356-020-08903-0\nhttps://linkinghub.elsevier.com/retrieve/pii/S0301479714003545\nhttps://linkinghub.elsevier.com/retrieve/pii/S0301479714003545\nhttps://jb.asm.org/content/169/8/3853\nhttp://link.springer.com/10.1007/s13399-020-01259-y\nhttps://academic.oup.com/jimb/article/32/9/409-414/5992807\nhttps://academic.oup.com/jimb/article/32/9/409-414/5992807\nhttp://link.springer.com/10.1007/BF00422281\nhttp://link.springer.com/10.1007/BF00422281\nhttp://doi.wiley.com/10.1111/j.1365-2672.2007.03429.x\nhttp://doi.wiley.com/10.1111/j.1365-2672.2007.03429.x\nhttps://linkinghub.elsevier.com/retrieve/pii/004313549500093Z\nhttp://www.tandfonline.com/doi/abs/10.1080/10643389.2011.627022\nhttp://www.tandfonline.com/doi/abs/10.1080/10643389.2011.627022\nhttp://link.springer.com/10.1007/s11274-019-2638-5\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852405002725\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852405002725\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830512000935\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830512000935\nhttp://link.springer.com/10.1007/s11270-007-9520-z\nhttps://linkinghub.elsevier.com/retrieve/pii/S001191640700776X\nhttps://linkinghub.elsevier.com/retrieve/pii/S001191640700776X\nhttps://linkinghub.elsevier.com/retrieve/pii/S001191640700776X\nhttps://www.tandfonline.com/doi/full/10.1016/j.hbrcj.2015.06.003\nhttps://www.tandfonline.com/doi/full/10.1016/j.hbrcj.2015.06.003\nhttp://link.springer.com/10.1007/s11356-019-06035-8\nhttps://www.scitechnol.com/peer-review/bioremediation-of-tannery-wastewater-using-immobilized-marine-microalga-tetraselmis-sp-experimental-studies-and-pseudosecond-order-Uoxt.php?article_id=3505\nhttps://www.scitechnol.com/peer-review/bioremediation-of-tannery-wastewater-using-immobilized-marine-microalga-tetraselmis-sp-experimental-studies-and-pseudosecond-order-Uoxt.php?article_id=3505\nhttps://www.scitechnol.com/peer-review/bioremediation-of-tannery-wastewater-using-immobilized-marine-microalga-tetraselmis-sp-experimental-studies-and-pseudosecond-order-Uoxt.php?article_id=3505\nhttps://www.scitechnol.", "start_char_idx": 85174, "end_char_idx": 87648, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "739cb038-98c2-4f0b-bcd4-f8cc68258aa1": {"__data__": {"id_": "739cb038-98c2-4f0b-bcd4-f8cc68258aa1", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "116a3d4a-fa98-4008-8949-59b4835931a5", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "4330fec7388bdacb4c0c98310fe5bbf6dc366f0bc66523b9f7a7f505980b49e5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "72a3d983-09e3-4d65-8095-31bbe2cae96f", "node_type": "1", "metadata": {}, "hash": "ab59a0e98e1566d08ee9fa624071ea6c94dcdd1477ceeb723de6b642855a465d", "class_name": "RelatedNodeInfo"}}, "text": "php?article_id=3505\nhttps://www.scitechnol.com/peer-review/bioremediation-of-tannery-wastewater-using-immobilized-marine-microalga-tetraselmis-sp-experimental-studies-and-pseudosecond-order-Uoxt.php?article_id=3505\nhttp://link.springer.com/10.1007/s10811-007-9153-z\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407002506\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407002506\nhttp://www.tandfonline.com/doi/abs/10.1081/SS-100100663\nhttp://www.tandfonline.com/doi/abs/10.1081/SS-100100663\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  72 \n\n113.  Deng L, Wang H, Deng N. Photoreduction of chromium(VI) in the \npresence of algae, Chlorella vulgaris. J Hazard Mater [Internet]. \n2006 Nov 16 [cited 2020 Apr 15];138(2):288\u201392. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940600629\n7 \n\n114.  Han X, Wong YS, Wong MH, Tam NFY. Biosorption and \nbioreduction of Cr(VI) by a microalgal isolate, Chlorella miniata. J \nHazard Mater [Internet]. 2007 Jul [cited 2020 Apr 9];146(1\u20132):65\u2013\n72. Available from: https://linkinghub.elsevier.com/retrieve \n/pii/S030438940601418X \n\n115.  Costa IGF, Terra NM, Cardoso VL, Batista FRX, Reis MHM. \nPhotoreduction of chromium(VI) in microstructured ceramic \nhollow fibers impregnated with titanium dioxide and coated with \ngreen algae Chlorella vulgaris. J Hazard Mater [Internet]. 2019 \nNov [cited 2021 Sep 16];379:120837. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438941930790\n3 \n\n116.  Dittert IM, de Lima Brand\u00e3o H, Pina F, da Silva EAB, de Souza \nSMAGU, de Souza AAU, et al. Integrated reduction/oxidation \nreactions and sorption processes for Cr(VI) removal from aqueous \nsolutions using Laminaria digitata macro-algae. Chem Eng J \n[Internet]. 2014 Feb [cited 2021 Aug 3];237:443\u201354. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S138589471301369\n7 \n\n117.  Adki VS, Jadhav JP, Bapat VA. Nopalea cochenillifera, a potential \nchromium (VI) hyperaccumulator plant. Environ Sci Pollut Res \n[Internet]. 2013 Feb [cited 2020 Apr 24];20(2):1173\u201380. Available \nfrom: http://link.springer.com/10.1007/s11356-012-1125-4 \n\n118.  Ranieri E, Gikas P. Effects of Plants for Reduction and Removal of \nHexavalent Chromium from a Contaminated Soil. Water Air Soil \nPollut [Internet]. 2014 Jun [cited 2020 Apr 24];225(6):1981. \nAvailable from: http://link.springer.com/10.1007/s11270-014-\n1981-2 \n\n119.  Shanker A, Djanaguiraman M, Sudhagar R, Chandrashekar C, \nPathmanabhan G. Differential antioxidative response of ascorbate \nglutathione pathway enzymes and metabolites to chromium \nspeciation stress in green gram ( (L.) R.Wilczek. cv CO 4) roots.", "start_char_idx": 87605, "end_char_idx": 90289, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "72a3d983-09e3-4d65-8095-31bbe2cae96f": {"__data__": {"id_": "72a3d983-09e3-4d65-8095-31bbe2cae96f", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "739cb038-98c2-4f0b-bcd4-f8cc68258aa1", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "87e12567acacde9e22cd2c1414be3ec95f4678f00bc67445dc97c5cad2b888bc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2c5b4798-2a5c-46f5-89a6-0a513fd1b5e7", "node_type": "1", "metadata": {}, "hash": "1b95288f8d13c7766a3f587af28ca4d828f938468e3c64bfda25216bc6893d10", "class_name": "RelatedNodeInfo"}}, "text": "R.Wilczek. cv CO 4) roots. \nPlant Sci [Internet]. 2004 Apr [cited 2020 Apr 29];166(4):1035\u201343. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0168945203005247 \n\n120.  Ma H-L, Zhang Y, Hu Q-H, Yan D, Yu Z-Z, Zhai M. Chemical \nreduction and removal of Cr(vi) from acidic aqueous solution by \nethylenediamine-reduced graphene oxide. J Mater Chem \n[Internet]. 2012 [cited 2020 Apr 15];22(13):5914. Available from: \nhttp://xlink.rsc.org/?DOI=c2jm00145d \n\n121.  Graham MC, Farmer JG, Anderson P, Paterson E, Hillier S, \nLumsdon DG, et al. Calcium polysulfide remediation of \nhexavalent chromium contamination from chromite ore \nprocessing residue. Sci Total Environ [Internet]. 2006 Jul [cited \n2020 May 21];364(1\u20133):32\u201344. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004896970500838\n7 \n\n122.  Park D, Yun Y-S, Park JM. Studies on hexavalent chromium \nbiosorption by chemically-treated biomass of Ecklonia sp. \nChemosphere [Internet]. 2005 Sep [cited 2020 May 21];60(10):1356\u2013\n64. Available from: https://linkinghub.elsevier.com \n/retrieve/pii/S0045653505002766 \n\n123.  Barrera\u2010D\u00edaz C, Palomar\u2010Pardav\u00e9 M, Romero\u2010Romo M, Mart\u00ednez \nS. Chemical and electrochemical considerations on the removal \nprocess of hexavalent chromium from aqueous media. J Appl \nElectrochem [Internet]. 2003 [cited 2020 May 21];33(1):61\u201371. \nAvailable from: http://link.springer.com/10.1023/ \nA:1022983919644 \n\n124.  Wang Y, Fang Z, Liang B, Tsang EP. Remediation of hexavalent \nchromium contaminated soil by stabilized nanoscale zero-valent \niron prepared from steel pickling waste liquor. Chem Eng J \n[Internet]. 2014 Jul [cited 2020 May 22];247:283\u201390. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S138589471400285\nX \n\n125.  Qin G, McGuire MJ, Blute NK, Seidel C, Fong L. Hexavalent \nChromium Removal by Reduction with Ferrous Sulfate, \nCoagulation, and Filtration: A Pilot-Scale Study. Environ Sci \n\nTechnol [Internet]. 2005 Aug 1 [cited 2021 Aug 7];39(16):6321\u20137. \nAvailable from: https://pubs.acs.org/doi/10.1021/es050486p \n\n126.  Rahaman A, Hosen MdR, Hena MA, Naher UHB, Moniruzzaman \nM. A Study on removal of chromium from tannery effluent \ntreatment of chrome tanning waste water using tannery solid \nwaste. Int J Hum Cap Urban Manag [Internet]. 2016 Oct [cited 2020 \nApr 9];1(4). Available from: \nhttp://doi.org/10.22034/ijhcum.2016.04.001 \n\n127.  Krishna D, Sree RP. Artificial Neural Network and Response \nSurface Methodology Approach for Modeling and Optimization \nof Chromium (VI) Adsorption from Waste Water using Ragi Husk \nPowder. Indian Chem Eng [Internet]. 2013 Sep [cited 2020 Apr \n9];55(3):200\u201322. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/00194506.2013.8\n29257 \n\n128.", "start_char_idx": 90263, "end_char_idx": 92992, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2c5b4798-2a5c-46f5-89a6-0a513fd1b5e7": {"__data__": {"id_": "2c5b4798-2a5c-46f5-89a6-0a513fd1b5e7", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "72a3d983-09e3-4d65-8095-31bbe2cae96f", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "7a4ede97b8c6f4d5e3528ed1838cfc36b854245422aae412796a54fb9d6d1b74", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "db78f5bb-ddc7-4634-a93d-319e1e838fe1", "node_type": "1", "metadata": {}, "hash": "155e7c5159c93570f672cf124a0eb9d97aa9542e83b38077b140093eddf2b6ac", "class_name": "RelatedNodeInfo"}}, "text": "Garg UK, Kaur MP, Garg VK, Sud D. Removal of hexavalent \nchromium from aqueous solution by agricultural waste biomass. J \nHazard Mater [Internet]. 2007 Feb [cited 2020 Apr 12];140(1\u20132):60\u2013\n8. Available from: https://linkinghub.elsevier.com/retrieve/pii/ \nS030438940600690X \n\n129.  Saha R, Saha B. Removal of hexavalent chromium from \ncontaminated water by adsorption using mango leaves ( Mangifera \nindica ). Desalination Water Treat [Internet]. 2014 Mar 21 [cited \n2020 Apr 19];52(10\u201312):1928\u201336. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.8\n04458 \n\n130.  Shi T, Wang Z, Liu Y, Jia S, Changming D. Removal of hexavalent \nchromium from aqueous solutions by D301, D314 and D354 anion-\nexchange resins. J Hazard Mater [Internet]. 2009 Jan [cited 2021 Jul \n19];161(2\u20133):900\u20136. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940800574\n8 \n\n131.  Chen J-H, Hsu K-C, Chang Y-M. Surface Modification of \nHydrophobic Resin with Tricaprylmethylammonium Chloride for \nthe Removal of Trace Hexavalent Chromium. Ind Eng Chem Res \n[Internet]. 2013 Aug 21 [cited 2021 Jul 19];52(33):11685\u201394. \nAvailable from: https://pubs.acs.org/doi/10.1021/ie401233r \n\n132.  Bansal M, Garg U, Singh D, Garg VK. Removal of Cr(VI) From \nAqueous Solutions Using Pre-Consumer Processing Agricultural \nWaste: A Case Study of Rice Husk. J Hazard Mater. 2008 Jun \n1;162:312\u201320.  \n\n133.  Setshedi KZ, Bhaumik M, Songwane S, Onyango MS, Maity A. \nExfoliated polypyrrole-organically modified montmorillonite clay \nnanocomposite as a potential adsorbent for Cr(VI) removal. Chem \nEng J [Internet]. 2013 Apr [cited 2021 Aug 7];222:186\u201397. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/ \nS1385894713002271 \n\n134.  Wang X, Lu J, Cao B, Liu X, Lin Z, Yang C, et al. Facile synthesis of \nrecycling Fe3O4/graphene adsorbents with potassium humate for \nCr(VI) removal. Colloids Surf Physicochem Eng Asp [Internet]. \n2019 Jan [cited 2021 Aug 7];560:384\u201392. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S092777571831028\n8 \n\n135.  Hokkanen S, Bhatnagar A, Repo E, Lou S, Sillanp\u00e4\u00e4 M. Calcium \nhydroxyapatite microfibrillated cellulose composite as a potential \nadsorbent for the removal of Cr(VI) from aqueous solution. Chem \nEng J [Internet]. 2016 Jan [cited 2021 Aug 7];283:445\u201352. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/ \nS1385894715010013 \n\n136.  Padmavathy KS, Madhu G, Haseena PV. A study on Effects of pH, \nAdsorbent Dosage, Time, Initial Concentration and Adsorption \nIsotherm Study for the Removal of Hexavalent Chromium (Cr \n(VI)) from Wastewater by Magnetite Nanoparticles. Procedia \nTechnol [Internet]. 2016 [cited 2021 Aug 7];24:585\u201394.", "start_char_idx": 92994, "end_char_idx": 95702, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "db78f5bb-ddc7-4634-a93d-319e1e838fe1": {"__data__": {"id_": "db78f5bb-ddc7-4634-a93d-319e1e838fe1", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2c5b4798-2a5c-46f5-89a6-0a513fd1b5e7", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "af4f172108b2264063217bbdf529b1d768869b693ecd8df31bea1b4e94f7019e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "64eb03f3-2b6d-49f3-8590-bddfcf52ff9e", "node_type": "1", "metadata": {}, "hash": "e0d9950bd7bbac1878add102e4301863064b99662a377bd9008a24a920f7c03f", "class_name": "RelatedNodeInfo"}}, "text": "2016 [cited 2021 Aug 7];24:585\u201394. Available \nfrom: https://linkinghub.elsevier.com/retrieve/pii/ \nS221201731630216X \n\n137.  Cherdchoo W, Nithettham S, Charoenpanich J. Removal of Cr(VI) \nfrom synthetic wastewater by adsorption onto coffee ground and \nmixed waste tea. Chemosphere [Internet]. 2019 Apr [cited 2021 Jun \n3];221:758\u201367. Available from: https://linkinghub. \nelsevier.com/retrieve/pii/ S0045653519301109 \n\n138.  Dakiky M, Khamis M, Manassra A, Mer\u2019eb M. Selective adsorption \nof chromium(VI) in industrial wastewater using low-cost \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389406006297\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389406006297\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389419307903\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389419307903\nhttps://linkinghub.elsevier.com/retrieve/pii/S1385894713013697\nhttps://linkinghub.elsevier.com/retrieve/pii/S1385894713013697\nhttp://link.springer.com/10.1007/s11356-012-1125-4\nhttp://link.springer.com/10.1007/s11270-014-1981-2\nhttp://link.springer.com/10.1007/s11270-014-1981-2\nhttp://xlink.rsc.org/?DOI=c2jm00145d\nhttps://linkinghub.elsevier.com/retrieve/pii/S0048969705008387\nhttps://linkinghub.elsevier.com/retrieve/pii/S0048969705008387\nhttps://linkinghub.elsevier.com/retrieve/pii/S138589471400285X\nhttps://linkinghub.elsevier.com/retrieve/pii/S138589471400285X\nhttps://pubs.acs.org/doi/10.1021/es050486p\nhttp://doi.org/10.22034/ijhcum.2016.04.001\nhttp://www.tandfonline.com/doi/abs/10.1080/00194506.2013.829257\nhttp://www.tandfonline.com/doi/abs/10.1080/00194506.2013.829257\nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.804458\nhttp://www.tandfonline.com/doi/abs/10.1080/19443994.2013.804458\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389408005748\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389408005748\nhttps://pubs.acs.org/doi/10.1021/ie401233r\nhttps://linkinghub.elsevier.com/retrieve/pii/S0927775718310288\nhttps://linkinghub.elsevier.com/retrieve/pii/S0927775718310288\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  73 \n\nabundantly available adsorbents. Adv Environ Res [Internet]. 2002 \nOct [cited 2021 Aug 7];6(4):533\u201340. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S109301910100079\nX \n\n139.  Mdlalose L, Balogun M, Setshedi K, Chimuka L, Chetty A. \nPerformance evaluation of polypyrrole\u2013montmorillonite clay \ncomposite as a re-usable adsorbent for Cr(VI) remediation. Polym \nBull [Internet]. 2021 Aug [cited 2021 Sep 11];78(8):4685\u201397.", "start_char_idx": 95668, "end_char_idx": 98198, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "64eb03f3-2b6d-49f3-8590-bddfcf52ff9e": {"__data__": {"id_": "64eb03f3-2b6d-49f3-8590-bddfcf52ff9e", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "db78f5bb-ddc7-4634-a93d-319e1e838fe1", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "16348972fe1b2fe63014964b7c8bedc52b032e2673ab66affa64699944bd54c8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a18ade22-49df-4ecb-9eb1-f88fc89bfcdb", "node_type": "1", "metadata": {}, "hash": "2064da6b44a1ad66c93b38dc53b42fbfbcb3a988db23e73f93434a0ab19bcfb4", "class_name": "RelatedNodeInfo"}}, "text": "Available from: https://link.springer.com/10.1007/s00289-020-\n03338-6 \n\n140.  Tariq MA, Nadeem M, Iqbal MM, Imran M, Siddique MH, Iqbal Z, \net al. Effective sequestration of Cr (VI) from wastewater using \nnanocomposite of ZnO with cotton stalks biochar: modeling, \nkinetics, and reusability. Environ Sci Pollut Res [Internet]. 2020 Sep \n[cited 2021 Sep 11];27(27):33821\u201334. Available from: \nhttps://link.springer.com/10.1007/s11356-020-09481-x \n\n141.  Saxena D, Levin R, Firer MA. Removal of chromate from industrial \neffluent by a new isolate of Staphylococcus cohnii. Water Sci \nTechnol [Internet]. 2000 Jul 1 [cited 2020 Apr 9];42(1\u20132):93\u20138. \nAvailable from: https://iwaponline.com/wst/article/42/1-\n2/93/9910/Removal-of-chromate-from-industrial-effluent-by-a \n\n142.  Yao Y, Hu L, Li S, Zeng Q, Zhong H, He Z. Exploration on the \nbioreduction mechanisms of Cr(VI) and Hg(II) by a newly isolated \nbacterial strain Pseudomonas umsongensis CY-1. Ecotoxicol \nEnviron Saf. 2020 Sep 1;201:110850.  \n\n143.  Tan H, Wang C, Zeng G, Luo Y, Li H, Xu H. Bioreduction and \nbiosorption of Cr(VI) by a novel Bacillus sp. CRB-B1 strain. J \nHazard Mater [Internet]. 2020 Mar [cited 2021 Aug 7];386:121628. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0304389419315821 \n\n144.  Huang X-N, Min D, Liu D-F, Cheng L, Qian C, Li W-W, et al. \nFormation mechanism of organo-chromium (III) complexes from \nbioreduction of chromium (VI) by Aeromonas hydrophila. \nEnviron Int [Internet]. 2019 Aug [cited 2021 Aug 7];129:86\u201394. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0160412019308232 \n\n145.  Sun Y, Lan J, Du Y, Guo L, Du D, Chen S, et al. Chromium(VI) \nbioreduction and removal by Enterobacter sp. SL grown with \nwaste molasses as carbon source: Impact of operational conditions. \nBioresour Technol [Internet]. 2020 Apr [cited 2021 Sep \n12];302:121974. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096085241931204\n0 \n\n146.  Princy S, Sathish SS, Cibichakravarthy B, Prabagaran SR. \nHexavalent chromium reduction by Morganella morganii (1Ab1) \nisolated from tannery effluent contaminated sites of Tamil Nadu, \nIndia. Biocatal Agric Biotechnol [Internet]. 2020 Jan [cited 2021 Sep \n12];23:101469. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S187881811931684\n6 \n\n147.  Gao J, Wu S, Liu Y, Wu S, Jiang C, Li X, et al. Characterization and \ntranscriptomic analysis of a highly Cr(VI)-resistant and -reductive \nplant-growth-promoting rhizobacterium Stenotrophomonas \nrhizophila DSM14405T. Environ Pollut [Internet]. 2020 Aug [cited \n2021 Sep 12];263:114622. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S026974911935521\n6 \n\n148.", "start_char_idx": 98200, "end_char_idx": 100896, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a18ade22-49df-4ecb-9eb1-f88fc89bfcdb": {"__data__": {"id_": "a18ade22-49df-4ecb-9eb1-f88fc89bfcdb", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "64eb03f3-2b6d-49f3-8590-bddfcf52ff9e", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "ed2991cbded589b5638446a10897d40af4a6dc1cb4c28bb058803e26829ff6e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b0cd4dbd-3c82-4d42-a65d-689f866db098", "node_type": "1", "metadata": {}, "hash": "6197788b8ed93edd5cfb0a3e4fbd2267f26ad612bfb09ec66d3c87866fb28582", "class_name": "RelatedNodeInfo"}}, "text": "Tirry N, Tahri Joutey N, Sayel H, Kouchou A, Bahafid W, Asri M, \net al. Screening of plant growth promoting traits in heavy metals \nresistant bacteria: Prospects in phytoremediation. J Genet Eng \nBiotechnol [Internet]. 2018 Dec [cited 2021 Sep 12];16(2):613\u20139. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS1687157X18300635 \n\n149.  Elmeihy R, Shi X-C, Tremblay P-L, Zhang T. Fast removal of toxic \nhexavalent chromium from an aqueous solution by high-density \nGeobacter sulfurreducens. Chemosphere [Internet]. 2021 Jan [cited \n2021 Sep 12];263:128281. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004565352032476\n0 \n\n150.  An Q, Deng S, Xu J, Nan H, Li Z, Song J-L. Simultaneous reduction \nof nitrate and Cr(VI) by Pseudomonas aeruginosa strain G12 in \nwastewater. Ecotoxicol Environ Saf [Internet]. 2020 Mar [cited 2021 \nJul 27];191:110001. Available from: \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S014765131931332\n6 \n\n151.  Cheng J, Gao J, Zhang J, Yuan W, Yan S, Zhou J, et al. Optimization \nof Hexavalent Chromium Biosorption by Shewanella putrefaciens \nUsing the Box-Behnken Design. Water Air Soil Pollut [Internet]. \n2021 Mar [cited 2021 Sep 12];232(3):92. Available from: \nhttp://link.springer.com/10.1007/s11270-020-04947-7 \n\n152.  Ma L, Xu J, Chen N, Li M, Feng C. Microbial reduction fate of \nchromium (Cr) in aqueous solution by mixed bacterial consortium. \nEcotoxicol Environ Saf [Internet]. 2019 Apr [cited 2021 Aug \n7];170:763\u201370. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S014765131831331\n9 \n\n153.  Prabhakaran D, Subramanian S. Studies on the Bioremediation of \nChromium from Aqueous Solutions Using C. paurometabolum. \nTrans Indian Inst Met. 2016 Nov 22;70.  \n\n154.  Karthik C, Vijayan SR, Pugazhendhi A, Kumar G, Arulselvi P. \nBiosorption and biotransformation of Cr(VI) by novel \nCellulosimicrobium funkei strain AR6. J Taiwan Inst Chem Eng. \n2017 Jan 26;17:0\u20130.  \n\n155.  Sathishkumar K, Murugan K, Benelli G, Higuchi A, Rajasekar A. \nBioreduction of hexavalent chromium by Pseudomonas stutzeri \nL1 and Acinetobacter baumannii L2. Ann Microbiol [Internet]. \n2017 Jan [cited 2021 Aug 7];67(1):91\u20138. Available from: \nhttp://link.springer.com/10.1007/s13213-016-1240-4 \n\n156.  Chen C-Y, Cheng C-Y, Chen C-K, Hsieh M-C, Lin S-T, Ho K-Y, et \nal. Hexavalent chromium removal and bioelectricity generation by \nOchrobactrum sp. YC211 under different oxygen conditions. J \nEnviron Sci Health Part A [Internet]. 2016 May 11 [cited 2021 Aug \n7];51(6):502\u20138. Available from: http://www.tandfonline.com \n/doi/ full/10.1080/10934529.2015.1128731 \n\n157.", "start_char_idx": 100898, "end_char_idx": 103518, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b0cd4dbd-3c82-4d42-a65d-689f866db098": {"__data__": {"id_": "b0cd4dbd-3c82-4d42-a65d-689f866db098", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a18ade22-49df-4ecb-9eb1-f88fc89bfcdb", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "f467aa34bcbad7438e68a55cdbf58c8b3bfd8b846491025264b387534e253ea0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0166515f-7091-415f-83fd-884328c67105", "node_type": "1", "metadata": {}, "hash": "e0dc9b0ce442502b6eaba5a3df6f2eaead69c1d6fb46f5d0706d9cd823cb6fc0", "class_name": "RelatedNodeInfo"}}, "text": "Sharma S, Adholeya A. Detoxification and accumulation of \nchromium from tannery effluent and spent chrome effluent by \nPaecilomyces lilacinus fungi. Int Biodeterior Biodegrad [Internet]. \n2011 Mar [cited 2021 Aug 7];65(2):309\u201317. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051000211\n8 \n\n158.  Pal S, Y V. Bioremediation of Chromium from Fortified Solutions \nby Phanerochaete Chrysosporium (MTCC 787). J Bioremediation \nBiodegrad [Internet]. 2011 [cited 2021 Aug 7];02(05). Available \nfrom: https://www.omicsonline.org/bioremediation-of-\nchromium-from-fortified-solutions-by-phanerochaete-\nchrysosporium-mtcc-787-2155-6199.1000127.php?aid=2103 \n\n159.  Carol D, Kingsley S, Vincent S. Hexavalent chromium removal \nfrom aqueous solutions by Pleurotus ostreatus spent biomass. Int \nJ Eng Sci. 2012 Jan 1;4:7\u201322.  \n\n160.  Chen R, Cheng Y, Wang P, Liu Z, Wang Y, Wang Y. High efficient \nremoval and mineralization of Cr(VI) from water by \nfunctionalized magnetic fungus nanocomposites. J Cent South \nUniv [Internet]. 2020 May [cited 2021 Sep 16];27(5):1503\u201314. \nAvailable from: https://link.springer.com/10.1007/s11771-020-\n4386-y \n\n161.  Husien Sh, Labena A, El-Belely EF, Mahmoud HM, Hamouda AS. \nAbsorption of hexavalent chromium by green micro algae \nChlorella sorokiniana: live planktonic cells. Water Pract Technol \n[Internet]. 2019 Sep 1 [cited 2020 Apr 17];14(3):515\u201329. Available \nfrom: \nhttps://iwaponline.com/wpt/article/14/3/515/67497/Absorpti\non-of-hexavalent-chromium-by-green-micro \n\n162.  Deng L, Zhang Y, Qin J, Wang X, Zhu X. Biosorption of Cr(VI) from \naqueous solutions by nonliving green algae Cladophora albida. \nMiner Eng [Internet]. 2009 Mar [cited 2020 Apr 9];22(4):372\u20137. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0892687508002525 \n\n163.  Kumaraguru K, Saravanan P, Rajesh kannan R, Saravanan V. A \nsystematic analysis of hexavalent chromium adsorption and \nelimination from aqueous environment using brown marine algae \n(Turbinaria ornata). Biomass Convers Biorefinery [Internet]. 2021 \nAug 6 [cited 2021 Sep 16]; Available from: \nhttps://link.springer.com/10.1007/s13399-021-01795-1 \n\n164.  Mari S, Vrane J. Characteristics and significance of microbial \nbiofilm formation. Period Biol.", "start_char_idx": 103520, "end_char_idx": 105777, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0166515f-7091-415f-83fd-884328c67105": {"__data__": {"id_": "0166515f-7091-415f-83fd-884328c67105", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b0cd4dbd-3c82-4d42-a65d-689f866db098", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "d8450439e9dd9b709868e4364b7f3bb50f3c952caea5dc314791ed3ee4f4d7d9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f2cfdad2-5dd1-49ad-8f2f-80a8a2c617c4", "node_type": "1", "metadata": {}, "hash": "647d584b883fdb8aac62d28c8acb5b5677fd3c68fbc1484f28957aa942b24d3b", "class_name": "RelatedNodeInfo"}}, "text": "Period Biol. 2007;109(2):7.  \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S109301910100079X\nhttps://linkinghub.elsevier.com/retrieve/pii/S109301910100079X\nhttps://link.springer.com/10.1007/s00289-020-03338-6\nhttps://link.springer.com/10.1007/s00289-020-03338-6\nhttps://link.springer.com/10.1007/s11356-020-09481-x\nhttps://iwaponline.com/wst/article/42/1-2/93/9910/Removal-of-chromate-from-industrial-effluent-by-a\nhttps://iwaponline.com/wst/article/42/1-2/93/9910/Removal-of-chromate-from-industrial-effluent-by-a\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852419312040\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852419312040\nhttps://linkinghub.elsevier.com/retrieve/pii/S1878818119316846\nhttps://linkinghub.elsevier.com/retrieve/pii/S1878818119316846\nhttps://linkinghub.elsevier.com/retrieve/pii/S0269749119355216\nhttps://linkinghub.elsevier.com/retrieve/pii/S0269749119355216\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653520324760\nhttps://linkinghub.elsevier.com/retrieve/pii/S0045653520324760\nhttps://linkinghub.elsevier.com/retrieve/pii/S0147651319313326\nhttps://linkinghub.elsevier.com/retrieve/pii/S0147651319313326\nhttp://link.springer.com/10.1007/s11270-020-04947-7\nhttps://linkinghub.elsevier.com/retrieve/pii/S0147651318313319\nhttps://linkinghub.elsevier.com/retrieve/pii/S0147651318313319\nhttp://link.springer.com/10.1007/s13213-016-1240-4\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830510002118\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830510002118\nhttps://www.omicsonline.org/bioremediation-of-chromium-from-fortified-solutions-by-phanerochaete-chrysosporium-mtcc-787-2155-6199.1000127.php?aid=2103\nhttps://www.omicsonline.org/bioremediation-of-chromium-from-fortified-solutions-by-phanerochaete-chrysosporium-mtcc-787-2155-6199.1000127.php?aid=2103\nhttps://www.omicsonline.org/bioremediation-of-chromium-from-fortified-solutions-by-phanerochaete-chrysosporium-mtcc-787-2155-6199.1000127.php?aid=2103\nhttps://link.springer.com/10.1007/s11771-020-4386-y\nhttps://link.springer.com/10.1007/s11771-020-4386-y\nhttps://iwaponline.com/wpt/article/14/3/515/67497/Absorption-of-hexavalent-chromium-by-green-micro\nhttps://iwaponline.com/wpt/article/14/3/515/67497/Absorption-of-hexavalent-chromium-by-green-micro\nhttps://link.springer.com/10.1007/s13399-021-01795-1\nhttps://link.springer.com/10.1007/s13399-021-01795-1\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  74 \n\n165.  Toyofuku M, Inaba T, Kiyokawa T, Obana N, Yawata Y, Nomura \nN. Environmental factors that shape biofilm formation. Biosci \nBiotechnol Biochem [Internet].", "start_char_idx": 105765, "end_char_idx": 108378, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f2cfdad2-5dd1-49ad-8f2f-80a8a2c617c4": {"__data__": {"id_": "f2cfdad2-5dd1-49ad-8f2f-80a8a2c617c4", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0166515f-7091-415f-83fd-884328c67105", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "7bd2c7876601196aaa7fdc34e2ff38943899a5cd18886e42459932382a434799", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8736f12b-25c9-46a1-b4a8-be2858bd64f5", "node_type": "1", "metadata": {}, "hash": "047747005c0503849062b1bdf3d1bd17dc85d899adebeee2d130aa7795177886", "class_name": "RelatedNodeInfo"}}, "text": "Biosci \nBiotechnol Biochem [Internet]. 2016 Jan 2 [cited 2019 Feb \n5];80(1):7\u201312. Available from: https://www.tandfonline \n.com/doi/full/10.1080/09168451.2015.1058701 \n\n166.  Evans LV. Biofilms recent advances in their study and control. \nAmsterdam: Taylor & Francis e-Library; 2004.  \n\n167.  O\u2019Toole G, Kaplan HB, Kolter R. Biofilm Formation as Microbial \nDevelopment. Annu Rev Microbiol [Internet]. 2000 Oct [cited 2020 \nApr 9];54(1):49\u201379. Available from: \nhttp://www.annualreviews.org/ \ndoi/10.1146/annurev.micro.54.1.49 \n\n168.  Edwards SJ, Kjellerup BV. Applications of biofilms in \nbioremediation and biotransformation of persistent organic \npollutants, pharmaceuticals/personal care products, and heavy \nmetals. Appl Microbiol Biotechnol [Internet]. 2013 Dec [cited 2020 \nMay 18];97(23):9909\u201321. Available from: \nhttp://link.springer.com/10.1007/s00253-013-5216-z \n\n169.  Das N, Basak LVG, Salam JA. Application of Biofilms on \nRemediation of Pollutants \u2013 An Overview. 2012;  \n\n170.  Shukla SK, Mangwani N, Rao TS, Das S. Biofilm-Mediated \nBioremediation of Polycyclic Aromatic Hydrocarbons. In: \nMicrobial Biodegradation and Bioremediation [Internet]. Elsevier; \n2014 [cited 2020 May 18]. p. 203\u201332. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/B978012800021200\n008X \n\n171.  Asri M, El Ghachtouli N, Elabed S, Ibnsouda Koraichi S, Elabed A, \nSilva B, et al. Wicherhamomyces anomalus biofilm supported on \nwood husk for chromium wastewater treatment. J Hazard Mater \n[Internet]. 2018 Oct [cited 2020 Jun 1];359:554\u201362. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438941830406\n0 \n\n172.  Herath HMLI, Rajapaksha AU, Vithanage M, Seneviratne G. \nDeveloped fungal\u2013bacterial biofilms as a novel tool for bioremoval \nof hexavelant chromium from wastewater. Chem Ecol [Internet]. \n2014 Jul 4 [cited 2020 Jun 1];30(5):418\u201327. Available from: \nhttp://www.tandfonline.com/doi/abs/10.1080/02757540.2013.8\n61828 \n\n173.  Smith WL, Gadd GM. Reduction and precipitation of chromate by \nmixed culture sulphate-reducing bacterial biofilms. J Appl \nMicrobiol [Internet]. 2000 Jun [cited 2020 Jun 1];88(6):983\u201391. \nAvailable from: http://doi.wiley.com/10.1046/j.1365-\n2672.2000.01066.x \n\n174.  Yong P, Liu W, Zhang Z, Beauregard D, Johns ML, Macaskie LE. \nOne step bioconversion of waste precious metals into Serratia \nbiofilm-immobilized catalyst for Cr(VI) reduction. Biotechnol Lett \n[Internet]. 2015 Nov [cited 2020 Jun 1];37(11):2181\u201391. Available \nfrom: http://link.springer.com/10.1007/s10529-015-1894-1 \n\n175.  Tripathi AG A. Bioremediation of toxic chromium from \nelectroplating effluent by chromate-reducing Pseudomonas \naeruginosa A2Chr in two bioreactors.", "start_char_idx": 108340, "end_char_idx": 111034, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8736f12b-25c9-46a1-b4a8-be2858bd64f5": {"__data__": {"id_": "8736f12b-25c9-46a1-b4a8-be2858bd64f5", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f2cfdad2-5dd1-49ad-8f2f-80a8a2c617c4", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "cdf80efd4ed03f8630f12ddce6b16765273d51cff6b89a74a7d775783c1a2f01", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d28b0ca0-989e-47c0-aad5-7f5f965b41c1", "node_type": "1", "metadata": {}, "hash": "80f55cb8e894c6e3085049e20036d44d0d403613ebfe87a0f0b5c683e6572ca1", "class_name": "RelatedNodeInfo"}}, "text": "Appl Microbiol Biotechnol \n[Internet]. 2002 Mar 1 [cited 2020 Apr 9];58(3):416\u201320. Available \nfrom: http://link.springer.com/10.1007/s00253-001-0871-x \n\n176.  Gabr RM, Gad-Elrab SMF, Abskharon RNN, Hassan SHA, Shoreit \nAAM. Biosorption of hexavalent chromium using biofilm of E. coli \nsupported on granulated activated carbon. World J Microbiol \nBiotechnol [Internet]. 2009 Oct [cited 2020 Jun 2];25(10):1695\u2013703. \nAvailable from: http://link.springer.com/10.1007/s11274-009-\n0063-x \n\n177.  Pan X, Liu Z, Chen Z, Cheng Y, Pan D, Shao J, et al. Investigation \nof Cr(VI) reduction and Cr(III) immobilization mechanism by \nplanktonic cells and biofilms of Bacillus subtilis ATCC-6633. Water \nRes [Internet]. 2014 May [cited 2020 Jun 1];55:21\u20139. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S004313541400108\n0 \n\n178.  Ar\u0131ca MY, Bayramo\u011flu G. Cr(VI) biosorption from aqueous \nsolutions using free and immobilized biomass of Lentinus sajor-\ncaju: preparation and kinetic characterization. Colloids Surf \nPhysicochem Eng Asp [Internet]. 2005 Feb [cited 2021 Aug \n6];253(1\u20133):203\u201311. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S092777570400890\n8 \n\n179.  Carmona M, Silva M, Leite S, Vasco-Echeverri O, Ocampo-Lopez \nC. Packed bed redistribution system for Cr(III) and Cr(VI) \nbiosorption by Saccharomyces cerevisiae. J Taiwan Inst Chem Eng. \n2011 Jan 1;43.  \n\n180.  Sanghi R, Srivastava A. Long-term chromate reduction by \nimmobilized fungus in continuous column. Chem Eng J. 2010 Aug \n1;162:122\u20136.  \n\n181.  Ahmad A, Bhat AH, Buang A. Enhanced biosorption of transition \nmetals by living Chlorella vulgaris immobilized in Ca-alginate \nbeads. Environ Technol [Internet]. 2019 Jun 20 [cited 2021 Aug \n6];40(14):1793\u2013809. Available from: \nhttps://www.tandfonline.com/doi/full/10.1080/09593330.2018.\n1430171 \n\n182.  Akhtar N, Iqbal M, Zafar SI, Iqbal J. Biosorption characteristics of \nunicellular green alga Chlorella sorokiniana immobilized in loofa \nsponge for removal of Cr(III). J Environ Sci [Internet]. 2008 Feb \n[cited 2021 Jul 16];20(2):231\u20139. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S100107420860036\n4 \n\n183.  Baykal \u00d6ZER T, A\u00e7ikg\u00f6z Erkaya \u0130, Udoh A, \u00d6zer T, Akbulut A, \nBayramoglu G, et al. Biosorption of Cr(VI) by free and \nimmobilized Pediastrum boryanum biomass: Equilibrium, kinetic, \nand thermodynamic studies. Environ Sci Pollut Res Int. 2012 Feb \n29;19:2983\u201393.  \n\n184.  petrovi\u010d A, Simoni\u010d M. Removal of heavy metal ions from \ndrinking water by alginate-immobilised Chlorella sorokiniana. Int \nJ Environ Sci Technol. 2016 May 18;13.  \n\n185.  Wong Y-S, Tam NFY, editors. Wastewater Treatment with Algae \n[Internet].", "start_char_idx": 111035, "end_char_idx": 113715, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d28b0ca0-989e-47c0-aad5-7f5f965b41c1": {"__data__": {"id_": "d28b0ca0-989e-47c0-aad5-7f5f965b41c1", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8736f12b-25c9-46a1-b4a8-be2858bd64f5", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "90cb8b60a9f7cdd8c5e3b5cd1f7a1244ca61cd0c7cf1b42499879e564d9c7922", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a7527e67-21b0-411f-9566-7df61982901e", "node_type": "1", "metadata": {}, "hash": "42d2591fd4cf015710bcf65d812ff7b3750b8699e0b3da55ce1ad3a7d0b00e77", "class_name": "RelatedNodeInfo"}}, "text": "Wastewater Treatment with Algae \n[Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 1998 \n[cited 2021 Aug 6]. Available from: \nhttp://link.springer.com/10.1007/978-3-662-10863-5 \n\n186.  Abinandan S, Subashchandrabose SR, Venkateswarlu K, Megharaj \nM. Microalgae\u2013bacteria biofilms: a sustainable synergistic \napproach in remediation of acid mine drainage. Appl Microbiol \nBiotechnol [Internet]. 2018 Feb [cited 2021 Sep 27];102(3):1131\u201344. \nAvailable from: http://link.springer.com/10.1007/s00253-017-\n8693-7 \n\n187.  Qu W, Zhang C, Chen X, Ho S-H. New concept in swine \nwastewater treatment: development of a self-sustaining synergetic \nmicroalgae-bacteria symbiosis (ABS) system to achieve \nenvironmental sustainability. J Hazard Mater [Internet]. 2021 Sep \n[cited 2021 Sep 27];418:126264. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438942101228\n0 \n\n188.  Sun J, Xu W, Cai B, Huang G, Zhang H, Zhang Y, et al. High-\nconcentration nitrogen removal coupling with bioelectric power \ngeneration by a self-sustaining algal-bacterial biocathode photo-\nbioelectrochemical system under daily light/dark cycle. \nChemosphere [Internet]. 2019 May [cited 2021 Sep 27];222:797\u2013809. \nAvailable from: https://linkinghub.elsevier.com/retrieve/pii/ \nS0045653519302139 \n\n189.  Roestorff M., Chirwa E. Bacterial cr(vi) reduction with internal \ncarbon recirculation using freshwater algae as primary producers. \nChem Eng Trans [Internet]. 2018 May [cited 2020 Jun 4];64:457\u201362. \nAvailable from: http://doi.org/10.3303/CET1864077 \n\n190.  Quintelas C, Fonseca B, Silva B, Figueiredo H, Tavares T. \nTreatment of chromium(VI) solutions in a pilot-scale bioreactor \nthrough a biofilm of Arthrobacter viscosus supported on GAC. \nBioresour Technol [Internet]. 2009 Jan [cited 2020 Jun \n3];100(1):220\u20136. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096085240800442\n2 \n\n191.  Morales DK, Ocampo W, Zambrano MM. Efficient removal of \nhexavalent chromium by a tolerant Streptomyces sp. affected by \nthe toxic effect of metal exposure: Chromium tolerant \nStreptomyces sp. J Appl Microbiol [Internet]. 2007 Aug 30 [cited \n2021 Jul 19];103(6):2704\u201312. Available from: \nhttps://onlinelibrary.wiley.com/doi/10.1111/j.1365-\n2672.2007.03510.x \n\n192.  C\u00f3rdoba A, Vargas P, Dussan J. Chromate reduction by \nArthrobacter CR47 in biofilm packed bed reactors. J Hazard Mater \n[Internet]. 2008 Feb [cited 2020 Jun 3];151(1):274\u20139.", "start_char_idx": 113671, "end_char_idx": 116107, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a7527e67-21b0-411f-9566-7df61982901e": {"__data__": {"id_": "a7527e67-21b0-411f-9566-7df61982901e", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d28b0ca0-989e-47c0-aad5-7f5f965b41c1", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "4d9cc133f6386254b28b636a3214672c72a172acb79203ea5e5600ae5e0b6cd4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a10b4037-3870-4da1-a56f-0c554b5a22f7", "node_type": "1", "metadata": {}, "hash": "87128209c3364d90d0316a6576a1437c46d4ca356c7a0bee29b0311e4afb7729", "class_name": "RelatedNodeInfo"}}, "text": "Available from: \n\nhttp://link.springer.com/10.1007/s00253-013-5216-z\nhttps://linkinghub.elsevier.com/retrieve/pii/B978012800021200008X\nhttps://linkinghub.elsevier.com/retrieve/pii/B978012800021200008X\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389418304060\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389418304060\nhttp://www.tandfonline.com/doi/abs/10.1080/02757540.2013.861828\nhttp://www.tandfonline.com/doi/abs/10.1080/02757540.2013.861828\nhttp://doi.wiley.com/10.1046/j.1365-2672.2000.01066.x\nhttp://doi.wiley.com/10.1046/j.1365-2672.2000.01066.x\nhttp://link.springer.com/10.1007/s10529-015-1894-1\nhttp://link.springer.com/10.1007/s00253-001-0871-x\nhttp://link.springer.com/10.1007/s11274-009-0063-x\nhttp://link.springer.com/10.1007/s11274-009-0063-x\nhttps://linkinghub.elsevier.com/retrieve/pii/S0043135414001080\nhttps://linkinghub.elsevier.com/retrieve/pii/S0043135414001080\nhttps://linkinghub.elsevier.com/retrieve/pii/S0927775704008908\nhttps://linkinghub.elsevier.com/retrieve/pii/S0927775704008908\nhttps://www.tandfonline.com/doi/full/10.1080/09593330.2018.1430171\nhttps://www.tandfonline.com/doi/full/10.1080/09593330.2018.1430171\nhttps://linkinghub.elsevier.com/retrieve/pii/S1001074208600364\nhttps://linkinghub.elsevier.com/retrieve/pii/S1001074208600364\nhttp://link.springer.com/10.1007/978-3-662-10863-5\nhttp://link.springer.com/10.1007/s00253-017-8693-7\nhttp://link.springer.com/10.1007/s00253-017-8693-7\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389421012280\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389421012280\nhttp://doi.org/10.3303/CET1864077\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852408004422\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852408004422\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852408004422\nhttps://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03510.x\nhttps://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03510.x\nhttps://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03510.x\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  75 \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940701555\n5 \n\n193.  Sarioglu OF, Celebioglu A, Tekinay T, Uyar T. Bacteria-\nimmobilized electrospun fibrous polymeric webs for hexavalent \nchromium remediation in water. Int J Environ Sci Technol \n[Internet]. 2016 Aug [cited 2020 Jun 3];13(8):2057\u201366.", "start_char_idx": 116108, "end_char_idx": 118491, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a10b4037-3870-4da1-a56f-0c554b5a22f7": {"__data__": {"id_": "a10b4037-3870-4da1-a56f-0c554b5a22f7", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a7527e67-21b0-411f-9566-7df61982901e", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "df1b1c89b9c9e6219274a59baf27253257301db5d2fed62811a592a20f88ed4e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5adddd51-92e7-4bcb-9949-fa3be161988c", "node_type": "1", "metadata": {}, "hash": "e774c6d6c46d780508ffe08ad378d531696ff9579b4ca936c61f44ac85c4125f", "class_name": "RelatedNodeInfo"}}, "text": "Available \nfrom: http://link.springer.com/10.1007/s13762-016-1033-0 \n\n194.  Dey S, Paul AK. Influence of metal ions on biofilm formation by \nArthrobacter sp. SUK 1205 and evaluation of their Cr(VI) removal \nefficacy. Int Biodeterior Biodegrad [Internet]. 2018 Aug [cited 2020 \nJun 3];132:122\u201331. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051731046\n6 \n\n195.  Focardi S, Pepi M, Landi G, Gasperini S, Ruta M, Di Biasio P, et al. \nHexavalent chromium reduction by whole cells and cell free \nextract of the moderate halophilic bacterial strain Halomonas sp. \nTA-04. Int Biodeterior Biodegrad [Internet]. 2012 Jan [cited 2020 \nJun 3];66(1):63\u201370. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051100230\n7 \n\n196.  Ravikumar KVG, Kumar D, Kumar G, Mrudula P, Natarajan C, \nMukherjee A. Enhanced Cr(VI) Removal by Nanozerovalent Iron-\nImmobilized Alginate Beads in the Presence of a Biofilm in a \nContinuous-Flow Reactor. Ind Eng Chem Res [Internet]. 2016 May \n25 [cited 2020 Jun 3];55(20):5973\u201382. Available from: \nhttps://pubs.acs.org/doi/10.1021/acs.iecr.6b01006 \n\n197.  Ait-Meddour A, Abbas N, Ouled-Haddar H, Sifour M, \nBendjeddou K, Idoui T. Biofilm Formation by the Hexavalent \nChromium Removing Strain Streptococcus salivarius: in Vitro \nApproach on Abiotic Surfaces. Pollution [Internet]. 2020 Apr [cited \n2020 Jun 4];6(2). Available from: \nhttp://doi.org/10.22059/poll.2020.288349.685 \n\n198.  Chirwa EMN, Wang Y-T. Chromium(VI) Reduction by  \nPseudomonas fluorescens  LB300 in Fixed-Film Bioreactor. J \nEnviron Eng [Internet]. 1997 Aug [cited 2020 Jun 4];123(8):760\u20136. \nAvailable from: \nhttp://ascelibrary.org/doi/10.1061/%28ASCE%290733-\n9372%281997%29123%3A8%28760%29 \n\n199.  Naeem A, Batool R, Jamil N. Cr(VI) reduction by \nCellulosimicrobium sp. isolated from tannery effluent. Turk J Biol. \n2013;8.  \n\n200.  Quintelas C, Rocha Z, Silva B, Fonseca B, Figueiredo H, Tavares T. \nRemoval of Cd(II), Cr(VI), Fe(III) and Ni(II) from aqueous \nsolutions by an E. coli biofilm supported on kaolin. Chem Eng J \n[Internet]. 2009 Jul 1 [cited 2021 Sep 22];149(1\u20133):319\u201324. Available \nfrom: \nhttps://linkinghub.elsevier.com/retrieve/pii/S138589470800735\n3 \n\n201.  Husien S, Labena A, El-Belely E, Mahmoud H, Hamouda A. \nApplication of Nostoc sp. for hexavalent chromium [Cr(VI)] \nremoval: planktonic and biofilm. Int J Environ Anal Chem \n[Internet]. 2020 Jun 5 [cited 2021 Sep 22];1\u201322. Available from: \nhttps://www.tandfonline.com/doi/full/10.1080/03067319.2020.\n1773454 \n\n202.", "start_char_idx": 118492, "end_char_idx": 121013, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5adddd51-92e7-4bcb-9949-fa3be161988c": {"__data__": {"id_": "5adddd51-92e7-4bcb-9949-fa3be161988c", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a10b4037-3870-4da1-a56f-0c554b5a22f7", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "4f02e28762bfda8915b4686b73187c3a0e19b2535494d01604773ba23644e80c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "68c2bbd3-feda-4951-b7c2-f1f27e627c11", "node_type": "1", "metadata": {}, "hash": "bbc83a42fc3bce252df830942d5c03b51b38f22fc5f4fdd29a28467561cfc313", "class_name": "RelatedNodeInfo"}}, "text": "1773454 \n\n202.  Zinicovscaia I, Safonov A, Boldyrev K, Gundorina S, Yushin N, \nPetuhov O, et al. Selective metal removal from chromium-\ncontaining synthetic effluents using Shewanella xiamenensis \nbiofilm supported on zeolite. Environ Sci Pollut Res [Internet]. \n2020 Apr [cited 2021 Sep 22];27(10):10495\u2013505. Available from: \nhttp://link.springer.com/10.1007/s11356-020-07690-y \n\n203.  Hussain S, Quinn L, Li J, Casey E, Murphy CD. Simultaneous \nremoval of malachite green and hexavalent chromium by \nCunninghamella elegans biofilm in a semi-continuous system. Int \nBiodeterior Biodegrad [Internet]. 2017 Nov [cited 2021 Sep \n22];125:142\u20139. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051730884\n3 \n\n204.  Dey S, Paul AK. Magnesium-induced biofilm development in \nArthrobacter sp. SUK 1201 and removal of hexavalent chromium. \nSoil Sediment Contam Int J [Internet]. 2018 Jul 4 [cited 2021 Sep \n22];27(5):383\u201392. Available from: \nhttps://www.tandfonline.com/doi/full/10.1080/15320383.2018.\n1484688 \n\n205.  Kumar H, Sinha SK, Goud VV, Das S. Removal of Cr(VI) by \nmagnetic iron oxide nanoparticles synthesized from extracellular \npolymeric substances of chromium resistant acid-tolerant \nbacterium Lysinibacillus sphaericus RTA-01. J Environ Health Sci \nEng [Internet]. 2019 Dec [cited 2021 Sep 22];17(2):1001\u201316. \nAvailable from: http://link.springer.com/10.1007/s40201-019-\n00415-5 \n\n206.  Tandon S, Jha M, Dudhwadkar S. Study on Ochrobactrum \npseudintermedium ADV31 for the removal of hexavalent \nchromium through different immobilization techniques. SN Appl \nSci [Internet]. 2020 Feb [cited 2021 Sep 22];2(2):296. Available from: \nhttp://link.springer.com/10.1007/s42452-020-2103-y \n\n207.  Pratush A, Kumar A, Hu Z. Adverse effect of heavy metals (As, \nPb, Hg, and Cr) on health and their bioremediation strategies: a \nreview. Int Microbiol [Internet]. 2018 Sep [cited 2021 Sep \n11];21(3):97\u2013106. Available from: \nhttp://link.springer.com/10.1007/s10123-018-0012-3 \n\n208.  Azubuike CC, Chikere CB, Okpokwasili GC. Bioremediation \ntechniques\u2013classification based on site of application: principles, \nadvantages, limitations and prospects. World J Microbiol \nBiotechnol [Internet]. 2016 Nov [cited 2021 Jul 26];32(11):180. \nAvailable from: http://link.springer.com/10.1007/s11274-016-\n2137-x \n\n209.  Ahmad WA, Venil CK, Nkhalambayausi Chirwa EM, Wang Y-T, \nSani MohdH, Samad AFA, et al. Bacterial Reduction of Cr(VI): \nOperational Challenges and Feasibility. Curr Pollut Rep [Internet]. \n2021 Jun [cited 2021 Sep 28];7(2):115\u201327. Available from: \nhttps://link.springer.com/10.1007/s40726-021-00174-8 \n\n210.  Apte A, Tare V, Bose P. Extent of oxidation of Cr(III) to Cr(VI) \nunder various conditions pertaining to natural environment.", "start_char_idx": 120999, "end_char_idx": 123757, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "68c2bbd3-feda-4951-b7c2-f1f27e627c11": {"__data__": {"id_": "68c2bbd3-feda-4951-b7c2-f1f27e627c11", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5adddd51-92e7-4bcb-9949-fa3be161988c", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "da2d14dd420f5849d33b29434f3d4d358133264a84285011a4b23de3936206e3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "06d0d982-b9b9-4640-be55-9da5907fbc98", "node_type": "1", "metadata": {}, "hash": "ad623777e6972dd463f639fd5d54426cb4250a386012e152605291fa35843b92", "class_name": "RelatedNodeInfo"}}, "text": "J \nHazard Mater [Internet]. 2006 Feb 6 [cited 2021 Sep 17];128(2\u2013\n3):164\u201374. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438940500455\n3 \n\n211.  Lindsay DR, Farley KJ, Carbonaro RF. Oxidation of CrIII to CrVI \nduring chlorination of drinking water. J Environ Monit [Internet]. \n2012 [cited 2021 Sep 17];14(7):1789. Available from: \nhttp://xlink.rsc.org/?DOI=c2em00012a \n\n212.  Espinoza-S\u00e1nchez MA, Ar\u00e9valo-Ni\u00f1o K, Quintero-Zapata I, \nCastro-Gonz\u00e1lez I, Almaguer-Cant\u00fa V. Cr(VI) adsorption from \naqueous solution by fungal bioremediation based using Rhizopus \nsp. J Environ Manage [Internet]. 2019 Dec [cited 2021 Sep \n28];251:109595. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030147971931313\n1 \n\n213.  Benila Smily JRM, Sumithra PA. Optimization of Chromium \nBiosorption by Fungal Adsorbent, Trichoderma sp. BSCR02 and its \nDesorption Studies. HAYATI J Biosci [Internet]. 2017 Apr [cited \n2021 Sep 28];24(2):65\u201371. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S197830191630434\nX \n\n214.  Xu X, Zhang Z, Huang Q, Chen W. Biosorption Performance of \nMultimetal Resistant Fungus Penicillium chrysogenum XJ-1 for \nRemoval of Cu 2+ and Cr 6+ from Aqueous Solutions. Geomicrobiol \nJ [Internet]. 2018 Jan 2 [cited 2021 Sep 28];35(1):40\u20139. Available \nfrom: \nhttps://www.tandfonline.com/doi/full/10.1080/01490451.2017.\n1310331 \n\n215.  Mondal NK, Samanta A, Roy P, Das B. Optimization study of \nadsorption parameters for removal of Cr(VI) using Magnolia leaf \nbiomass by response surface methodology. Sustain Water Resour \nManag [Internet]. 2019 Dec [cited 2021 Sep 28];5(4):1627\u201339. \nAvailable from: http://link.springer.com/10.1007/s40899-019-\n00322-5 \n\n216.  Vendruscolo F, da Rocha Ferreira GL, Antoniosi Filho NR. \nBiosorption of hexavalent chromium by microorganisms. Int \nBiodeterior Biodegrad [Internet]. 2017 Apr [cited 2020 Apr \n9];119:87\u201395. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096483051630505\n4 \n\n217.  Antony GS, Manna A, Baskaran S, Puhazhendi P, Ramchary A, \nNiraikulam A, et al. Non-enzymatic reduction of Cr (VI) and it\u2019s \neffective biosorption using heat-inactivated biomass: A \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407015555\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389407015555\nhttp://link.springer.com/10.1007/s13762-016-1033-0\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830517310466\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830517310466\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830511002307\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830511002307\nhttps://pubs.acs.org/doi/10.", "start_char_idx": 123758, "end_char_idx": 126388, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "06d0d982-b9b9-4640-be55-9da5907fbc98": {"__data__": {"id_": "06d0d982-b9b9-4640-be55-9da5907fbc98", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "68c2bbd3-feda-4951-b7c2-f1f27e627c11", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "796e60d1d16bdcfccb5f886228ff0fbc1693c6aafb1bd25f83487aa70ceaabbd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7710c75c-8bac-4b4d-b6ed-a67704ae4e36", "node_type": "1", "metadata": {}, "hash": "1d5e57a7be3203dd0aa456ecee706ccf4bc1d0efd12256da9ec97c84a55d15f2", "class_name": "RelatedNodeInfo"}}, "text": "acs.org/doi/10.1021/acs.iecr.6b01006\nhttp://doi.org/10.22059/poll.2020.288349.685\nhttp://ascelibrary.org/doi/10.1061/%28ASCE%290733-9372%281997%29123%3A8%28760%29\nhttp://ascelibrary.org/doi/10.1061/%28ASCE%290733-9372%281997%29123%3A8%28760%29\nhttps://linkinghub.elsevier.com/retrieve/pii/S1385894708007353\nhttps://linkinghub.elsevier.com/retrieve/pii/S1385894708007353\nhttps://www.tandfonline.com/doi/full/10.1080/03067319.2020.1773454\nhttps://www.tandfonline.com/doi/full/10.1080/03067319.2020.1773454\nhttp://link.springer.com/10.1007/s11356-020-07690-y\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830517308843\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830517308843\nhttps://www.tandfonline.com/doi/full/10.1080/15320383.2018.1484688\nhttps://www.tandfonline.com/doi/full/10.1080/15320383.2018.1484688\nhttp://link.springer.com/10.1007/s40201-019-00415-5\nhttp://link.springer.com/10.1007/s40201-019-00415-5\nhttp://link.springer.com/10.1007/s42452-020-2103-y\nhttp://link.springer.com/10.1007/s10123-018-0012-3\nhttp://link.springer.com/10.1007/s11274-016-2137-x\nhttp://link.springer.com/10.1007/s11274-016-2137-x\nhttps://link.springer.com/10.1007/s40726-021-00174-8\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389405004553\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389405004553\nhttp://xlink.rsc.org/?DOI=c2em00012a\nhttps://linkinghub.elsevier.com/retrieve/pii/S0301479719313131\nhttps://linkinghub.elsevier.com/retrieve/pii/S0301479719313131\nhttps://linkinghub.elsevier.com/retrieve/pii/S0301479719313131\nhttps://linkinghub.elsevier.com/retrieve/pii/S197830191630434X\nhttps://linkinghub.elsevier.com/retrieve/pii/S197830191630434X\nhttps://www.tandfonline.com/doi/full/10.1080/01490451.2017.1310331\nhttps://www.tandfonline.com/doi/full/10.1080/01490451.2017.1310331\nhttp://link.springer.com/10.1007/s40899-019-00322-5\nhttp://link.springer.com/10.1007/s40899-019-00322-5\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830516305054\nhttps://linkinghub.elsevier.com/retrieve/pii/S0964830516305054", "start_char_idx": 126373, "end_char_idx": 128391, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7710c75c-8bac-4b4d-b6ed-a67704ae4e36": {"__data__": {"id_": "7710c75c-8bac-4b4d-b6ed-a67704ae4e36", "embedding": null, "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-243", "node_type": "4", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "52c187f24ddb926d3298410ef7de0358a95af428d50f1ed54ff0c53d4ac437f6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "06d0d982-b9b9-4640-be55-9da5907fbc98", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "2e98c4747e8fd858b2a75eebfa7a6a9725e475d73f7d8bab2feeeda82c8aedaa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3aab2d2e-dc87-4049-8ae9-6140574347ff", "node_type": "1", "metadata": {}, "hash": "0e55cfe47eb9697652409efc432fafbf5db4c15e26b2e25a715f55cfde2a5f10", "class_name": "RelatedNodeInfo"}}, "text": "elsevier.com/retrieve/pii/S0964830516305054\n\n\nNepal J Biotechnol. 2 0 2 2 D e c ; 1 0 : 57-76    Aththanayake et al.  \n\n\u00a9NJB, BSN  76 \n\nfermentation waste material. J Hazard Mater [Internet]. 2020 Jun \n[cited 2021 Sep 28];392:122257. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S030438942030245\n4 \n\n218.  Daneshvar E, Zarrinmehr MJ, Kousha M, Hashtjin AM, Saratale \nGD, Maiti A, et al. Hexavalent chromium removal from water by \nmicroalgal-based materials: Adsorption, desorption and recovery \nstudies. Bioresour Technol [Internet]. 2019 Dec [cited 2021 Sep \n28];293:122064. Available from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S096085241931294\n5 \n\n219.  Sukumar C, Janaki V, Kamala-Kannan S, Shanthi K. Biosorption of \nchromium(VI) using Bacillus subtilis SS-1 isolated from soil \nsamples of electroplating industry. Clean Technol Environ Policy \n[Internet]. 2014 Feb [cited 2021 Jul 16];16(2):405\u201313. Available \nfrom: http://link.springer.com/10.1007/s10098-013-0636-0 \n\n220.  Sathvika T, Manasi, Rajesh V, Rajesh N. Adsorption of chromium \nsupported with various column modelling studies through the \nsynergistic influence of Aspergillus and cellulose. J Environ Chem \nEng [Internet]. 2016 Sep [cited 2021 Sep 28];4(3):3193\u2013204. \nAvailable from: \nhttps://linkinghub.elsevier.com/retrieve/pii/S221334371630238\nX \n\n221.  Aravindhan R, Aafreen Fathima, Selvamurugan M, Raghava Rao \nJ, Balachandran UN. Adsorption, desorption, and kinetic study on \nCr(III) removal from aqueous solution using Bacillus subtilis \nbiomass. Clean Technol Environ Policy [Internet]. 2012 Aug [cited \n2021 Jul 19];14(4):727\u201335. Available from: \nhttp://link.springer.com/10.1007/s10098-011-0440-7 \n\n  \n\n \n\n \n\n \n\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389420302454\nhttps://linkinghub.elsevier.com/retrieve/pii/S0304389420302454\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852419312945\nhttps://linkinghub.elsevier.com/retrieve/pii/S0960852419312945\nhttp://link.springer.com/10.1007/s10098-013-0636-0\nhttps://linkinghub.elsevier.com/retrieve/pii/S221334371630238X\nhttps://linkinghub.elsevier.com/retrieve/pii/S221334371630238X\nhttps://linkinghub.elsevier.com/retrieve/pii/S221334371630238X\nhttp://link.springer.com/10.1007/s10098-011-0440-7\nhttp://link.springer.com/10.1007/s10098-011-0440-7", "start_char_idx": 128348, "end_char_idx": 130656, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3aab2d2e-dc87-4049-8ae9-6140574347ff": {"__data__": {"id_": "3aab2d2e-dc87-4049-8ae9-6140574347ff", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7710c75c-8bac-4b4d-b6ed-a67704ae4e36", "node_type": "1", "metadata": {"identifier": "njb-243", "author": "Aththanayake, A.M.K.C.B.; Rathnayake, I.V.N.; Deeyamulla, M.P.", "title": "Detoxification and Removal of Hexavalent Chromium in Aquatic Systems: Applications of Bioremediation", "date": "2022-12-31", "file": "njb-243.pdf"}, "hash": "34a7f57814853c62dde34d63233fee09cda9d0ae31fb4acd6a0fb834ed89cf56", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3624c09c-3d6f-477a-a55a-0722ba4bdd1c", "node_type": "1", "metadata": {}, "hash": "ed92114791d10ebd27bb347a5dadcb685a61a4244fd8922b547645b8bd1eccb4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 33-36   ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    33        Nepjol.info/index.php/njb \n\n \n\nHormonal Effect on Mandarin Orange (Citrus reticulata Blanco) \nMicro-propagation \n\nResham Babu Amgai1*, Hari Kumar Prasai1, Yama Raj Pandey1 \n1Regional Agriculture Research Station-Lumle, Nepal Agricultural Research Council, Kaski, Nepal. \n\nAbstract \nTissue culture is the best option to produce disease free seedling of the fruit crop rapidly.  Micro-propagation \n\nand use of the in-vitro grafting (micro-grafting) is very helpful for production of virus free planting materials in \n\nmandarin. Different levels of the in-vitro hormone affect the success of callusing, shooting and plant \n\nregeneration in mandarin. Shoot bud, flower bud and in-vitro seedling epicotyl was used as explants to study \n\nthe hormonal effect on mandarin micro-propagation. Similarly, 10 levels of BAP and IAA combination on MS \n\nmedia for mandarin tissue culture were used. Observation was done for 100 test tubes per treatment \n\ncombination after 4, 8 and 12 weeks of culture. Data was arc sine transformed for analysis. Shooting from \n\nexplants was significantly higher (71.72%) on medium level of the BAP (0.5 mg/L) and IAA (0.2 mg/L) using \n\nin-vitro seedling stem as explant, however, it was 27.91% for stem bud as explant. Stem bud showed higher \n\nlevel of callusing (6.15%, p<0.001) in mandarin orange. However, flower bud didn\u2019t develop shoot in mandarin \n\ntissue culture. Increment of the in-vitro regeneration of the shooting and callusing was observed by the \n\nincrement of the in-vitro incubation duration in mandarin orange tissue culture. \n\nKeywords: mandarin orange, Citrus reticulata, in-vitro shooting, hormone, callusing. \n\n*Corresponding Author \n\nEmail: reshamamgain@yahoo.com \n\nIntroduction \nCitrus is the major fruit in Nepal that shares 26.84% \n\nof the total fruit growing area in the country. \n\nMandarin orange (Citrus reticulata Balnco) is \n\npredominant occupying 26495 ha i.e. 71.65% of total \n\ncitrus growing area in Nepal [1]. Nepalese \n\nMandarin industry is facing a lot of citrus decline \n\nrelated problem that can be overcomed through use \n\nof disease free planting materials. Seed propagation \n\nis 85-90% in Mandarin; however, grafting is gaining \n\npopularity [2].  \n\nCertain pathogens are difficult to be eliminated \n\nfrom mother plant like citrus exocortis and \n\nstubborn, which might be eliminated by a process of \n\nshoot tip grafting in-vitro. Indian Citrus Ringspot \n\nVirus (ICRSV) can also be eliminated by using the \n\nshoot tip (0.7mm) of sprouts of cultured nodal \n\nsegment of Kinnow through in-vitro shoot tip \n\ngrafting (micro-grafting) [3]. \n\nSimilarly, plant obtained by micro-grafting do not \n\nhave the same problems as nucellar plants such as \n\nreversion to juvenile state, excessive thorniness, \n\nvigorous and upright habit of growth, slowness to \n\nfruiting, alternate bearing in early years and \n\nphysical differences in fruit characteristics [4]. The \n\nscion taken from in-vitro cultured buds produced \n\nhealthy plants than scions from whole trees [5]. \n\nSimilarly, it reduced the dependency for the \n\nseasonal flush of the mandarin for scion during \n\nmicro-grafting. \n\nYoutsey [6] reported that rate of successful grafts (as \n\nhigh as 50%) often influenced by the condition of \n\nthe plant material and time of year.", "start_char_idx": 47, "end_char_idx": 3522, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3624c09c-3d6f-477a-a55a-0722ba4bdd1c": {"__data__": {"id_": "3624c09c-3d6f-477a-a55a-0722ba4bdd1c", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3aab2d2e-dc87-4049-8ae9-6140574347ff", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "c685d09105fd01b671b2126e3cc56f01b700e14b358a3d529c8f7e4e52f69a29", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9d466d71-7a26-403f-9f3d-f7f8336cdd79", "node_type": "1", "metadata": {}, "hash": "a28add05bbdf5eb10ae790993168070e07ee69c07dbbcabb67f7c18dc2ed88eb", "class_name": "RelatedNodeInfo"}}, "text": "Therefore, the \n\ncontinue supply of the in-vitro scion material will \n\nenhance the micro-grafting efficiency of citrus for all \n\nyear around. \n\nSimilarly, in-vitro callusing, regeneration and \n\nconservation of mandarin is very important. Martin \n\net al. [7] also established a protocol by using nodal \n\nstem segments of sweet orange for in-vitro \n\nconservation for two years and its recovery that was \n\nsuccessfully applied on mandarin. Therefore, this \n\nstudy was conducted to identify the suitable in-vitro \n\nculture media for mandarin in-vitro shooting and \n\ncallusing. \n\nMaterials and Methods \nVariety and Ex-plant Selection \nMandarin orange variety \u2018Manakamana Local\u2019 was \n\nused in this study. Immature shoot with single bud, \n\nmature flower bud and in-vitro seedling epicotyl (2 \n\ncm long) was used as ex-plant. In-vitro seedling of 8 \n\nweek was used to harvest epicotyl. Explants were \n\ncollected during citrus flowering season. Immature \n\nshoot with single bud was used as explant to study \n\nthe shooting from callus. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 33-36  Amgai et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    34        Nepjol.info/index.php/njb \n\n \n\nIn-vitro culture media \nDifferent hormonal composition was applied for the \n\nMurasige and Skoog (MS) basal media [8] (as in \n\nTable 1) \n\nEx-plant culture and incubation \nThe explant was sterilized with 4% sodium \n\nhypochlorite solution for 3 minutes. It was washed \n\nwith distilled water for 3 times and cultured on test \n\ntube with 20 ml specified media (Table 1). The test \n\ntubes were incubated at 22\u00b12oC with 16:8 hours \n\nlight: dark. \n\n 4. Observation and data analysis \n\nObservation was taken after 4 weeks, 8 weeks and \n\n12 weeks after incubation. Three replications were \n\nused for each treatment. Observation for each \n\ntreatment consists of 100 test tubes. Each test tube \n\nis observed for callusing and shooting directly \n\nfrom ex-plant or from callus. Data was angular \n\ntransformed for variance analysis. \n\nResults and discussion \nShooting \n\n \nFlower bud don\u2019t show shooting directly, however, \n\nit showed highest callusing rate (81.77%). Similarly, \n\nhighest shooting directly from explants (Figure 1) \n\n(31.195%) was observed from in-vitro epicotyle \n\n(Table 2). Mendes et al. [9] also observed that \n\nepicotyl explants from 35-day-old seedlings \n\nproduced significantly more shoots per explants in \n\nsweet orange variety \u2018Para\u2019.  \n\nNo shooting from callus was observed on MS basal \n\nmedia+BAP 4 mg/l with/without IAA.  Similarly, \n\nhighest shooting from callus (24%) was observed on \n\nMS basal media+0.5 mg/l \n\nBAP. Highest callusing was \n\nobserved on MS basal \n\nmedia+ BAP 2mg/l \n\nwith/without IAA 0.2mg/l \n\n(Table 3). MS basal \n\nmedia+1mg/l BAP gave \n\nhighest shooting (49.81%)  \n\ndirectly from explant. \n\nShooting directly from explant and from callus was \n\nfound increased by increment of the incubation \n\nperiod (Figure 2). Similarly, significant interaction \n\nbetween explant and media, explant and incubation \n\nperiod was observed for callusing from explant and \n\nshooting directly from explant. No interaction was \n\nobserved among explant, media and incubation  \n\nCallusing \n\nFlower bud gave highest callusing for all media \n\ncomposition (Figure 3). It was highest on MS basal \n\nmedia+0.5 mg/l BAP with or without 0.2 mg/l IAA \n\nand MS basal media+2 mg/l BAP from flower bud \n\nexplant. No callusing was observed under hormonal \n\nlevel 1 mg/l BAP in in-vitro shoot tip.", "start_char_idx": 3523, "end_char_idx": 7019, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9d466d71-7a26-403f-9f3d-f7f8336cdd79": {"__data__": {"id_": "9d466d71-7a26-403f-9f3d-f7f8336cdd79", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3624c09c-3d6f-477a-a55a-0722ba4bdd1c", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "4571a1013eebf808aa98badf5748f8d04460859e09f814829f1cb75d45f0b307", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9a21dfe1-2101-41d7-a0e1-05534f9eb32d", "node_type": "1", "metadata": {}, "hash": "70d2c9d94e643dc8ecf3918de6e726eeb35c19a1f7d3622b6bcc1b9d9b5325ce", "class_name": "RelatedNodeInfo"}}, "text": "Highest \n\ncallusing from immature shoot with single bud was \n\nobserved on MS basal media+1 mg/l BAP+0.2 mg/l \n\nIAA (Table 5). \n\nTable 1: Different level of growth hormone used in the in-vitro culture of mandarin \n[T denotes media composition with pH 5.70] \n\nHormone \n\nConcentration (mg/l) \n\nT1 T2 T3 T4 T5 T6 T7 T8 T9 T10 \n\n6-Benzylaminopurine \n(BAP) \n\n0 0 0.5 0.5 1.0 1.0 2.0 2.0 4.0 4.0 \n\nIndole-3-acetic acid \n(IAA) \n\n0 0.2 0 0.2 0 0.2 0 0.2 0 0.2 \n\nTable 2: Mean Percentage of the callusing and shooting directly \nfrom explants observed according to different plant parts used as \nexplants. [Value in parentheses is angular transformation; letters \nafter mean is the comparisons using Duncan's Multiple Range Test \n(DMRT) for mean differences] \n\nExplant \nCallusing from \n\nexplant \nShooting directly \nfrom explant \n\nImmature shoot with \nsingle bud \n\n21.043 (21.175)b 19.69 (19.002)b \n\nFlower bud 81.77 (71.872)a No Shooting \n\nIn-vitro shoot tip \n (2 cm long) \n\n21.216 (20.347)b 31.195 (33.431)a \n\nProbability <0.001 <0.001 \n\nLSD at 5% 4.829 4.494 \n\nCV% 3.522 5.807 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 33-36  Amgai et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    35        Nepjol.info/index.php/njb \n\n \n\n \n\nFigure 4. Effect of different level of hormones on \n\nmandarin orange immature-shoot with bud explant \n\n \n\n \n\nShooting directly from ex-plant \nFlower bud didn\u2019t show any direct shooting nor \n\ndoes it produce any shoot after callusing. MS basal \n\nmedia+4mg/l BAP+0.2mg/l IAA don\u2019t produce any \n\nshooting directly from immature shoot with single \n\nbud (Table 6). It didn\u2019t produce shoot from callus \n\ntoo (Table 3). Highest shooting was observed on MS \n\nbasal media+1 mg/l BAP from in-vitro epicotyle \n\n(71.722%) and from immature shoot with single bud \n\n(27.906%) (Figure 1 and Figure 4). Similarly, \n\nimmature shoot with single bud produced highest \n\nshooting in MS basal media+0.20mg/l IAA (Table \n\n6).  However, Mendes et al. [9] observed that the \n\npercentage of shoots that produced roots in sweet \n\norange variety \u2018Para\u2019 was significantly higher in \n\nmedia with NAA and IBA than with NAA alone. \n\nConclusion \nSince the requirement of BAP for shoot \n\ndevelopment was genotype specific [9], for \n\nmandarin orange cv. \u2018Manakamana Local\u2019 to \n\nproduce in-vitro scion for micro-grafting the MS \n\nbasal media+0.20mg/l IAA in immature shoot is \n\n \n\nTable 3: Mean Percentage of the callusing, shooting directly from explants and shooting from callus observed according to different \nmedia composition. [Value in parentheses is angular transformation; letters after mean is the comparisons using Duncan's Multiple Range \nTest (DMRT) for mean differences.", "start_char_idx": 7020, "end_char_idx": 9717, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9a21dfe1-2101-41d7-a0e1-05534f9eb32d": {"__data__": {"id_": "9a21dfe1-2101-41d7-a0e1-05534f9eb32d", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9d466d71-7a26-403f-9f3d-f7f8336cdd79", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "4d3af0917ff1945c87688c5e656339aed8f383e86df50ac714c2c11c23b86b18", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "08ac1fb9-4afc-4573-a307-a0b77000757e", "node_type": "1", "metadata": {}, "hash": "3f9b29d30067f8bd4fad063420f726bf3ad330aba593723f7a2d80323de35d4a", "class_name": "RelatedNodeInfo"}}, "text": "MS-Murashige and Skoog [8], BAP-Benzayl amino phosphate, IAA-Indole acetic acid] \n\nMedia composition \nCallusing from explant Shooting from callus Shooting directly from explant \n\nMedia BAP, mg/L \nIAA, \nmg/L \n\nMS 0.00 0.00 33.02(30.244)c 6.1(10.310)a 20.574(22.875)bc \n\nMS 0.00 0.20 33.844(30.398)c 12.4(17.720)ab 27.355(28.907)b \n\nMS 0.50 0.00 39.441(35.993)bc 24(25.360)b 21.726(24.37)bc \n\nMS 0.50 0.20 41.316(37.254)abc 8.4(12.260)a 31.18731.838b \n\nMS 1.00 0.00 40.879(36.210)bc 8.3(12.130)a 49.814(44.503)a \n\nMS 1.00 0.20 49.201(46.483)a 11.9(17.310)a 36.316(33.575)b \n\nMS 2.00 0.00 50.396(45.978)abc 8.3(12.000)a 23.46(24.727)bc \n\nMS 2.00 0.20 42.652(38.895)abc 8.3(12.200)a 20.392(22.105)bc \n\nMS 4.00 0.00 39.136(36.189)bc No shooting 11.666(14.584)c \n\nMS 4.00 0.20 43.545(40.336)abc No shooting 11.938(14.678)c \n\nProbability 0.0031 0.018 <0.001 \n\nLSD at 5% 8.817 7.964 10.05 \n\nCV% 3.522 3.540 5.807 \n\nTable 4: F-Probability value for interaction between explants, media and incubation period. \n\nInteraction \nCallusing from \nexplant \n\nShooting from callus \nShooting directly from \nexplant \n\nExplant X Media <0.001 Only immature shoot used as explant 0.0379 \n\nExplant X incubation period <0.001 Only immature shoot used as explant <0.001 \n\nMedia X incubation period 0.240 0.255 >0.650 \n\nExplant X Media X incubation \nperiod \n\n>0.650 Only immature shoot used as explant >0.650 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 33-36  Amgai et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    36        Nepjol.info/index.php/njb \n\n \n\nbest for high shooting. Similarly, the MS basal \n\nmedia+0.5 mg/l BAP is suitable for shooting from \n\ncallus (24%) on this cultivar. \n\nReferences \n1. MOAD: Statistical Information on Nepalese \n\nAgriculture 2012/13. Government of Nepal. Ministry \n\nof Agriculture Development. Agriculture Business \n\nPromotion and Statistical Division, Statistics Section, \n\nSinghadurbar, Kathmandu, Nepal, 2013.  \n\n2. FAO: Training manual for combating citrus \n\ndecline problem in Nepal. Directorate of \n\nAgriculture, Ministry of Agriculture and \n\nCooperatives, Government of Nepal and Food and \n\nAgriculture Organization of United Nations, 2011, \n\nTCP/NEP/3302:(D). \n\n3. Sharma S, Singh B, Rani G, Zaidi AA, Hallan V, \n\nNagpal A, Virk GS: Production of Indian citrus \n\nringspot virus free plants of Kinnow employing \n\nchemotherapy coupled with shoot tip grafting. J  \n\nCen Eur Agri. 2007, 8(1): 1-8. \n\n4.", "start_char_idx": 9718, "end_char_idx": 12162, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "08ac1fb9-4afc-4573-a307-a0b77000757e": {"__data__": {"id_": "08ac1fb9-4afc-4573-a307-a0b77000757e", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9a21dfe1-2101-41d7-a0e1-05534f9eb32d", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "971f69fc52ea02dacdcf4d2a21782ecc27fbb0af695c4d75374d48c78e69e6a0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7298332b-493b-4b62-89b1-ddedfe640ba2", "node_type": "1", "metadata": {}, "hash": "ea24515e7d0a50e9327827e8a62321b1c27bf0fe2a61fd7d7e80b8cf8277a346", "class_name": "RelatedNodeInfo"}}, "text": "2007, 8(1): 1-8. \n\n4. Nauer EM, Roistacher CN, Carson TL, Murashigue \n\nT: In-vitro shoot tip grafting to eliminate citrus  \n\n \n\n \n\n \n\n \n\n \n\n \n\nviruses and virus-like pathogens produces \n\nuniform budliness. Hortscience 1983, 18: 308-309. \n\n5. Navarro L: Shoot-tip grafting in-vitro (STG) and \n\nits application: A review. Proceeding of the \n\nInternational Society of Citriculture 1981, 1: 452-456. \n\n6. Youtsey CO: A method for virus-free propagation \n\nof citrus: shoot tip grafting. Citrus Industry 1978, \n\n59:39-47. \n\n7. Martin ML, Duran-Vila N: Conservation of citrus \n\ngermplasm in-vitro. J Amer Soc Hort Sci. 1991, 116 \n\n(4): 740-746. \n\n8. Murasige T, Skoog F: A revised medium for rapid \n\ngrowth and bioassays with tobacco tissue cultures. \n\nPhysiol. Plant. 1962, 15: 473-497. \n\n9. Mendes AFS, Cidade LC, Manzoli GN, Otoni WC, \n\nFilho WSS, Costa MGC: Tissue culture parameters \n\nin sweet orange cultivars. Pesq. Agropec. Bras, \n\nBrasilia 2008, 43(8): 1093-1096. \n\nTable 5: Mean Percentage of the callusing from explants observed with respect to different media composition and \nexplants used (LSD = 15.27 at 5%). [Value in parentheses is angular transformation; letters after mean is the comparisons \nusing Duncan's Multiple Range Test (DMRT) for mean differences. MS-Murashige and Skoog [8], BAP-Benzayl amino \nphosphate, IAA-Indole acetic acid] \n\nMedia composition \nImmature shoot with single bud Flower bud \n\nIn-vitro shoot tip (2 \ncm long) Media BAP mg/L IAA mg/L \n\nMS 0.00 0.00 33.434(30.385)ghi 65.622(60.061)bcd 0.000(0.287)l \n\nMS 0.00 0.20 20.677(21.556)hijk 80.853(69.352)abc 0.000(0.287)l \n\nMS 0.50 0.00 25.618(26.992)ghij 92.704(80.700)a 0.000(0.287)l \n\nMS 0.50 0.20 30.737(30.366)ghi 93.208(81.108)a 0.000(0.287)l \n\nMS 1.00 0.00 31.998(31.180)ghi 90.635(77.164)ab 0.000(0.287)l \n\nMS 1.00 0.20 34.999(33.592)fgh 88.644(77.054)ab 23.958(28.802)ghij \n\nMS 2.00 0.00 13.196(15.258)ijkl 90.703(79.234)a 47.289(43.443)efg \n\nMS 2.00 0.20 8.097(10.009)kl 60.832(56.382)cde 59.028(50.294)def \n\nMS 4.00 0.00 0.000(0.287)l 77.197(68.928)abc 40.208(39.354)fg \n\nMS 4.00 0.20 11.668(12.125)jkl 77.300(68.737)abc 41.667(40.146)efg \n \n\nTable 6: Mean Percentage of the shooting directly from explants observed with respect to different media composition and \nexplants used (LSD = 14.21 at 5%). [Value in parentheses is angular transformation; letters after mean is the comparisons \nusing Duncan's Multiple Range Test (DMRT) for mean differences.", "start_char_idx": 12141, "end_char_idx": 14584, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7298332b-493b-4b62-89b1-ddedfe640ba2": {"__data__": {"id_": "7298332b-493b-4b62-89b1-ddedfe640ba2", "embedding": null, "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-25", "node_type": "4", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "7f1ad188f7685a936c05f62d9975e3faa264c72287f6bb3788514e507e5a1db7", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "08ac1fb9-4afc-4573-a307-a0b77000757e", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "1b699069c7a68a18fc37f1d34d8270173d68ca85067ced2daa39bb4d046e8c0b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6261b087-346d-4879-9f3d-88b2e9088f9a", "node_type": "1", "metadata": {}, "hash": "b2eb1106bd68ddbd81c65a1c3dab8858d37ce320ca171910d4d89f922585e9a4", "class_name": "RelatedNodeInfo"}}, "text": "MS-Murashige and Skoog [8], BAP-Benzayl amino \nphosphate, IAA-Indole acetic acid] \n\nMedia composition \nImmature shoot with single bud In-vitro shoot tip (2 cm long) \n\nMedia \nBAP, \nmg/L \n\nIAA, \nmg/L \n\nMS 0.00 0.00 12.938(13.974)efg 28.209(31.775)bcd \n\nMS 0.00 0.20 27.117(26.317)bcdef 27.593(31.497)bcd \nMS 0.50 0.00 26.236(24.501)cdef 17.216(24.240)cdef \nMS 0.50 0.20 26.930(27.202)bcde 35.444(36.475)bc \nMS 1.00 0.00 27.906(28.415)bcde 71.722(60.590)a \nMS 1.00 0.20 26.314(24.306)cdef 46.319(42.844)b \nMS 2.00 0.00 17.356(16.732)def 29.565(32.721)bcd \nMS 2.00 0.20 22.224(18.764)def 18.561(25.446)cdef \nMS 4.00 0.00 9.878(9.519)fg 13.453(19.648)cdef \nMS 4.00 0.20 0.000(0.287)g 23.874(29.069)bcde", "start_char_idx": 14585, "end_char_idx": 15282, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6261b087-346d-4879-9f3d-88b2e9088f9a": {"__data__": {"id_": "6261b087-346d-4879-9f3d-88b2e9088f9a", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7298332b-493b-4b62-89b1-ddedfe640ba2", "node_type": "1", "metadata": {"identifier": "njb-25", "author": "Amgai, Resham Babu; Prasai, Hari Kumar; Pandey, Yama Raj", "title": "Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation", "date": "2016-12-31", "file": "njb-25.pdf"}, "hash": "3bc471dc0ca1ece10e8d29d5c828650e35c1a666589431e919152f5c88905608", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e9961c62-fa60-4cf7-8d38-dd75f887f3a5", "node_type": "1", "metadata": {}, "hash": "5c8665fa1c1d7699a60996ec7e634eabf020fcaaa732f241f0f8bdc9f12fd8a8", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:   26-32 ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    26        Nepjol.info/index.php/njb \n\nEvaluation of the XpertMTB/RIF for the Diagnosis of \nPulmonary Tuberculosis Among the Patients Attending DOTS \n\nCenter Parsa District of Nepal \nRoshan Kurmi1*, Ramanuj Rauniyar1, Krishna Das Manandhar2, Birendra Prasad Gupta2 \n\n1Bhawani Hospital, Birgunj, Parsa, Nepal. \n2Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. \n\nAbstract \nTuberculosis diagnosis and monitoring rely in Sputum microscopy of National Tuberculosis Programme, Nepal \n\nbecause of its low cost and easier to perform. Direct sputum microscopy is popular worldwide. Currently, there \n\nare 533 microscopy centres catering for sputum microscopy examination throughout the country. Most of the \n\nmicroscopy centres are established within government jurisdiction and remaining are established as non-\n\ngovernmental organization as well as private sectors.  \n\nA cross-sectional study was conducted from July 2013 to January 2015. A total of 2091 patients were enrolled in \n\nthe study who were attending the DOTS Centre in Parsa District of Public Health Office, Nepal. Smears stained \n\nwith ZN stain methods were examined microscopically followed by the GeneXpert MTB/RIF assay.  \n\nOut of 2091 suspected pulmonary TB patients enrolled for sputum microscopy and GeneXpert MTB/RIF for the \n\nconfirmation of TB, the 1301(62.21%) were male and 790 (37.78%) were female. The maximum TB cases were \n\nfrom Parsa district (555, 26.5%). The comparative study of different diagnostic tools reveals the sensitivity of \n\nMTB/RIF was 95.50% (91.87, 97.82) and significantly higher than smear microscopy performed on the same fluid, \n\nwhich had a sensitivity of 61.97% (55.41, 68.21). Five of 127 smear-negative cases had MTB/RIF-positive un-\n\ncentrifuged sputum, resulting in a specificity of 81.23% (75.95, 85.78), which was similar to smear microscopy \n\n98.29 % (97.34, 98.97; p=0.121). The positive predictive value (PPV) and negative predictive value (NPV) of \n\nMTB/RIF were 96.85% (93.61, 98.72) and 94.95 % (93.52, 96.14), respectively. HIV co-infection did not impact \n\nsensitivity, specificity or liquid culture time to positivity (TTP). When MTB/RIF accuracy was evaluated using \n\ncomposite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those \n\nobtained in the primary analysis using either definite TB versus possible and non-TB combined; definite and \n\npossible TB combined versus non-TB.  \n\nKeywords: Mycobacterium tuberculosis, Prevalence, GeneXpert, Nepal \n\n*Corresponding Authors  \n\nE-mail: dr_roshankurmi@hotmail.com   \n\nIntroduction \nTuberculosis (TB) is a leading cause of death \n\nworldwide. In the early 1990s, the government of \n\nNepal brought up a program to control TB at \n\nnational scale called DOTS (Directly Observed \n\nTreatment Short Course) [1]. DOTS program was \n\ninitiated in April 2001 with a goal to diagnose and \n\ncure TB patients which has treated more than 20  \n\nthousand patients till date [2]. Until December of \n\n2014 there are 554 microscopy centres and 22 \n\nGenXpert location in the country which specializes \n\nin diagnosis of TB [3].", "start_char_idx": 47, "end_char_idx": 3380, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e9961c62-fa60-4cf7-8d38-dd75f887f3a5": {"__data__": {"id_": "e9961c62-fa60-4cf7-8d38-dd75f887f3a5", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6261b087-346d-4879-9f3d-88b2e9088f9a", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "a0a4ea7b467855f1d82af5b973153e4da43aba2702145e0d71907a70b4da45d0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bcdf9372-7f9c-4db0-8e6e-cd6890afad6c", "node_type": "1", "metadata": {}, "hash": "847b97d18997be6a878c12e89edd5a182fd5811093030f56d31f79f68c661bf8", "class_name": "RelatedNodeInfo"}}, "text": "The expansion of DOTS \n\nprogram has proven its efficacy in reducing the \n\nmortality and morbidity in Nepal, however, \n\ndespite of available diagnosis and treatment \n\nregimes, approximately 3000-5000 people are still \n\ndying per year  of TB [4]. In a DOTS centre in \n\nKathmandu, prevalence of Multi Drug Resistant \n\n(MDR) case of Tuberculosis was found to be 3.6% \n\n[5], which could have been mitigated by early \n\ndiagnosis and rapid identification of anti-TB agents \n\nresistance. Recent data from East Nepal suggests \n\nthat 3.3% of all patients, who were smear \n\nmicroscopy negative, have RMP resistant TB [6]. \n\nDespite of DOTS and available diagnosis, \n\ncomplications due to MDR are emerging and also \n\nthere hasn\u2019t been overall increment in number of \n\npeople on anti tuberculosis treatment [6]. Sputum \n\nsmear light microscopy, patients symptoms \n\ncombined with CXR results is the preferred \n\nalgorithm for TB diagnosis in Nepal.  Smear test is \n\nneither relevant nor sensitive enough for the \n\ndiagnosis of TB alone and needs additional \n\ndiagnosis as well clinical suspecting to decide \n\neither patients should be enrolled on antibiotic or \n\nnot [7].  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    27        Nepjol.info/index.php/njb \n\nThis study aims to clarify if current algorithm of TB \n\ndiagnosis is flexible enough to trace the TB rates at \n\nGeneXpert centre in Parsa district of Public Health \n\nOffice, Nepal. Our aims were to determine \n\nepidemiological characteristics, HIV status of \n\npatients with tuberculosis and comparative study \n\nof different test routinely used for diagnosis of TB \n\nat a large outpatient clinic in Parsa of Nepal.  \n\nMaterials and Methods \nStudy sites and population \nStudy was conducted in Parsa district in the \n\nNarayani Zone of southern Nepal which lies \n\n283 km (176 mi) south of the capital Kathmandu, \n\n3 km north of the border of the Indian state of Bihar \n\nwith population 315,011 in 2011. The district is \n\ndivided into 86 Village Development Committees \n\nand one municipality which is one of the relatively \n\nwell developed districts in the country located on \n\nthe flat Terai lowland. The public health sector has \n\n49 facilities including 1 zonal hospital, 1 public \n\nhealth office, 3 primary health centres, 9 health \n\nposts and 35 sub-health posts, additionally there \n\nare 6 private sector facilities thus resulting in a total \n\nof 55 facilities providing DOTS services in Parsa \n\ndistrict. The reported notification rate of new \n\npulmonary positive tuberculosis in Parsa district \n\nwas 69 per 100,000 populations in 2014 [3].  \n\nPatient classification and diagnosis \n\nalgorithm  \nPatients with suspected TB (\u226518 ye of age) were \n\nenrolled between April 2014 and May 2015. WHO \n\nalgorithm was basis for the classification of \n\nsuspects of TB. Based on WHO algorithm, two \n\nsubgroups were formed to classify TB suspects; 1) \n\npatients with cough for \u2265 2 weeks and 2) patients \n\nwithout cough for \u2265 2 weeks but having symptoms \n\nlike weight loss accompanied with night fever \n\nT>37.5\u00b0C, pleural effusion or pericarditis induced \n\nbreathlessness, chronic headache, swelled armpit \n\n(>2cm) or abnormal chest radiogram, thus referred \n\nas, \u201cwith cough or without cough\u2019\u2019. Patients with \n\nWHO defined danger sign with respiratory rate  \n\n>30/minute, fever >39\u00b0C, pulse rate >120/minute \n\nand unambulatory patients were excluded from the \n\nstudy.  \n\nSmear positive were not enrolled for GenXpert thus \n\nwere directly enrolled for anti TB regime. \n\nRemaining patients with smear negative were \n\nenrolled in GenXpert and further confirmation was \n\ndone by liquid culture for MTB positive and RIF \n\nresistant samples. Those cultures were further \n\nscreened for MDR against first line and second line \n\ndrugs.", "start_char_idx": 3381, "end_char_idx": 7233, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bcdf9372-7f9c-4db0-8e6e-cd6890afad6c": {"__data__": {"id_": "bcdf9372-7f9c-4db0-8e6e-cd6890afad6c", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e9961c62-fa60-4cf7-8d38-dd75f887f3a5", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "ea259b4530f56f17035cfc3a528a5b30e7d6eb9e0edc20de79e7598b237b14e9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "757b50ab-a4a7-48ff-a8b8-5a041d8f2f33", "node_type": "1", "metadata": {}, "hash": "b6363fa9f7cf6ec5e57108cda914a4dbd591c469ffdfc58194b50eec23f00d91", "class_name": "RelatedNodeInfo"}}, "text": "Patients with sputum negative for TB were \n\nclassified as possible TB (if they are on anti-TB \n\ntreatment program) or as non-TB (if they are not on \n\nanti-TB treatment program). Prospective patients \n\n(\u226520 years of age) with suspected pulmonary TB \n\n(n=2091) were enrolled in the study who were \n\nattending the DOTS Centre in Parsa District of \n\nPublic Health Office, Nepal from Jul 2013 to Jan \n\n2015. Patients had at least one symptom of TB \n\nincluding chest radiograph with infiltrates and \n\nsmear microscopy negative results in duplicate.   \n\nSpecimen collection and laboratory \n\nprocedures \nSputum specimens were processed using \n\nstandardized protocols. The length of time between \n\nsample collection and results being issued to the \n\nclinic was also recorded. Smears stained with ZN \n\nstain methods were examined microscopically \n\nfollowing the standard protocol. At least 200 fields \n\nwere evaluated before reporting  negative. Bacillary \n\ndensity was graded as scanty, 1+, 2+, and 3+, and \n\nall such smears were defined as \u2018\u2018smear-positive\u2019. \n\nFollowing decontamination with N-acetyl-L-\n\ncysteine and sodium hydroxide, centrifuged \n\nsputum deposits underwent microscopy, and \n\nfollowing re-suspension in phosphate buffer, equal \n\nvolumes were tested by the GeneXpert MTB/RIF \n\nassay. The results of all tests were read by \n\ntechnologists blinded to the outcomes of the other \n\nassays. Sputum pellets were also tested by trained \n\ntechnologists using the GeneXpert MTB/RIF assay.  \n\n GeneXpert MTB/RIF and patient \n\nmanagement \nThe collected sputum sample was aliquoted in two \n\nequal volumes; first half for immediate primary \n\nclinical works and other for reference for future \n\nworks at -20\u00b0C. From the first aliquot, the specimen \n\nwas used for smear tests followed by GeneXpert \n\nMTB/RIF assay. The diagnostic assays were \n\nfollowed by a single specialized clinical research \n\nassociate blind to the patient's diagnosis in \n\nhttp://en.wikipedia.org/wiki/Narayani_Zone\nhttp://en.wikipedia.org/wiki/Nepal\nhttp://en.wikipedia.org/wiki/Kathmandu\nhttp://en.wikipedia.org/wiki/India\nhttp://en.wikipedia.org/wiki/States_and_territories_of_India\nhttp://en.wikipedia.org/wiki/Bihar", "start_char_idx": 7237, "end_char_idx": 9422, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "757b50ab-a4a7-48ff-a8b8-5a041d8f2f33": {"__data__": {"id_": "757b50ab-a4a7-48ff-a8b8-5a041d8f2f33", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bcdf9372-7f9c-4db0-8e6e-cd6890afad6c", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "bfd7c158bda82dda50293d4fcbb57b5c1b9f5b98d44c4a81b00f63904f3e2d03", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b41af851-90db-4d5a-bb97-728eda59f2ce", "node_type": "1", "metadata": {}, "hash": "0cf5008f3f4290983993fb512e475dc32729a52d969d50d28727aedbe66ae597", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    28        Nepjol.info/index.php/njb \n\nreference standard and index test, throughout the \n\nstudy period. \n\nStatistical analysis \nCollected data were analyzed and interpreted \n\nstatistically using graphPad prism version 6.0 and \n\nSPSS 17.0. All the values are expressed as mean\u00b1SD \n\nand are analyzed using Student\u2019s t test which is \n\nparametric as well Mann-Whitney test wherever \n\napplicable. P value (P<0.05), was considered \n\nsignificant unless stated otherwise.  \n\nResults \nStudy population and TB diagnoses \n\nFigure 1: District wise number of Tuberculosis Patients \n\nvisited at GeneXpert Centre in Parsa  \n\nThe suspected pulmonary TB patients (n= 2091) \n\nwere enrolled for sputum microscopy and \n\nGeneXpert MTB/RIF for the confirmation of TB. \n\nAmong them, 1301(62.21%) were male and 790 \n\n(37.78%) were female. All the patients were \n\nreferred to this centre by local doctors and \n\nphysicians after having tuberculosis symptoms and \n\nabnormal chest radiograph for sputum microscopy \n\nand GeneXpert MTB/RIF for the confirmation of \n\nMakwanpur, Mahottari, Panchthar, Saptari, Siraha, \n\nMorang, Sindhuli, Rasuwa and Chitwan (Figure 1). \n\nSince the study conducted at Parsa district, the \n\nactual number of case presented here does not \n\nhighlight the prevalence of TB in the respective \n\ndistrict.  \n\n As Bara is the adjacent district to Parsa, the \n\nnumber of patients visited is more than other \n\ndistrict. The prevalence of TB in Parsa district is 3 \n\ntimes higher than the prevalence in the Bara \n\ndistrict. \n\n TB. The clinical symptoms showed most of the \n\npatients have fever, cough, and weight loss and \n\nnight sweat (Table 4). \n\nThe maximum TB cases were from Parsa district \n\n(555, 26.5%) followed by Bara, Rautahat, Sarlahi,  \n\nDiagnostic accuracy of different method \n\nand MTB/RIF performed on un-\n\ncentrifuged sputum \nDirect sputum microscopy is popular worldwide. \n\nIn Nepal, the Tuberculosis diagnosis and \n\nmonitoring rely in Sputum microscopy because of \n\nits low cost and easier to perform. At present there \n\nare 533 microscopy centres catering the sputum \n\nmicroscopy examination throughout the country. \n\nThe region wise distribution is given in Table 1. \n\nMost of the microscopy centres are established in \n\ngovernment setting and few are established in non-\n\ngovernmental organization and private sectors.  \n\nNTC-National  \n\n \nFigure 2: MDR Pattern in TB Patients  \n\nFigure 3: Gene Xpert MTB RIF Pattern in TB patient \n\n0\n\n200\n\n400\n\n600\n\n800\n\n1000\n\n1200\n\n1400\n1357\n\n476\n\n11890\n17 12 6 4 2 1 1 1 1 5\n\n0\n500\n\n1000\n1500\n2000\n2500 S-ve S+ve Not done Total\n\nTable 4: Clinical Feature of Tuberculosis \n\nClinical Feature Present Absent \n\nFever 1885 206 \n\nCough 1741 350 \n\nWeight Loss 1471 620 \n\nNight sweat 1655 436 \n\nLoss of appetite 1376 715 \n\nMalaise 1117 974 \n\nChest Pain 1744 347 \n\n0\n\n500\n\n1000\n\n1500\n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No.", "start_char_idx": 9425, "end_char_idx": 12450, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b41af851-90db-4d5a-bb97-728eda59f2ce": {"__data__": {"id_": "b41af851-90db-4d5a-bb97-728eda59f2ce", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "757b50ab-a4a7-48ff-a8b8-5a041d8f2f33", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "bdd5f5316937fd00ccdeaee5a3ab442588719695650ce45298e11bf3165d42e1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5e6128f6-a75f-47ee-ae35-9e4e901665bc", "node_type": "1", "metadata": {}, "hash": "ba4a096f8280626e6d70ce7d40d78bae1aaa65632b48f0732c8cd5f6a59b697c", "class_name": "RelatedNodeInfo"}}, "text": "2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    29        Nepjol.info/index.php/njb \n\nReference Laboratory and GENETUP are providing \n\nculture and in the beginning of 2012, a new \n\nmolecular diagnostic tool; Gene Xpert MTB/RIF \n\nmachines for rapid diagnosis of DR TB had been \n\nintroduced to nine health facilities (7 in Eastern \n\nRegion and 2 in Central Region) in collaboration \n\nwith NTP Nepal and International Organization for \n\nMigration (IOM) through TB Reach funding. Gene \n\nXpert MTB RIF Pattern in TB patient and MDR \n\nPattern in TB Patients are shown in Figure 2 and \n\nFigure 3 respectively. \n\nTable 1: Number of DOTS and Microscopy Centres by \nRegion \nDevelopment \nRegion/ \nCentres \n\nEDR CDR WD\nR \n\nMWD\nR \n\nFW\nDR \n\nTotal \n\nNo. of \ntreatment \ncentres \n\n237 366 239 199 143 1184 \n\nNo. of Sub-\ncentres \n\n709 1008 667 406 284 3074 \n\nDR TB \nTreatment \ncentres \n\n2 4 3 1 3 13 \n\nDR TB \nTreatment \nsub-centres \n\n11 37 15 5 3 71 \n\nMicroscopy \ncentres \n\n122 198 82 73 79 554 \n\n* Showed the number of DOTS and Microscopy centres in which \n\nnumber of sub-centres are more followed by number of \n\ntreatment centres and microscopy centres. There is few DR TB \n\ntreatment centres. (EDR: Eastern Development Region. CDR: \n\nCentral Development Region; WDR: Western Development \n\nRegion; MWDR: Mid Western Development Region; FWDR: Far \n\nWestern Development Region) \n\nTable 3:  Comparison of different test for diagnosis of \npulmonary Tuberculosis \n\nTest Result \n\nSmear Positive 585 \n\nSmear Negative 1506 \n\nGene Xpert Positive 788 \n\nGene Xpert Negative 1303 \n \n\nThe comparative study of different diagnostic tools \n\nis given in Table 3.  \n\nOf the 2091 patients enrolled in this study, the \n\nsensitivity of MTB/RIF was 95.50% (91.87, 97.82) \n\nand significantly higher than smear microscopy \n\nperformed on the same fluid, which had a \n\nsensitivity of 61.97% (55.41, 68.21, Table 5). Five of \n\n127 smear-negative cases had MTB/RIF-positive \n\nun-centrifuged sputum, resulting in a specificity of \n\n81.23% (75.95, 85.78), which was similar to smear \n\nmicroscopy 98.29 % (97.34, 98.97; p=0.121). The \n\npositive predictive value (PPV) and negative \n\npredictive value (NPV) of MTB/RIF were 96.85% \n\n(93.61, 98.72) and 94.95 % (93.52, 96.14), \n\nrespectively. HIV co-infection did not impact \n\nsensitivity, specificity (Table 6) or TTP. When \n\nMTB/RIF accuracy was evaluated using composite \n\nreference standard culture positivity from sputum, \n\nthe sensitivity and specificity were similar to those \n\nobtained in the primary analysis using either \n\ndefinite TB versus possible and non-TB combined; \n\ndefinite and possible TB combined versus non-TB.  \n\nDiscussion \nIn Nepal, the estimated incidence of TB is 163 per \n\n100,000 with a prevalence rate of 241 per 100,000 \n\npopulations. In 2014-15, the Nepal Tuberculosis \n\nProgramme (NTP) registered 17,788 sputum smear-\n\npositive cases and 8,367 sputum smear negative \n\ncases [6, 8]. A total of 2091 individuals from \n\nthirteen districts of Nepal were included in the \n\npresent study and the prevalence of smear positive \n\npulmonary tuberculosis was found to be 788 out of \n\n2091(37.06 %).", "start_char_idx": 12430, "end_char_idx": 15601, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5e6128f6-a75f-47ee-ae35-9e4e901665bc": {"__data__": {"id_": "5e6128f6-a75f-47ee-ae35-9e4e901665bc", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b41af851-90db-4d5a-bb97-728eda59f2ce", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "21f108743fa830ffd0e46a18fb939f05f5634576d1bf2fb02c37cf53c2bc9a88", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1402e86e-1a0f-4554-817c-12fbbccf3974", "node_type": "1", "metadata": {}, "hash": "483dc27ee1c6ef5d7131e8e3813ffe3b055c71daefb45044ffdae326cfc50101", "class_name": "RelatedNodeInfo"}}, "text": "We found the most of the patients \n\nwere passively participated in the study and almost \n\nall patients are visited Government hospital for the \n\ntreatment. Approximately 66.76% of the patient \n\nhad the cough duration for last 2 days. Although, \n\nonly 37.09 % of patients had AFB positive on \n\nsmear, out of which 97.8 % were new  \n\nTable 5: Pattern of sputum culture with active TB \n\nParameters  \n\nCohort \n\n(n=2091) \n\nSputum smear \n\npositive with \n\nactive TB \n\n(n=672) \n\nSputum smear \n\nnegative \n\ncompatible with \n\nactive TB \n\n(n=1419) \n\nMedian age in \n\nyears \n\n37.3 (26.1-\n\n48.3) \n\n33.8 (28.1-\n\n42.8) \n\n37.4 (29.9-47.1) \n\nMale 1301 464 725 \n\nFemale 790 208 694 \n\nHIV infected 65 22 43 \n\nMedian CD4 \n\ncounts (If HIV \n\npositive) \n\n98 78 20 \n\nPrevious TB \n\ntreatment \n89 9 80 \n\nSmokers (Past \n\nor current) \n\n201 118 83 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    30        Nepjol.info/index.php/njb \n\ncases and only 2.2. % is repeated cases; the Chest X \n\nray reveals 99.43% had abnormal finding in chest. \n\nThe GeneXpert result shows large number of \n\npatients diagnosed with Tuberculosis.  This might \n\nbe that the AFB is not present in initial few days \n\nbecause most of the patient has visited less than 5 \n\ndays of appearance of cough. The similar study \n\ncarried by Shagufta Iram et al[9], shows the \n\nGeneXpert MTB/RIF is a sensitive method for \n\nrapid diagnosis of Tuberculosis, especially in smear \n\nnegative cases and in PTB as compared to the \n\nconventional ZN staining. For countries endemic \n\nfor TB GeneXpert can serve as a sensitive and time \n\nsaving diagnostic modality for pulmonary TB. Our \n\nstudy is consistent with other studies which have \n\nsuggested the benefit of GeneXpert in smear-\n\nnegative patients in developing countries [10, \n\n11].The majority of these studies have been carried \n\nout in Africa where there is a substantially higher \n\nHIV burden than in South Asia. Three studies have \n\nbeen reported from low-HIV prevalence regions, \n\nincluding Peru and two hospitals (Hinduja \n\nHospital and Christian Medical College Hospital) \n\nin India [11, 12].   \n\nIt is not clear how many of the patients testing \n\npositive for MTB by smear, culture and Gene Xpert \n\nin this study would have been started on TB \n\ntreatment based on the judgement of the treating \n\nclinician if GeneXpert testing was not available. In \n\nthe previous year (2013-2014) at Parsa district, 2245 \n\npatients were treated for TB, approximately one-\n\nthird of those detected by GeneXpert during the \n\nstudy period. It is possible that the availability of \n\nGeneXpert increased the recognition of TB \n\nsymptoms, although we can claim that in this \n\nendemic setting TB awareness is consistently high \n\namong clinicians. Conversely, it is possible that a \n\nnegative smear test may have discouraged \n\nTable 6: Accuracy of Xpert  MTB/RIF for the detection of Smear positive TB in sputum \n\n All patients \nn=2091 \n\n HIV uninfected \nn=2026 \n\n HIV infected \nn=65 \n\n \n\n Sensitivity, % \n(95% CI) \n\nSpecificity, % \n(95% CI) \n\nSensitivity, % \n(95% CI) \n\nSpecificity, % \n(95% CI) \n\nSensitivity, % \n(95% CI) \n\nSpecificity, \n% (95% CI) \n\nSmear microscopy 61.97% \n(55.41,68.21) \n\n96.48 % (95.24, \n97.48) \n\n78.38% \n(71.74, 84.08) \n\n92.", "start_char_idx": 15602, "end_char_idx": 18910, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1402e86e-1a0f-4554-817c-12fbbccf3974": {"__data__": {"id_": "1402e86e-1a0f-4554-817c-12fbbccf3974", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5e6128f6-a75f-47ee-ae35-9e4e901665bc", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "003eae64b1fb9ddc4baf1d67dd9cfe20fdd45a6ce1a3e42905866ce639b11f6d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ce2d90db-73b8-4095-be02-da4743d31016", "node_type": "1", "metadata": {}, "hash": "ecf6af5b51204958342e2c29ec4aef550fd3fb7074acbad92e7ebaad8c0c3046", "class_name": "RelatedNodeInfo"}}, "text": "38% \n(71.74, 84.08) \n\n92.50 % \n(90.85,93.93) \n\n62.36% \n(51.93,64.82) \n\n95.23 % \n(93.84, 97.41) \n\nMTB/RIF \nperformed on \nuncentrifuged \n\nsputum \n\n95.50% \n(91.87, 97.82) \n\n94.69 % \n(93.23, 95.92) \n\n77.66% \n(72.24, 82.46) \n\n \n\n99.09 % \n(98.33, 99.56) \n\n \n\n94.13% \n(90.16, 95.28) \n\n92.47 % \n(90.67, 94.54) \n\nMTB/RIF \nperformed on \nuncentrifuged \n\nsputum in patients \nwho were smear \n\nnegative \n\n81.23% \n(75.95, 85.78) \n\n98.29 % \n(97.34, 98.97) \n\n91.77% \n(87.45, 94.98) \n\n95.70 % \n(94.36, 96.80) \n\n83.82% \n(76.72, 86.26) \n\n94.78 % \n(91.23, 97.76) \n\nMTB/RIF \nperformed on a \n\nresuspended pellet \nof centrifuged \n\nsputum \n\n51.89% \n(44.94,58.78) \n\n93.79 % \n(92.24, 95.11) \n\n60.44% \n(52.94, 67.60) \n\n91.42 % \n(89.68, 92.95) \n\n52.62% \n(46.73, 56.28) \n\n91.72% \n(89.91, 93.63) \n\n PPV\u2020 \n(95% CI) \n\nNPV\u2020 \n(95% CI) \n\nPPV\u2020 \n(95% CI) \n\nNPV\u2020 \n(95% CI) \n\nPPV\u2020 \n(95% CI) \n\nNPV\u2020 \n(95% CI) \n\nSmear microscopy 47.44% \n(40.61,54.34) \n\n92.73 % \n(91.08, 94.16) \n\n54.84 % \n(47.39, 62.13) \n\n90.46 % \n(88.65, 92.08) \n\n85.95% \n(80.92, 90.07) \n\n98.76 % \n(97.93, 99.32) \n\nMTB/RIF \nperformed on \nuncentrifuged \n\nsputum \n\n96.85% \n(93.61,98.72) \n\n \n\n94.95 % \n(93.52, 96.14) \n\n78.75% \n(73.42, 83.45) \n\n99.36 % \n(98.69,99.74) \n\n93.69% \n(89.65, 96.51) \n\n97.04 % \n(95.89,97.94) \n\nMTB/RIF \nperformed on \nuncentrifuged \n\nsputum in patients \nwho were smear \n\nnegative \n\n62.61% \n(56.12,68.77) \n\n97.09 % \n(95.93, 97.99) \n\n81.87% \n(75.49, 87.18) \n\n92.51 % \n(90.87, 93.95) \n\n51.89% \n(44.94, 58.78) \n\n94.48 % \n(93.00, 95.72) \n\nMTB/RIF \nperformed on a \n\nresuspended pellet \nof centrifuged \n\nsputum \n\n47.44% \n(40.61,54.34) \n\n92.73% \n(91.08, 94.16) \n\n54.84% \n(47.39, 62.13) \n\n90.46 % \n(88.65, 92.08) \n\n90.13% \n(87.25,92.47) \n\n52.43% \n(49.10, 53.91)", "start_char_idx": 18885, "end_char_idx": 20583, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ce2d90db-73b8-4095-be02-da4743d31016": {"__data__": {"id_": "ce2d90db-73b8-4095-be02-da4743d31016", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1402e86e-1a0f-4554-817c-12fbbccf3974", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "fc069b067687c917073b82bca70529a882c3a8cacb16ae80ed9d99ef9b40ce60", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2ba9195b-1bd6-499f-a3f2-69086728f556", "node_type": "1", "metadata": {}, "hash": "17d14946d6cb8ac4957f278657ef938f9b6517cdf856f6560b38848be8202352", "class_name": "RelatedNodeInfo"}}, "text": "47) \n\n52.43% \n(49.10, 53.91) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    31        Nepjol.info/index.php/njb \n\ntreatment in some cases which would otherwise \n\nhave been treated, although clinicians were \n\ninformed that a negative smear test does not \n\nexclude a TB diagnosis. Patients with a strong \n\npossibility of pulmonary TB despite a negative \n\nsmear test were referred for further investigations \n\nincluding culture at a tertiary centre. The rate of \n\nsmear positivity at Parsa is 15.0 % which is \n\nconsistent with the results in other areas of similar \n\nendemicity; it is therefore unlikely that the \n\nGeneXpert diagnosis was inflated due to low smear \n\nmicroscopy confirmation [13-15].  \n\nIf we look on test selection, smear microscopy, due \n\nto its simplicity, speed, and low cost, is used widely \n\nin low-resource settings; the low sensitivity \n\nprecludes it from being an ideal test. The \n\nrequirement for a rapid, simple TB diagnostic is \n\nevidenced by the widespread application of \n\ncommercial serological tests which are inaccurate. \n\nThese tests are widely provided at a cost to the \n\npatient and used to determine medical treatment.  \n\nAlthough the GeneXpert has several limitations, \n\nincluding requirement for stable electricity supply, \n\nlimited temperature range, availability of \n\nmaintenance, and bulky consumables, wider \n\navailability of the accurate GeneXpert assay may \n\ncounter the use of these serological tests by \n\nproviding a viable alternative to the patient and \n\nhealthcare provider. It is highly probable that a \n\nsmall number of cases of TB were missed in this \n\nstudy as GeneXpert does not have as high \n\nsensitivity as culture. There is a danger that \n\nclinicians will \u201cexclude\u201d a TB diagnosis on the \n\nbasis of a negative GeneXpert test and it is \n\nimportant that education is carried out to ensure \n\nclinicians are aware of the test limitations prior to \n\nthe test being implemented. However, it is not \n\nsustainable to implement TB culture facilities at \n\ngeneral hospitals in South Asia and the long \n\nturnaround of results means loss to follow-up in \n\nthe diagnostic pathway is high. This study was not \n\nan assessment of GeneXpert sensitivity and \n\nspecificity, as this has been comprehensively \n\nevaluated in comparison with culture by others. \n\nConclusion  \nThe prevalence of TB is high in Parsa DOTS centre \n\nin comparable to other districts of Nepal. However, \n\nthe relatively higher rate of RIF resistance observed \n\nin our study signals the danger of increasing \n\nMDRTB in the study areas in the future in Nepal. \n\nOn the other hand, MTB RIF positivity among \n\nsputum negative, RIF resistant (DR TB) among \n\nsputum negative and RIF resistant among \n\nretreatment cases is large in this study. The higher \n\nlevel of MDR-TB among previously treated patients \n\nsuggests the need to strengthen the DOTS strategy \n\nand the capacity of laboratories to undertake \n\neffective treatment especially among previously \n\ntreated patients in Nepal. The GeneXpet is more \n\nspecific and sensitive that smear AFB detection. \n\nThe Government of Nepal Ministry of Health and \n\npopulation should implement GeneXpert in each \n\nDOTS centre in Nepal for better diagnosis. \n\nAbbreviations  \n CXR: Chest X-Ray; DOTS: Directly observed \n\ntreatment short course; DST: Drug susceptibility \n\ntesting; HIV: Human Immunodeficiency Virus; \n\nMDR: Multidrug-resistance; NTP: National \n\nTuberculosis Program; PPV:  Positive Predictive \n\nValue; NPV: Negative Predictive Value. \n\nAuthors\u2019 contributions \nRK, BPG was involved in the study conception and \n\ndesign, data analysis and drafting of the \n\nmanuscript.  AA, RR and KDM were involved in \n\nthe design and reviewing of the manuscript. BPG \n\nwas involved in reviewing of the manuscript. All \n\nauthors have read and approved the final version \n\nof the manuscript. \n\nConflict of Interests  \nThe authors declare no competing interests \n\nregarding the publication of this paper.", "start_char_idx": 20555, "end_char_idx": 24595, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2ba9195b-1bd6-499f-a3f2-69086728f556": {"__data__": {"id_": "2ba9195b-1bd6-499f-a3f2-69086728f556", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ce2d90db-73b8-4095-be02-da4743d31016", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "5021f2c64239f0e42723f7ffd97a21d10e1dc24714a665ab43472214b23b666b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f3f0a6fb-66f7-4ec1-8dd6-cd058932631f", "node_type": "1", "metadata": {}, "hash": "b25d327ad0e7c3020f53fa838e33e46a6f6936ecedfba2d4447470bf5efcd022", "class_name": "RelatedNodeInfo"}}, "text": "Acknowledgment  \nAuthors acknowledge the role of the staff of DOTS \n\nCentre, Parsa during the conduct of this study for \n\ntheir invaluable help during the identification of \n\ncases and collection of data. \n\nReferences \n1. His Majesty\u2019s Government of Nepal Ministry of \n\nHealth and the World Health Organization. \nTuberculosis Control in Nepal 1995\u20131999. A \ndevelopment plan for the National Tuberculosis \nProgramme. Kathmandu, Nepal, 1995. \n\n2. National Tuberculosis Programme. Tuberculosis \ncontrol in Nepal. Status report. Thimi, Nepal: \nNational Tuberculosis Centre. 1997. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 26-32    Kurmi et al..\n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    32        Nepjol.info/index.php/njb \n\n3. Goverment of Nepal. National Tuberculosis \nCenter 2014. http://www.nepalntp.gov.np/ \nindex.php?view=publication  \n\n4. . \n5. Bhatt CP, Bhatt AB, Shrestha B: Drug resistant \n\ncases of tuberculosis in directly observed \ntreatment short course. Journal of Nepal Health \nResearch Council 2010, 8(1):44-47. \n\n6. Creswell J, Rai B, Wali R, Sudrungrot S, Adhikari \nLM, Pant R, Pyakurel S, Uranw D, Codlin AJ: \nIntroducing new tuberculosis diagnostics: the \nimpact of Xpert((R)) MTB/RIF testing on case \nnotifications in Nepal. The international journal of \ntuberculosis and lung disease : the official journal of the \nInternational Union against Tuberculosis and Lung \nDisease 2015, 19(5):545-551. \n\n7. Desikan P: Sputum smear microscopy in \ntuberculosis: is it still relevant? The Indian journal \nof medical research 2013, 137(3):442-444. \n\n8. Banjara MR, Kroeger A, Huda MM, Kumar V, \nGurung CK, Das ML, Rijal S, Das P, Mondal D: \nFeasibility of a combined camp approach for \nvector control together with active case detection \nof visceral leishmaniasis, post kala-azar dermal \nleishmaniasis, tuberculosis, leprosy and malaria \nin Bangladesh, India and Nepal: an exploratory \nstudy. Transactions of the Royal Society of Tropical \nMedicine and Hygiene 2015, 109(6):408-415. \n\n9. Iram S, Zeenat A, Hussain S, Wasim Yusuf N, \nAslam M: Rapid diagnosis of tuberculosis using \nXpert MTB/RIF assay - Report from a developing \ncountry. Pakistan journal of medical sciences 2015, \n31(1):105-110. \n\n10. Rachow A, Zumla A, Heinrich N, Rojas-Ponce G, \nMtafya B, Reither K, Ntinginya EN, O'Grady J, \nHuggett J, Dheda K et al: Rapid and accurate \ndetection of Mycobacterium tuberculosis in \nsputum samples by Cepheid Xpert MTB/RIF \nassay--a clinical validation study. PloS one 2011, \n6(6):e20458. \n\n11. Boehme CC, Nicol MP, Nabeta P, Michael JS, \nGotuzzo E, Tahirli R, Gler MT, Blakemore R, \nWorodria W, Gray C et al: Feasibility, diagnostic \naccuracy, and effectiveness of decentralised use of \nthe Xpert MTB/RIF test for diagnosis of \ntuberculosis and multidrug resistance: a \nmulticentre implementation study. Lancet 2011, \n377(9776):1495-1505. \n\n12.", "start_char_idx": 24598, "end_char_idx": 27491, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f3f0a6fb-66f7-4ec1-8dd6-cd058932631f": {"__data__": {"id_": "f3f0a6fb-66f7-4ec1-8dd6-cd058932631f", "embedding": null, "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-26", "node_type": "4", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "05c83f15ab66e24e54cb41fa9993a7af3c25f8ce1159d740eed83d46415f91e1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2ba9195b-1bd6-499f-a3f2-69086728f556", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "9457cdff8db83f65be52880a930b915d8b500cfa0e870bed87fe6a165dc1ae53", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "deee63f4-3f24-4a6b-81c5-1b001583e998", "node_type": "1", "metadata": {}, "hash": "f3afee804093026285eb28b95bcfeddb26e92b0150988375163ba8928b0b7c00", "class_name": "RelatedNodeInfo"}}, "text": "12. Vassall A, van Kampen S, Sohn H, Michael JS, John \nKR, den Boon S, Davis JL, Whitelaw A, Nicol MP, \nGler MT et al: Rapid diagnosis of tuberculosis \nwith the Xpert MTB/RIF assay in high burden \ncountries: a cost-effectiveness analysis. PLoS \nmedicine 2011, 8(11):e1001120. \n\n13. Hamid S, Hussain SA, Ali I: Comparative analysis \nof case screening with varying cough duration \nand sputum samples for diagnosis of tuberculosis \nin patients attending the OPD at a tertiary care \nhospital at Srinagar, India. Nigerian journal of \nclinical practice 2012, 15(4):430-435. \n\n14. Malhotra S, Zodpey SP, Chandra S, Vashist RP, \nSatyanaryana S, Zachariah R, Harries AD: Should \nsputum smear examination be carried out at the \nend of the intensive phase and end of treatment in \nsputum smear negative pulmonary TB patients? \nPloS one 2012, 7(11):e49238. \n\n15. Hooja S, Pal N, Malhotra B, Goyal S, Kumar V, \nVyas L: Comparison of Ziehl Neelsen & Auramine \nO staining methods on direct and concentrated \nsmears in clinical specimens. The Indian journal of \ntuberculosis 2011, 58(2):72-76.", "start_char_idx": 27488, "end_char_idx": 28565, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "deee63f4-3f24-4a6b-81c5-1b001583e998": {"__data__": {"id_": "deee63f4-3f24-4a6b-81c5-1b001583e998", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f3f0a6fb-66f7-4ec1-8dd6-cd058932631f", "node_type": "1", "metadata": {"identifier": "njb-26", "author": "Kurmi, Roshan; Rauniyar, Ramanuj; Manandhar, Krishna Das; Gupta, Birendra Prasad", "title": "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal", "date": "2016-12-31", "file": "njb-26.pdf"}, "hash": "a731fc91cd0a32c2afc79dd758235d0a7fbef941a68a78284fa9521dec4f5e5e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "be07f8ba-7227-401e-8db8-27af24d77c4a", "node_type": "1", "metadata": {}, "hash": "1321805b0bbc10ee212bd16aaf02f8582e1dc70f9cbd0def69143232547b63aa", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:  18-25  ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    18      Nepjol.info/index.php/njb \n\n \n\nDetection of Latent HIV-1 Infection and Drug Resistant Mutation \nTesting in Nepal: HIV-1 env V3 DNA Sequence and RT Gene \n\n(M184V) Mutation \nRupendra Shrestha1,2*, Sundar Khadka1,2, Susbin Raj Wagle1,2, Alisha Sapkota1,2 \n\n1Department of Laboratory Medicine, Nobel College, Sinamangal, Kathmandu, Nepal. \n2Center for Molecular Dynamics Nepal (CMDN), Thapathali-11, Kathmandu, Nepal. \n\nAbstract \nHIV-1 resistance to antiretroviral therapy (ART) is a crucial issue, despite various effective drugs are available \n\nfor the treatment.  Although the viral RNA is suppressed below the detection limit (<50 copies/ml) with the use \n\nof potent antiviral drugs, the mutation can be archived in the cellular reservoir as proviral DNA. The detection \n\nof proviral DNA and mutation screening in HIV 1 RNA for genotypic resistance is the sole basis for monitoring \n\nthe effectiveness of ART. Our study aim to access the extent of latent HIV infections by detecting env V3 DNA \n\nand also testing of M184V (meth184val; ATG - GTG substitution at 184th codon) specific mutations in HIV-1 RT \n\ngene to monitor the effectiveness of ART. The HIV-1 env V3 DNA sequence was amplified using multiple \n\nupstream and downstream primes to show the latent HIV infections, whereas polymerase chain reaction- \n\nrestriction fragment digestion assay (PCR-RFDA) was used for testing M184V mutation in HIV-1 RT gene. In the \n\nstudy, out of 15 HIV infected patient blood samples, 12 shows amplification of env V3 DNA, confirming the latent \n\nHIV infections while 3 were negative for env V3 DNA. HIV-1 RT gene tested for M184V mutation in all 15 samples \n\nshowed wild type after analysis using PCR-RFDA. After digestion with CviAII, three bands were observed in \n\nwild type whereas in mutant only two bands. Although the study shows negative for the M184V resistance \n\nmutation, screening of various panels of drug resistance mutations should be performed in recently infected HIV-\n\n1 patients for planning the effective ART strategy. The data is not enough to compare the overall scenario of the \n\nNepal thus warrant urgency for large scale study with standard genotypic tools. \n\nKeywords: HIV-1, Antiretroviral Therapy, Resistance Mutation, env V3 DNA, RT gene. \n\n*Corresponding Author \nE-mail: dph.rupendra@gmail.com \n\nIntroduction \nHIV/AIDS is the gradually increasing global \n\nepidemic threat with an estimated 36.7 million HIV \n\npositive people worldwide. Among them 5.1 \n\nmillion people are in Asia including 3,00,000 newly \n\ninfected and 1,80,000 AIDS-related deaths occurred \n\nin Asia per year [1]. Till date 39,249 people are \n\nestimated to be living with HIV in Nepal [2]. \n\nThe HIV epidemic has changed due to use of  \n\nantiretroviral therapies (ARTs) that suppress the \n\nHIV-1 RNA load in plasma below the detection limit \n\n(<50 copies/ml). However, lifelong ART is \n\nnecessary for patients with latent HIV infections \n\ndue to decrease in level of CD4+ lymphocytes [3, 4]. \n\nThe decrease in level of CD4+ T cells caused by HIV \n\ninfection leads into AIDS and cause opportunistic \n\ninfections, like Pneumocystis Carinii pneumonia \n\nand Kaposi\u2019s sarcoma [5]. More than 50% of the \n\nworldwide epidemic are due to HIV-1 subtype C \n\nand are prone to genotypic resistance with \n\ntreatment failure.", "start_char_idx": 47, "end_char_idx": 3546, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "be07f8ba-7227-401e-8db8-27af24d77c4a": {"__data__": {"id_": "be07f8ba-7227-401e-8db8-27af24d77c4a", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "deee63f4-3f24-4a6b-81c5-1b001583e998", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "8593e3d396a20c50c55a0d656d27ae5f4d38003a20d9f346c5132e0783b2d1ea", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0f79d5e6-f8bc-4dea-9b67-f14fe49ba46a", "node_type": "1", "metadata": {}, "hash": "da55e5499a46b6741a61947aeabce8b1cec8de10b3ad60858c2d3cffdae130dd", "class_name": "RelatedNodeInfo"}}, "text": "HIV-1 has a higher rate of \n\nreplication with lack of 3\u2019 to 5\u2019 proof reading \n\nmechanism and highly error-prone to reverse \n\ntranscriptase [6-8]. The selection of drug specific \n\nresistance mutation in viral genome is due to \n\ntreatment failure and those mutations may be \n\narchived into the cellular reservoir (Proviral DNA). \n\nThus, mutations can be detected in proviral DNA \n\neven if HIV RNA is below detection limit [9].  HIV-\n\n1 resistance to ART is a major problem which is due \n\nto suboptimum treatment and transmission of \n\nresistant variant at the time of infection [10-12]. \n\nResistance mutation is associated with high \n\nreplicative activities of the viral genome during \n\ntherapy. Several drugs have been approved for the \n\ntreatment of HIV-1 infection such as nucleotide and \n\nnucleoside reverse transcriptase inhibitors (NRTIs), \n\nprotease inhibitors (PIs), nonnucleoside RT \n\ninhibitors (NNRTIs), and fusion inhibitors. \n\nHowever, drug resistance mutations in HIV-1 \n\nremain crucial, even though effective (ART) is \n\navailable in the global market. One of the commonly \n\nassociated drug resistance mutations in HIV-1 RT \n\ngene is M184V. The appearance of Lamivudine \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    19      Nepjol.info/index.php/njb \n\n \n\n(3TC) resistance mutation, M184I, was found to be \n\ntransiently proceeded to the outgrowth of M184V \n\nsubstitution [13-15]. The study of the nucleotide \n\nsequences of both 3TC-resistant variants shows that \n\nM184V (GTG) originates from wild-type Met (ATG) \n\nbut not from the M184I variant (ATA) [16]. Both \n\nvariants are generated from the wildtype ATG \n\nsequence by transitional substitutions (G to A for \n\n184I and A to G for 184V). The G to A substitution is \n\nthe type of mutation that most commonly occur \n\nduring replication of HIV-1 thus M184I appears \n\nbefore M184V [17, 18]. In addition, there have been \n\nno systematic studies of HIV-1 resistance mutation \n\nin Nepalese population. \n\nHere, we performed the preliminary study of \n\ncommon drug resistance mutation, M184V using a \n\nbasic molecular tool called PCR-RFDA and assessed \n\nthe detection of proviral DNA in HIV-1 infected \n\npatients with prolonged ART. \n\nMaterials and Methods \nPatients counselling and collection of \n\nsamples \nIndividual patient counselling was performed on \n\nthe background of HIV/AIDs, diagnosis, \n\nmonitoring, treatment, drug resistant and genotype \n\ntesting. A questionnaire was performed with \n\nregards to drug use, sexual behavior and travel \n\nhistory. 15 samples were collected from HIV-1 \n\ninfected patients undergoing long term ART \n\ncommonly with 2-3 NRTIs and 1-2 PIs or an NNRTI \n\nfor at least 3 - 4 years recruited in sparsha Nepal, \n\nsanepa, Kathmandu was included. Out of 15 cases, \n\nonly two were clinically approved as ART resistance \n\nand viral loads of each patients are shown in Table \n\n1. Data were collected as a part of a continuing study \n\nof antiretroviral treatment at the time of primary \n\nHIV-1 infection, which was approved by the \n\nDepartment of Laboratory Medicine, Research \n\nCommittee of Nobel College and Chief of Sparsha \n\nNepal.  \n\nExtraction of Nucleic Acid \nThe nucleic acid extraction procedure was \n\nperformed in Biosafety cabinet (BSL-2). HIV-1 RNA \n\nwas extracted using precision QIAamp Viral RNA \n\nmini kit (Qiagen, Hilden, Germany) while DNA was \n\nextracted using Shine Gene Whole Blood DNA \n\nextraction kit (Wuhe Road, Minhang District, \n\nShanghai, China) as per manufacturer's \n\ninstructions.", "start_char_idx": 3547, "end_char_idx": 7130, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0f79d5e6-f8bc-4dea-9b67-f14fe49ba46a": {"__data__": {"id_": "0f79d5e6-f8bc-4dea-9b67-f14fe49ba46a", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "be07f8ba-7227-401e-8db8-27af24d77c4a", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "0d79874a5613ff53f057ef2ec71f90f49bb32523e594eb6a338f0e8b4db2d8cd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aeaf6be3-2beb-4f94-bf92-729704ffd95d", "node_type": "1", "metadata": {}, "hash": "abd252d7763ee18ad28dbc47f66d51a7190384834b60a8df33655feab13307ea", "class_name": "RelatedNodeInfo"}}, "text": "Proviral DNA Detection: Nested PCR \n\nNested PCR for HIV-1 env gene \nThe nested PCR was performed in the env V3 DNA \n\nsequences of HIV-1 for the amplification using high-\n\nfidelity Pfu DNA Polymerase. The amplification \n\nwas performed as described in previous study [19]. \n\nThe total reaction volume of 20\u03bcl containing 1X PCR \n\nbuffer, 1.5 mM MgCl2, 200 \u03bcM dNTPs, and 0.25 U \n\nof Pfu enzyme and RNase free water. The first \n\nround PCR was performed with 0.5 \u03bcM of upstream \n\nprimers JA9AE (5\u2019-\n\nCACAGTACAATGCACACATG-3\u2019), JA9B (5\u2019-\n\nCACAGTACAATGTACACATG-3\u2019), and \n\ndownstream primers JA12A (5\u2019-\n\nGCAATAGAAAAATTCTCCTC-3\u2019), JA12B (5\u2019-\n\nACAGTAGAAAAATTCCCCTC-3\u2019). The reaction \n\nmixture was incubated at 95\u00b0C for 15 minutes, 94\u00b0C \n\nfor 30 Sec, 58\u00b0C for 30 Sec, 72\u00b0C for 30 Sec and 72\u00b0C \n\nfor 10 minutes, for 35 PCR cycles. 1 \u03bcl of the first-\n\nround PCR product was used as template for \n\nsecond-round PCR. Inner upstream primers used \n\nwere a mixture of 0.33 \u03bcM each of JA10UB (5\u2019-\n\nCTGTTAAATGGCAGTCTAGC-3\u2019), JA10UC (5\u2019-\n\nCTGTTAAATGGTAGTCTAGC-3\u2019), and JA10UG \n\n(5\u2019-CTGTTAAATGGCAGTTTAGC-3\u2019). Inner \n\ndownstream primers used were different for each \n\nreaction, namely, 0.33 \u03bcM each of JA11LAE (5\u2019-\n\nAATTTCTAGATCCCCTCCTG-3\u2019), JA11LB (5\u2019-\n\nAATTTCTGGGTCCCCTCCTG-3\u2019), and JA11LC (5\u2019-\n\nAATTTCTAGGTCCCCTCCTG-3\u2019). The reaction \n\nmixture was incubated at 95\u00b0C for 15 minutes, 94\u00b0C \n\nfor 30 Sec, 50\u00b0C for 30 Sec, 72\u00b0C for 30 Sec and 72\u00b0C \n\nfor 10 minutes, for 35 PCR cycles. The final PCR \n\nproduct was resolved in 1.7% agarose gel \n\nelectrophoresis and image was captured using gel \n\ndocumentation. \n\nHIV-1 RNA Mutational (M184V) \nAnalysis: PCR-RFDA \nRT gene amplification: Nested PCR \nThe amplification of HIV-1 RT gene was performed \n\nusing nested primer pairs which were reported in \n\nprevious study [20]. The isolated HIV-1 RNA was \n\nsubjected to OneStep RT-PCR (Qiagen, Hilden, \n\nGermany). The RT-PCR product was then used as \n\ntemplates for second round PCR. The QIAGEN  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    20      Nepjol.info/index.php/njb \n\n \n\nOneStep RT-PCR was performed as recommended \n\nby the manufacturer. The total volume of 50\u03bcl \n\nreaction was carried out containing RNase free \n\nwater, 1x OneStep RT-PCR buffer, 200 \u03bcM dNTP \n\nmix, 0.6 \u03bcM of each primers A1 (5\u2019-\n\nAATTTTCCCATTAGCCCTATT-3\u2019) and NE1 (5\u2019-\n\nTATGTCATTGACAGTCCAGCT-3\u2019), 2\u03bcl of \n\nOneStep RT-PCR enzyme mix, 5 units RNase \n\ninhibitor and 2 \u03bcg viral RNA for reverse-\n\ntranscriptase gene.", "start_char_idx": 7133, "end_char_idx": 9681, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aeaf6be3-2beb-4f94-bf92-729704ffd95d": {"__data__": {"id_": "aeaf6be3-2beb-4f94-bf92-729704ffd95d", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0f79d5e6-f8bc-4dea-9b67-f14fe49ba46a", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "147ae492cefc494d4c38561fdb7c76f58b67db8df010853fae353a722b2bb73c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1c1d1fd1-04c9-4e6a-ac2a-24a7fc1824d0", "node_type": "1", "metadata": {}, "hash": "68a585e854898e4c6e3a25080508776068b27f02d6f070554e1eadcc72efe48b", "class_name": "RelatedNodeInfo"}}, "text": "The reaction was incubated at \n\n48\u00b0C for 20 minutes (reverse transcription) followed \n\nby 94\u00b0C for 4 minutes, 94\u00b0C for 30 Sec, 52\u00b0C for 30 \n\nSec, 72\u00b0C for 30 Sec, and 72\u00b0C for 10 minutes, for 35 \n\nPCR cycles. The second round PCR was done by \n\nusing a QIAGEN master mix with 5 \u00b5l of the first \n\nRT-PCR product and primers NNA (5\u2019-\n\nAAGCCAGGAATGGATGGCCCA-3,) and E (5\u2019-\n\nCCATTTATCAGGATGGAGTTC-3\u2019). The reaction \n\nmixture was incubated at 95\u00b0C for 15 minutes, 94\u00b0C \n\nfor 30 Sec, 50\u00b0C for 30 Sec, 72\u00b0C for 45 Sec and 72\u00b0C \n\nfor 10 minutes, for 30 PCR cycles.  \n\nM184V Mutation Testing: Restriction \n\nFragment Digestion Assay (RFDA) \nThe amplified RT gene was tested for M184V \n\nmutation by digestion approach called RFDA. The \n\nRFDA was performed using restriction enzymes \n\nCviAII. The total volume of 50\u03bcl reaction was \n\nperformed containing 1X NEBuffer, 10 units\u2019 CviAII \n\nrestriction enzyme, and 1\u03bcg RT-PCR product at \n\n25oC for 1 hour. The digested product was resolved \n\nin 1.7% agarose gel electrophoresis and image were \n\ncaptured using gel documentation. \n\nResults \nIn the present study, detection of proviral DNA by \n\namplification of env V3 DNA sequences was done by \n\nnested PCR while testing of resistance mutation in \n\nthe HIV-1 RT (MET 184 VAL) gene was performed \n\nby PCR and RFDA. Individual band, resulting from \n\nfinal PCR product and enzyme digested product \n\nwas compared with ART resistance strain and \n\nmolecular size marker (100bp). The results are \n\ntabulated and is shown in Table 1. \n\nTable 1: Patient\u2019s information, ART and genotypic testing in the study population n-15 \n\nCode Gender/Age ART used Viral Load \n\ncopies/ml \n\nenv V3 DNA M184V Mutation Patient Status \n\nHRM1 Male/36 yrs AZT, 3TC, NFV 1500 Positive Negative Critical \n\nHRM2 Male/33 yrs AZT, 3TC, EFZ 1500 Positive Negative Critical \n\nHRM3 Male/26yrs AZT, 3TC, NFV 1500 Positive Negative Critical \n\nHRM4 Male/35yrs AZT, 3TC, NFV 1000 Positive Negative Expired \n\nHRM5 Male/23yrs AZT, 3TC, NFV 1000 Positive Negative ART \n\nHRM6 Male/32yrs AZT, 3TC, NFV 300 Negative Negative ART \n\nHRM7 Male/34yrs AZT, 3TC, NFV 800 Positive Negative ART \n\nHRM8 Male/25yrs AZT, 3TC, NFV 800 Positive Negative ART \n\nHRM9 Male/28yrs AZT, 3TC, EFZ 500 Positive Negative ART \n\nHRM10 Male/30yrs AZT, 3TC, NFV 250 Negative Negative ART \n\nHRM11 Male/36yrs AZT, 3TC, NFV 300 Negative Negative ART \n\nHRM12 Male/25yrs AZT, 3TC, NFV 750 Positive Negative ART \n\nHRM13 Male/27yrs AZT, 3TC, NFV 400 Positive Negative ART \n\nHRM14 Female/28yrs AZT, 3TC, EFZ 650 Positive Negative ART \n\nHRM15 Female/33yrs AZT, 3TC, NFV 550 Positive Negative ART \n\nKey: HRM-HIV-1 Resistance Mutation, ART-Anti Retroviral Therapy, AZT- Zidovudine, 3TC- Lamivudine, EFV-Efavirenz, NFV- \n\nNelfinavir \n\nTable 2: Most common mutations in RT gene and specific drugs resistance.", "start_char_idx": 9682, "end_char_idx": 12485, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1c1d1fd1-04c9-4e6a-ac2a-24a7fc1824d0": {"__data__": {"id_": "1c1d1fd1-04c9-4e6a-ac2a-24a7fc1824d0", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aeaf6be3-2beb-4f94-bf92-729704ffd95d", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "2b98dd6a77730baf8ba4b09f75254e6e4f2314e0d73b3b8f75e70635fe5b3ed4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c3d4c622-82ae-462d-a3d8-84ea8fb22adf", "node_type": "1", "metadata": {}, "hash": "6f6b0d97054fdafaaf25760b9f079592072c962c7b0613bf0b19dc8388375794", "class_name": "RelatedNodeInfo"}}, "text": "Resistance genes Resistance level \n\nMutation Gene High Intermediate Low \n\nM184V/I RT 3TC,  FTC - ABC, ddI \n\nK65R RT ABC, ddI, TNF, d4T 3TC, FTC  \n\nM41L RT AZT, d4T ddI, ABC TNF \n\nK103N RT EFV, NVP - - \n\nKey: 3TC- Lamivudine, FTC-emtricitabine, AZT- Zidovudine, d4T- Stavudine, EFV-Efavirenz, NVP- Nevirapine, ABC- Abacavir, ddI- \n\nDidanosine, TNF- Tenofovir", "start_char_idx": 12488, "end_char_idx": 12845, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c3d4c622-82ae-462d-a3d8-84ea8fb22adf": {"__data__": {"id_": "c3d4c622-82ae-462d-a3d8-84ea8fb22adf", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1c1d1fd1-04c9-4e6a-ac2a-24a7fc1824d0", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "75b2ac0d31f36617ad272e703e2179e968b44eb6f2522ae852fe3555f103c44f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "09ce9c5b-5505-4f54-bc61-07b47d1d28d9", "node_type": "1", "metadata": {}, "hash": "47036f731508e87cb86f3fd0cdbfd7d4ab6cfe9862730f39af513a5faef87cdf", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    21      Nepjol.info/index.php/njb \n\n \n\nIdentification of env V3 DNA \nThe env V3 DNA sequence was analyzed to rule out \n\nlatent HIV-1 infections. Out of 15 samples amplified \n\nfor env gene, only 12 of samples show positive for \n\nenv V3 DNA sequence by nested PCR. The size of \n\nthe product was 335bp which was a band of our  \n\nFigure 1: Nested PCR for env V3 DNA sequences. Marker (M) of 100bp was used to compare the molecular weight of \namplified product resolved in gel of 1.7%. a) Amplified product with band of interest (335bp) was visualized in all lane \n1-6. b) Amplified product was observed only in lane 7, 8, 10, 12 but not in lane 9 and 11.  Gel image of 3 positive samples \n\nis not shown.\n\ninterest and is illustrated in Figure 1 (a & b). The \n\nremaining 3 samples negative for the same gene was \n\nreamplified twice showed the similar results. The \n\nreason for env gene negative in those three samples \n\nmight be due to complete elimination of proviral \n\nDNA or due to its reduction below the detection \n\nlimit after prolonged ART. \n\nM184V mutation in HIV-1 RT gene \nHIV-1 RNA was tested for the most common \n\nmutation; M184V which emerges in patients using \n\nlamivudine (3TC) monotherapy or can be a \n\nphenotypic reversion of zidovudine (ZDV). PCR-\n\nRFDA molecular tool was used for testing the \n\nmutation in which gene was amplified by nested \n\nPCR and further final product was digested with \n\nCviAII restriction endonucleases. In the present \n\nstudy, we tested M184V mutation in 15 samples and \n\nall showed wildtype strain. The wildtype strains \n\nshow three fragment after digestion with band size \n\nof 453bp, 60bp and 168bp  are susceptible to 3TC \n\nwhile mutant type have 2 bands with size of 453bp \n\nand 228bp are considered as 3TC  resistance which \n\nmeans the M184V resistance mutation was emerged \n\nby modification of the 184 restriction site \n\nsubstituting CviAII enzymes. The virtual schematic \n\nillustration of Nested PCR and RFDA of both wild \n\ntype and mutant type is shown in Figure 2 (a & b). \n\nDiscussion \nEarly detection of drug resistance mutation in \n\nHIV-1 infected patients in developing countries is \n\na big challenge however, can aid clinicians in \n\nmaking proper ART strategy plans. Various \n\nmolecular tools like real time PCR and sequencing \n\nfacilities are still lacking. Also, such tools are \n\nexpensive and less sensitive to minor resistance \n\nvariants. The development of cheap and sensitive \n\ntools to screen resistance mutation can be a \n\nbreakthrough in the field of molecular biology for \n\nlow economic countries like Nepal. In the study, \n\nwe used nested PCR and PCR-RFDA to detect \n\nHIV-1 Infections and screen the prevalence of \n\nM184V mutation in HIV-1 patients underwent \n\nprolong ART. The cheap and sensitive methods \n\nfor detecting primary infection in infants and \n\nlatent HIV-1 infection is to identify proviral DNA. \n\nThe HIV proviral DNA is positive after 28 days \n\nwhile most healthy individuals are accustomed to \n\nwaiting 3 months for a conclusive result. We \n\nperformed nested PCR to identify the env V3 DNA \n\nsequences in 15 samples, out of which 12 showed \n\npositive for proviral DNA. The PCR amplification \n\non 3 negative samples was repeated twice with the \n\nsame primers for the env V3 DNA but consistently \n\nthe results were negative. The reason might be a \n\nlow copy number which is below the detection \n\nlimit or can be complete elimination of proviral \n\nDNA due to prolonged ART. The standard \n\nmethods for the detection of HIV-1 Proviral DNA \n\nare currently lacking and several discordant \n\nresults are still present in different studies [21]. In \n\nthe present study, we didn\u2019t study for the", "start_char_idx": 12851, "end_char_idx": 16650, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "09ce9c5b-5505-4f54-bc61-07b47d1d28d9": {"__data__": {"id_": "09ce9c5b-5505-4f54-bc61-07b47d1d28d9", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c3d4c622-82ae-462d-a3d8-84ea8fb22adf", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "fac61d21eada09132ac0084378e40259de1863b1fdb1b6f3b272f3c3ea546c99", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "48474daf-d160-4b41-9fbb-61662d041bcd", "node_type": "1", "metadata": {}, "hash": "5470a86a5222df16f55e835338b8d75758fb0fd7e192af0c07799b8d2a8ac5bc", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    22      Nepjol.info/index.php/njb \n\n \n\nFigure 2: Nested PCR and RFDA for detection of M184V resistance mutation. a) Wildtype stain with three bands of \n\nsize 453bp, 60bp, 168bp. b) Mutant strain with two bands of size 453bp and 228bp \n\nresistance mutation in HIV-1 proviral DNA. \n\nHowever, the presence of drug resistance \n\nmutations in the reservoir of latent HIV-1 provirus \n\nand their stability may impact future management \n\nand/or treatment possibilities.  The presence of \n\nlatent proviral DNA doesn\u2019t indicate the \n\nresistance to ART but the specific mutation like \n\nM184V, k65R, M41L and K103N etc. can be \n\n detected if the similar mutation is observed in \n\nHIV-1 RNA. This is due to retention of \n\nmutant from HIV-1 RNA into cDNA due to long \n\nterm suppressive ART that integrates into host \n\ngenome. The evolution of drug resistance mutation \n\nin globally epidemic HIV-1 is due to suboptimal \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    23      Nepjol.info/index.php/njb \n\n \n\ntreatment, a point mutation in viral genome and \n\nresistance variants leads to incomplete suppression \n\nof the viral genome. The development of drug \n\nresistance mutation threatens the success of the \n\nfuture therapy regimens. The challenge in treatment \n\nof HIV-1 is associated with the development of drug \n\nresistance mutation against antiretroviral drugs \n\nwhich can significantly reduce the viral RNA to \n\nundetectable levels in plasma [22]. The various \n\nresistance mutation pattern has been reported with \n\nmajorities in both nucleoside reverse transcriptase \n\ninhibitors (NRTIs) and non- nucleoside reverse \n\ntranscriptase inhibitors (NNRTIs) while only a few \n\nwith NNRTIs and also in minor ratio with protease \n\ninhibitors (PI) and 3- class resistance (NRTIs, \n\nNNRTIs and PIs). The genotyping of the reverse \n\ntranscriptase gene sequence revealed that almost \n\n90% resistance mutation were M184V [23] and such \n\nmutation emerges in patients receiving lamivudine \n\n(3TC) therapy, but also it could be associated with \n\nphenotypic reversion of zidovudine (ZDV) \n\nresistance [24]. In the present study, we screened for \n\nM184V resistance mutation in HIV-1 infected \n\npatients receiving 3TC in combination by PCR-\n\nRFDA methods. The only wild type strain was \n\nfound in all the sample analyzed which means the \n\nM184V mutation was negative. Our results \n\ncompletely contradict the other reports on M184V. \n\nIn the study though phenotypic resistance and high \n\nviral load were included, M184V mutation was not \n\ndetected which might be due to numerous reasons \n\nlike 1) duration of 3TC ART, 2) Possibilities of other \n\nmutation like M184I, K65R, etc  3) change in drugs  \n\n4) less likely to be technical error .  The commonly \n\nused drugs by the HIV-1 infected patients in Nepal \n\nare AZT, 3TC, EFV and NFV and is shown in Table \n\n1. The high level resistance to 3TC associated with \n\nM184V mutation is also resistance to other drugs \n\nlike FTC, ABC and ddI. In addition to this, resistance \n\nto 3TC is also associated with K65R mutation and its \n\nvariant forms but the level of resistance may vary \n\nfrom high-intermediate-low. The most common RT \n\ngene mutation and specific drug resistance is shown \n\nin Table 2. There are several mutations in genes of \n\nHIV-1 associated with resistance to specific drugs \n\n[25].", "start_char_idx": 16655, "end_char_idx": 20195, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "48474daf-d160-4b41-9fbb-61662d041bcd": {"__data__": {"id_": "48474daf-d160-4b41-9fbb-61662d041bcd", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "09ce9c5b-5505-4f54-bc61-07b47d1d28d9", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "243730035b9910fdd385d41156a0080817b8c254b8bdf515bcb5c5ff741746d1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a24f95ce-3ee5-49b9-bf24-b2ec9bd9ac02", "node_type": "1", "metadata": {}, "hash": "ef34690daf721ebe9dae5c1a761efec817757d69d88907b618ba0a82cde29ede", "class_name": "RelatedNodeInfo"}}, "text": "The M184V mutation was detected in lesser \n\nthan 10% by allele specific PCR but was consistently \n\nnegative by standard genotyping [26] While the \n\nratio of mutant and wild type is equal to 50:50 \n\nreported by almost half of the laboratories [27]. The \n\nprevalence of the M184V mutation is notably higher \n\nthan that of other mutation [28-30] and is \n\nsignificantly associated with 3TC [31].  \n\nThe early detection of resistance mutation in HIV-1 \n\ninfected patients would help to change the \n\ntreatment regimens susceptible to both Proviral \n\nDNA and viral RNA and control measures can be \n\ntaken to minimize the transmission of resistant \n\nvariants. An understanding of the molecular \n\nmechanism of drug resistance will enable us to \n\ndevelop improved tools for resistance screening.  \n\nConclusion \nIn the present study, we set out to identify the HIV-\n\n1 latent infections by detecting env V3 DNA \n\nsequences using nested PCR. In addition to this, the \n\nM184V resistance mutation was not observed in the \n\nsamples analyzed thus indicating as wild type \n\nstrain. Also, we optimized the PCR-RFDA protocol \n\nusing CviAII restriction enzymes for the HIV-1 RT \n\nstrains for mutation testing. This strategy was \n\nmeant to enhance assay discrimination between the \n\nspecific drug resistance mutation (mutant allele) \n\nand its wild type (wild type allele) using nested \n\nprimers and CviAII restriction enzymes.  \n\nDeclaration of interests \nThe authors declare that they have no competing \n\ninterests. \n\nAuthors\u2019 contributions \nRupendra came up with the study, frame \n\nexperimental work and prepared manuscript. All \n\nauthors have equally involved in completing the \n\nresearch experiment and data compiling. All \n\nauthors read and approved the final manuscript for \n\npublication. \n\nAcknowledgement \nThe authors would like to thank the Nobel College \n\nfor funding the projects and Center for Molecular \n\nDynamics Nepal (CMDN) for providing all the \n\nfacilities to perform the research experiment. We \n\nalso like to thank Dr. Sameer M. Dixit, Bhavesh \n\nMishra, Raunak Shrestha, Sulochana Manandhar, \n\nand Prajwal Rajbhandari for their valuable \n\ndiscussion and extensive support to make this work \n\ndone. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    24      Nepjol.info/index.php/njb \n\n \n\nReferences \n1. Global AIDS Update: UNAIDS Report. Geneva; \n\n2016.  \n2. Country Progress Report: National Centre for \n\nAIDS and STD Control (NCASC). Nepal; 2015. \n3. Finzi D, Hermankova M, Pierson T, Carruth LM, \n\nBuck C, Chaisson RE, Quinn TC, Chadwick K, \nMargolick J, Brookmeyer R, Gallant J, Markowitz \nM, Ho DD, Richman DD, Siliciano RF: \nIdentification of a reservoir for HIV-1 in patients \non highly active antiretroviral therapy. Science \n1997, 278(5341): 1295-1300. \n\n4. Siliciano JD, Kajdas J, Finzi D, Quinn TC, \nChadwick K, Margolick JB, Kovacs C, Gange SJ, \nSiliciano RF: Long-term follow-up studies \nconfirm the stability of the latent reservoir for \nHIV-1 in resting CD4+ T cells. Nature medicine \n2003, 9(6): 727-728. \n\n5. Levy JA, Hoffman AD, Kramer SM, Landis JA, \nShimabukuro JM, Oshiro LS: Isolation of \nlymphocytopathic retroviruses from San \nFrancisco patients with AIDS. Science 1984, \n225(4664): 840-842. \n\n6. Ho DD, Neumann AU, Perelson AS, Chen W, \nLeonard JM, Markowitz M: Rapid turnover of \nplasma virions and CD4 lymphocytes in HIV-1 \ninfection.", "start_char_idx": 20196, "end_char_idx": 23645, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a24f95ce-3ee5-49b9-bf24-b2ec9bd9ac02": {"__data__": {"id_": "a24f95ce-3ee5-49b9-bf24-b2ec9bd9ac02", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "48474daf-d160-4b41-9fbb-61662d041bcd", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "0a8554ade9ec2f897ce8c994df173ee192423557da1edb2bbf6aaf7b332eb90d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cd6ec6aa-cb2f-43f8-a4fc-23001a1a299d", "node_type": "1", "metadata": {}, "hash": "2bf1bd02764765500cd0d446938244fad181961c4686b785cd54fd6ecd1e9da4", "class_name": "RelatedNodeInfo"}}, "text": "Nature 1995, 373(6510): 123-126. \n\n7. Roberts JD, Bebenek K, Kunkel TA: The accuracy \nof reverse transcriptase from HIV-1. Science 1988, \n242(4882): 1171-1173. \n\n8. Preston BD, Poiesz BJ, Loeb LA: Fidelity of HIV-1 \nreverse transcriptase. Science 1988, 242(4882): 1168-\n1171. \n\n9. Chew CB, Potter SJ, Wang B, Wang YM, Shaw CO, \nDwyer DE, Saksena NK: Assessment of drug \nresistance mutations in plasma and peripheral \nblood mononuclear cells at different plasma viral \nloads in patients receiving HAART. J. of clin virol \n2005, 33(3): 206-216. \n\n10. Hirsch MS, Conway B, D'Aquila RT, Johnson VA, \nBrun-Vezinet F, Clotet B, Demeter LM, Hammer \nSM, Jacobsen DM, Kuritzkes DR, Loveday C, \nMellors JW, Vella S, Richman DD: Antiretroviral \ndrug resistance testing in adults with HIV \ninfection: implications for clinical management. \nInternational AIDS Society--USA Panel. Jama \n1998, 279(24): 1984-1991. \n\n11. Yerly S, Rakik A, Kinloch-de-Loes S, Erb P, \nVernazza P, Hirschel B, Perrin L: [Prevalence of \ntransmission of zidovudine-resistant viruses in \nSwitzerland. l'Etude suisse de cohorte VIH]. \nSchweiz med Wochenschr 1996, 126(43): 1845-1848. \n\n12. Hecht FM, Grant RM, Petropoulos CJ, Dillon B, \nChesney MA, Tian H, Hellmann NS, Bandrapalli \nNI, Digilio L, Branson B, Kahn JO: Sexual \ntransmission of an HIV-1 variant resistant to \nmultiple reverse-transcriptase and protease \ninhibitors. N Engl j of med 1998, 339(5): 307-311. \n\n13. Boucher CA, Cammack N, Schipper P, Schuurman \nR, Rouse P, Wainberg MA, Cameron JM: High-\nlevel resistance to (-) enantiomeric 2'-deoxy-3'-\nthiacytidine in vitro is due to one amino acid \n\nsubstitution in the catalytic site of human \nimmunodeficiency virus type 1 reverse \ntranscriptase. Antimicrobial agents and chemotherapy \n1993, 37(10): 2231-2234. \n\n14. Back NK, Nijhuis M, Keulen W, Boucher CA, Oude \nEssink BO, van Kuilenburg AB, van Gennip AH, \nBerkhout B: Reduced replication of 3TC-resistant \nHIV-1 variants in primary cells due to a \nprocessivity defect of the reverse transcriptase \nenzyme. The EMBO journal 1996, 15(15): 4040-4049. \n\n15. Larder BA, Kemp SD, Harrigan PR: Potential \nmechanism for sustained antiretroviral efficacy \nof AZT-3TC combination therapy. Science 1995, \n269(5224): 696-699. \n\n16. Keulen W, Back NK, van Wijk A, Boucher CA, \nBerkhout B. Initial appearance of the 184Ile \nvariant in lamivudine-treated patients is caused \nby the mutational bias of human \nimmunodeficiency virus type 1 reverse \ntranscriptase. J virol 1997, 71(4): 3346-3350. \n\n17. Cheynier R, Gratton S, Vartanian JP, Meyerhans A, \nWain-Hobson S: G --> A hypermutation does not \nresult from polymerase chain reaction.", "start_char_idx": 23646, "end_char_idx": 26297, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cd6ec6aa-cb2f-43f8-a4fc-23001a1a299d": {"__data__": {"id_": "cd6ec6aa-cb2f-43f8-a4fc-23001a1a299d", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a24f95ce-3ee5-49b9-bf24-b2ec9bd9ac02", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "a23ba3417668f591ea793a6deecfca234b83ce88f94d19d1626e2742e89721e9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0160aa42-31f0-4813-b744-10b68acde03c", "node_type": "1", "metadata": {}, "hash": "a61eed91764c5ca03acac860aee62b256aaad8089f34bcd5925da734c7f5ab44", "class_name": "RelatedNodeInfo"}}, "text": "AIDS \nresearch and human retroviruses 1997, 13(12): 985-\n986. \n\n18. Gunther S, Sommer G, Plikat U, Iwanska A, Wain-\nHobson S, Will H, Meyerhans A: Naturally \noccurring hepatitis B virus genomes bearing the \nhallmarks of retroviral G-->A hypermutation. \nVirology 1997, 235(1): 104-108. \n\n19. Khan S, Rai MA, Khanani MR, Khan MN, Ali SH: \nHIV-1 subtype A infection in a community of \nintravenous drug users in Pakistan. BMC \ninfectious diseases 2006, 6: 164. \n\n20. Yerly S, Kaiser L, Race E, Bru JP, Clavel F, Perrin L: \nTransmission of antiretroviral-drug-resistant \nHIV-1 variants. Lancet 1999, 354(9180): 729-733. \n\n21. Jangam SR, Yamada DH, McFall SM, Kelso DM: \nRapid, Point-of-Care Extraction of Human \nImmunodeficiency Virus Type 1 Proviral DNA \nfrom Whole Blood for Detection by Real-Time \nPCR. J Clin Microbiol 2009, 47(8): 2363-2368. \n\n22. De Rossi A, Zanchetta M, Vitone F, Antonelli G, \nBagnarelli P, Buonaguro L, Capobianchi MR, \nClementi M, Abbate I, Canducci F, Monachetti A, \nRiva E, Rozera G, Scagnolari C, Tagliamonte M, Re \nMC: Quantitative HIV-1 proviral DNA detection: \na multicentre analysis. The new microbiologica  2010, \n33(4): 293-302. \n\n23. Kandathil AJ, Kannangai R, Verghese VP, \nPulimood SA, Rupali P, Sridharan G, Grant P, \nPillay D, Abraham OC: Drug resistant mutations \ndetected by genotypic drug resistance testing in \npatients failing therapy in clade C HIV-1 infected \nindividuals from India. Ind j med microbio 2009, \n27(3): 231-236. \n\n24. Miller V, Phillips A, Rottmann C, Staszewski S, \nPauwels R, Hertogs K, de Bethune MP, Kemp SD, \nBloor S, Harrigan PR, Larder BA: Dual resistance \nto zidovudine and lamivudine in patients treated \nwith zidovudine-lamivudine combination \ntherapy: association with therapy failure. The \nJournal of infectious diseases 1998, 177(6): 1521-1532.", "start_char_idx": 26298, "end_char_idx": 28112, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0160aa42-31f0-4813-b744-10b68acde03c": {"__data__": {"id_": "0160aa42-31f0-4813-b744-10b68acde03c", "embedding": null, "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-27", "node_type": "4", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "cbd0712f907bee16724172ee8718b2f10e9b68a7047a070a93d4e755533598c1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cd6ec6aa-cb2f-43f8-a4fc-23001a1a299d", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "75274648514543340805d66d53bfd2e43119fa0b7b82f6d97482716797289f4b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "05aa4e20-6f4a-4d30-9106-aeb475941118", "node_type": "1", "metadata": {}, "hash": "dc6c9752426acd8e9c4d1a09cf582a26810b75e0fa776cc6ece93564c07ef4b9", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 18-25  Shrestha et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    25      Nepjol.info/index.php/njb \n\n \n\n25. Wensing AM, Calvez V, G\u00fcnthard HF, Johnson \nVA, Paredes R, Pillay D, Shafer RW, Richman DD: \n2014 Update of the drug resistance mutations in \nHIV-1. Topics in Antiviral Medicine 2014, 22(3): 642-\n650.  \n\n26. Paredes R, Marconi VC, Campbell TB, Kuritzkes \nDR: Systematic evaluation of allele-specific real-\ntime PCR for the detection of minor HIV-1 \nvariants with pol and env resistance mutations. \nJournal of virological methods 2007, 146(1-2): 136-146. \n\n27. Frater AJ, Dunn DT, Beardall AJ, Ariyoshi K, \nClarke JR, McClure MO, Weber JN: Comparative \nresponse of African HIV-1-infected individuals \nto highly active antiretroviral therapy. AIDS 2002, \n16(8): 1139-1146. \n\n28. Delaunay C, Brun-V\u00e9zinet F, Landman R, Collin G, \nPeytavin G, Trylesinski A, Flandre P, Miller M, \nDescamps D: Comparative Selection of the K65R \nand M184V/I Mutations in Human \nImmunodeficiency Virus Type 1-Infected \nPatients Enrolled in a Trial of First-Line Triple-\nNucleoside Analog Therapy (Tonus IMEA 021).  \nJournal of virology 2005, 79:9572-9578. \n\n29. Hira SK, Panchal K, Parmar PA, Bhatia VP: High \nresistance to antiretroviral drugs: the Indian \nexperience. International journal of STD & AIDS \n2004, 15(3): 173-177. \n\n30. Sen S, Tripathy SP, Patil AA, Chimanpure VM, \nParanjape RS: High prevalence of human \nimmunodeficiency virus type 1 drug resistance \nmutations in antiretroviral treatment-\nexperienced patients from Pune, India. AIDS \nresearch and human retroviruses 2007, 23(10): 1303-\n1308. \n\n31. Zaccarelli M, Perno CF, Forbici F, Cingolani A, \nLiuzzi G, Bertoli A, Trotta MP, Bellocchi MC, Di \nGiambenedetto S, Tozzi V, Gori C, D'Arrigo R, De \nLongis P, Noto P, Girardi E, De Luca A, Antinori \nA: Using a database of HIV patients undergoing \ngenotypic resistance test after HAART failure to \nunderstand the dynamics of M184V mutation. \nAntiviral therapy 2003, 8(1): 51-56.", "start_char_idx": 28117, "end_char_idx": 30165, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "05aa4e20-6f4a-4d30-9106-aeb475941118": {"__data__": {"id_": "05aa4e20-6f4a-4d30-9106-aeb475941118", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0160aa42-31f0-4813-b744-10b68acde03c", "node_type": "1", "metadata": {"identifier": "njb-27", "author": "Shrestha, Rupendra; Khadka, Sundar; Wagle, Susbin Raj; Sapkota, Alisha", "title": "Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation", "date": "2016-12-31", "file": "njb-27.pdf"}, "hash": "075eab23a1e9220329987836e854ab8f004a39b4760ab74f0412036c4847cc32", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "76c0aa49-e4ea-498f-9838-8beeed228a73", "node_type": "1", "metadata": {}, "hash": "522feff1fb621771f321a359a2a1f7a0a83a38b4aa0327ec332960332d07f9a3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1): 27-34         Research article  DOI: https://doi.org/10.54796/njb.v11i1.244  \n\n\u00a9NJB, BSN    27 \n\nEffect of Coconut Water and GA3 Concentrations on in vitro Clonal \nPropagation of Potato Cultivars from Nepal \nDurga Prasad Kafle1 , Sanam Parajuli1, Aastha Upreti1, Dhurva Prasad Gauchan1  \n1Department of Biotechnology, School of Science, Kathmandu University, Dhulikhel, Nepal \n\nReceived: 22 Jun 2023; Revised: 22 Jul 2023; Accepted: 24 Jul 2023; Published online: 31 Jul 2023 \n\nAbstract \nNodal propagation plays a crucial role in mass multiplication of potato plants. Growth regulators and media selection have \n\nan impact on the efficacy and quality of propagation. Both coconut water, a naturally occurring organic source of growth-\n\npromoting compounds, and the synthetic growth regulator GA3 (gibberellic acid-3), have the ability to accelerate plant \n\ngrowth.  The purpose of this study was to evaluate the effects of growth regulators and media, specifically GA3 and coconut \n\nwater (CW) in agar-based media (ABM) and Clarigel-based media (CBM) which is a Gellan gum-based media, on potato \n\nnodal propagation. In contrast to the control group, GA3 in ABM did not produce definitive results, however GA3 in CBM \n\nshowed a considerable level of efficacy. For the Janak Dev variety, CBM surpassed ABM in terms of root length, root hairs, \n\nleaf size, and dry biomass, whereas ABM demonstrated superior root length for the cardinal variety. In comparison to the \n\nGA3 alone at concentration ranges from 0 to 2 mg/L, adding CW at 200 mL/L to CBM or combining GA3 (0.25 mg/L) and \n\nCW (10 mL/L) substantially enhanced features including shoot length, leaf size, and root growth for both kinds. Acclimatized \n\nplantlets had a survival efficiency of 85% to 95%, with CBM supplemented with GA3 showing the highest survival rate and \n\nCBM supplemented with CW coming in second. These results highlight the significance of growth regulator and media choice \n\nin enhancing potato nodal propagation for improved plant quality and multiplication. \n\nKeywords: Potato, PBS, GA3, Clarigel, Agar, Coconut water, Micropropagation \n\n Corresponding author, email:  gauchan@ku.edu.np \n\nIntroduction \nMicropropagation has revolutionized plant propagation \n\nand is a key factor in the mass production of disease-free \n\nplants. This method entails growing plant cells, tissues, \n\nor organs in an aseptic environment in a controlled \n\nlaboratory setting [1]. The potato (Solanum tuberosum L.), \n\namong the plant species extensively researched utilizing \n\ntissue culture, stands out for its nutritional value and \n\ntolerance to varied agro-climatic situations [2]. The major \n\ngoal of potato tissue culture techniques is the disease-free \n\npropagation and maintenance of plants through \n\nmeristem culture [3]. This method requires isolating and \n\ncultivating the apical meristem, a tiny area near the \n\nplant's growing tip that contains cells that are actively \n\ndividing. Since meristems frequently have no viral \n\ninfections, meristem culture is especially useful for \n\ngetting rid of viruses. But the likelihood of getting virus-\n\nfree seedlings increases when chemo and thermotherapy \n\nare applied to the explants before meristem culture [4].  \n\nIn Nepal, potatoes have emerged as one of the most \n\nimportant cash crops and vegetables, covering a total of \n\n193,997 hectares of land and producing 3,112,947 metric \n\ntons annually as of 2019 [5]. Potatoes like Janakdev, \n\nKhumal seto, Cardinal, Kufri Jyoti, and Khumal rato-2, \n\nDesiree, are among the most popular kinds in Nepal. In \n\naddition to these, there are several domestic and foreign \n\nvariations. Janak Dev and Cardinal are the most popular \n\nin the Nepalese hills, where there are many others.", "start_char_idx": 48, "end_char_idx": 3837, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "76c0aa49-e4ea-498f-9838-8beeed228a73": {"__data__": {"id_": "76c0aa49-e4ea-498f-9838-8beeed228a73", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "05aa4e20-6f4a-4d30-9106-aeb475941118", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "8a04d0e5f1e85dac4768a36a67157ced8b37c59539881107180cd3bea435a5f6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "35cfad03-c902-4a49-ab2b-1f825c41bafa", "node_type": "1", "metadata": {}, "hash": "60e95cad48f3282b716d28e238cad7be71d456f947af8d1d2d979cfee2f90378", "class_name": "RelatedNodeInfo"}}, "text": "This \n\nwork focuses on improving the tissue culture conditions \n\nof these two types in that environment based on growth \n\nregulators and solidifying agents. \n\nThe success of commercial potato tissue culture depends \n\non the selection of the right culture media and \n\ntechnology. Plant performance in terms of growth can \n\nvary depending on the composition of the medium and \n\nthe container system. Since the beginning of tissue \n\nculture, the most widely used method for \n\nmicropropagating plants, including the potato itself, has \n\nbeen solid media-based tissue culture utilizing glass jars. \n\nThe choice of solidifying agent for an example has also \n\nbeen a factor in driving the results [6].  Agar is utilized to \n\nbuild a strong support system in the medium that aids in \n\nboth the support of the plants and the media's \n\ndiffusibility. Interest has been generated by gellan gum \n\n(Clarigel), an efficient replacement for the commonly \n\nemployed agar-based media. Clarigel has shown \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0009-0000-5369-9829\nhttps://orcid.org/0000-0002-3728-5076\nmailto:gauchan@ku.edu.np\nmailto:gauchan@ku.edu.np\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    28 \n\nencouraging results in terms of plants\u2019 shoot \n\nmultiplication and root growth [7]. \n\nIn plant tissue culture, the basal media compositions \n\nalmost remained unchanged since the development of \n\nMurashige and Skoog (MS) media in 1962 that supplies \n\nwith the macro-nutrients, micro-nutrients, iron, and \n\nvitamins [8]. The basic elements like nitrogen, phosphate, \n\npotassium, iron, and manganese are supplied by the \n\nbasal media. The basal media has to be enriched with \n\ncarbon sources (dextrose, sucrose etc.), inositol and \n\nhormones depending upon the plants\u2019 requirements [9].  \n\nApart from that, plant hormones including auxins, \n\ncytokinins and Gibberellic acid (GA3) are commonly \n\nemployed as plant growth regulators, that play a vital \n\nrole in regulating physiological processes during tissue \n\nculture. In potato tissue culture, GA3 is the most \n\nfrequently used hormone that promotes shoot elongation \n\nand multiplication of the in vitro plantlets [10]. However, \n\nthe high cost and limited availability of such hormones \n\nhave prompted researchers to explore alternative \n\ngrowth-promoting substances, such as coconut water \n\n(CW), which has shown potential as a natural substitute \n\n[11,12]. \n\nFigure 1: Selected potato varieties (a); dissection of sprouts to \nexpose meristem (b); meristem at the tip of surgical blade (c); \nmeristem with leaf primordia (d); meristem regenerated \nplantlets in jam jar (e); nodal sub culture to obtain enough \nplants for the experiment (f); clonal varieties from nodal sub-\nculture if the meristem cultured potato plants (g); acclimatized \nplants in a screenhouse (h); Well survived plantlet in the \nscreenhouse transplanted from the seedling tray (i); Janak dev \nvariety (left) with agar without growth regulators (A), with \nagar+GA3 (G+A) and with Clarigel and CW (C+C); Cardinal \nvariety (right) with agar without growth regulators (A), with \nagar+GA3 (G+A) and with Clarigel and CW (C+C); comparison \nof plant morphology of Janak dev and cardinal (L and M \nrespectively); comparison of leaf area using graph paper (n and \no). \n\nIn light of these key components, this study was \n\nconducted with an aim to explore the advantages of the \n\nCW over commercially available plant growth \n\nregulators, virus elimination, and growth performance in \n\ndifferent solidifying agents (agar vs. Clarigel) were \n\nexamined in two varieties of potato; Janak Dev and \n\nCardinal as these varieties are the most common ones for \n\nthe hilly regions of Nepal.", "start_char_idx": 3838, "end_char_idx": 7721, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "35cfad03-c902-4a49-ab2b-1f825c41bafa": {"__data__": {"id_": "35cfad03-c902-4a49-ab2b-1f825c41bafa", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "76c0aa49-e4ea-498f-9838-8beeed228a73", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "4a9ae307a15c884de2e8dc887495315d386bb2688e08d2ba6e911fd5835f5e49", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7a7dc66d-5ddc-45f3-9bc3-5c582755c11e", "node_type": "1", "metadata": {}, "hash": "6f3b83d82c3207abaeaf2d49173b6197c30794dec34eb1ca5c839b92f63fb209", "class_name": "RelatedNodeInfo"}}, "text": "Furthermore, the study was set \n\nto evaluate the effectiveness of GA3 versus CW as \n\ngrowth-promoting substances. Additionally, the use of \n\nthe MS basal medium will be investigated for its \n\neffectiveness in supporting potato tissue culture. It was \n\ndiscovered that GA3 (gibberellic acid) in CBM (coconut \n\nbroth medium) had notable efficacy while GA3 in ABM \n\nfailed to produce definitive results. When compared to \n\nABM, the Janak Dev variety outperformed CBM in terms \n\nof root length, root hairs, leaf size, and dry biomass. The \n\ncardinal variant, however, showed longer roots in ABM. \n\nAdditionally, both types' shoot length, leaf size, and root \n\ngrowth were greatly enhanced by the combination of \n\nGA3 and CW at particular concentrations. The CBM \n\nsupplemented with GA3 showed the best survival rate \n\nfor acclimatized plantlets, followed by the CBM \n\nsupplemented with CW. These results highlight the \n\nsignificance of using the proper growth regulator and \n\nmedium to improve potato nodal propagation for \n\nimproved plant quality and multiplication. Through this \n\nstudy, we aim to contribute to the optimization and \n\nadvancement of potato tissue culture techniques, \n\nultimately enhancing potato production and global food \n\nsecurity. \n\nMaterials and methods \n\nSelection of potato tubers and sprouting \n\nJanak Dev and Cardinal varieties of S. tuberosum that \n\nwere suspected to be Virus infected were chosen for the \n\nstudy as shown in Figure 1. Average sized tubers were \n\nlabelled separately and stored in warm dark chamber for \n\n8 to 10 days until they sprouted. Healthy sprouts \n\nmeasuring 1.0 to 2.0 cm in length were excised and  \n\nsurface sterilized as suggested by [13]. To begin, potato \n\nsprouts were carefully excised from the tubers. These \n\nsprouts underwent a thorough cleansing process using \n\ndistilled water, followed by a brief immersion in 70% \n\nalcohol for 30 seconds. Subsequently, they were rinsed \n\nagain with distilled water and subjected to sterilization \n\nwithin a laminar air flow cabinet using a 0.1% aqueous \n\nsolution of HgCl2 for a duration of 4 to 7 minutes. After \n\nsterilization, the sprouts were further washed with \n\nsterilized distilled water, repeating this procedure 4 to \n\n5bntimes to ensure their surface cleanliness and complete \n\nremoval of sterilant.  \n\nApical Meristem culture  \n\nMeristem culture procedure was followed as done by \n\n[14]. Primarily, surface sterilized sprouts\u2019 tip was \n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    29 \n\ncarefully removed, and the outer leaves and leaf \n\nprimordia were dissected under a dissecting microscope \n\nin a controlled laminar airflow cabinet. The resulting \n\nmeristem tip was then put onto test-tube with hormone \n\nfree potato culture media that was composed of \n\nMurashige and Skoog [8] media (MS media) \n\nsupplemented by 2 mg/L calcium d-pantothenate \n\n(HiMedia, India), 30 g/l plant tissue culture grade \n\nsucrose (Thermo Fisher, India) and 0.8% agar (HiMedia). \n\nThis meristem tip was made up of the apical dome and \n\nthe first pair of leaf primordia, which were roughly 0.1 to \n\n0.15 mm in length depending on the particular cultivar. \n\nIn order for the meristem tips to multiply and generate \n\nadventitious shoots measuring roughly 2 cm in length, \n\nthese culture tubes were put in a growth environment \n\nunder precise circumstances (22 \u00b1 1\u00b0C, 16/8 hours of \n\nlight/dark frequency per day with an intensity of 18 \n\nW/m2). The plants developed from meristem were again \n\nexplanted for another cycle of meristem culture and this \n\nprocess was repeated for two more times to make sure of \n\nviral sterility.", "start_char_idx": 7722, "end_char_idx": 11381, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7a7dc66d-5ddc-45f3-9bc3-5c582755c11e": {"__data__": {"id_": "7a7dc66d-5ddc-45f3-9bc3-5c582755c11e", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "35cfad03-c902-4a49-ab2b-1f825c41bafa", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "bf01126963876fbe141a3bc64a441ee776bc204afaa01c713efd5c7c4b46f8aa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e490664b-02d7-4766-b1d4-39d0b819c8ad", "node_type": "1", "metadata": {}, "hash": "5cb89edf40fce9c8d60fb0849f2edb5552b5f2d651f6e6ff245f20b6543cbfb5", "class_name": "RelatedNodeInfo"}}, "text": "Plant micropropagation  \n\nAfter the development of plants from cultured meristem, \n\nthey were micropropagated by single nodal sub-culture \n\ntechnique as suggested by [10,15] in jam jars using \n\npreviously used potato tissue culture agar media \n\nsupplemented by 0.25 Mg/L of GA3 provided by \n\nHiMedia, India and the media pH was maintained at 5.8 \n\nusing pH Thermo orion-420 pH meter. The process of \n\nsub-culture was repeated until the enough number of \n\nplants for the further experiment were achieved. \n\nMedia composition for optimization of plant\u2019s \n\ngrowth  \n\nMedia composition that remained unchanged: MS basal \n\nmedia [8], Sucrose (30g/l), 2 mg/L calcium d-\n\npantothenate and myo-inositol provided by HiMedia \n\n(0.1g/l). \n\nTwo experimental sets were prepared as described \n\nbelow: \n\ni) Unchanged media composition + varying \n\nconcentrations of GA3 + Agar (HiMedia) as gelling \n\nagent  \n\nThe media composition mentioned earlier that remained \n\nunchanged was prepared using an already prepared \n\nconcentrated stock. In order to generate GA3 \n\nconcentrations of 0, 0.1, 0.25, 0.5, 1, and 2 mg/L, each \n\npresent in triplicate media jam jars with 50 ml media for \n\neach potato variety, a stock solution of GA3 at a \n\nconcentration of 50 mg/L was first made and added to \n\nthe media. Agar, at a concentration of 8 g/l, was utilized \n\nas the solidifying agent.  \n\nSingle node with identical sized were cultured in the \n\nprepared media under Laminar Air Flow (LAF) \n\ncondition and the cultured jam jars were incubated at  25 \n\n\u00b1 2\u00b0C, 16/8 hours of light/dark frequency per day with \n\nan intensity of 18 W/m2 [16] for 30 days. Results for the \n\nplants\u2019 shoot length, root length, number of roots, \n\nnumber of leaves, number of nodes, internode length, \n\nleaf size and dry biomass were observed at the 30th day \n\nof incubation.  \n\nii) Unchanged media composition + varying \n\nconcentrations of coconut water (CW), 0.25 mg/L GA3 \n\nand combination of 0.25 mg/L GA3 and 10 mg/L \n\ncoconut water 2.5 g/l Gellan gum as gelling agent  \n\nThe unchanged media composition was supplemented in \n\ntriplicates for each variety by 0, 10, 20, 50, 100 and 200 \n\nmL/L filtered CW in jam jar with net 50 ml media. For \n\ncomparison, two other experimental sets of media, one \n\nwith 0.25 mg/L GA3 and the other with the combination \n\nof 0.25 mg/L GA3 and 10 mg/L CW were prepared in \n\nsimilar manner. Gellan gum (ClarigelTM, Plant tissue \n\nculture tested from HiMedia) at a concentration of 2.5 \n\ng/L, was utilized as the solidifying agent. Nodal culture \n\nand result observation was done same as in previous \n\nsection. \n\nAnalysis of plants growth performance \n\nAs plants growth indicators, following parameters were \n\nanalyzed as suggested by [17]: Shoot length, root length, \n\nnumber of root hairs, number of nodes, dry biomass, leaf \n\nsize and internode length. \n\nPlant hardening: \n\nIn vitro plantlets that were 30 days old in jam jars were \n\nbrought out of the incubation chamber and placed in \n\nnormal screen house temperature for 5 to 7 days. Then \n\nthe plants were transplanted into a plastic seedling tray \n\nwith a sterile sand and soil substrate in a 2:1:1 ratio. The \n\ntransplant procedure was carried out as suggested by [18] \n\nin an enclosed space (an aphid-proof screen house) with \n\nspecified soil composition and fertilizer combination. \n\nAfter properly cleaning and removing all media residue \n\nfrom the plant roots, Bavistin 200 mg/ml was applied for \n\n30 seconds to the roots. Prior to transplantation, soil was \n\nimproved with 2 g/kg of urea, 2 g/kg of DAP, and 1.2 \n\ng/kg of potassium fertilizer, taking into account our soil \n\nfor 0.2 m2 area. The plants were irrigated with 5 ml sterile \n\nwater on daily basis.", "start_char_idx": 11384, "end_char_idx": 15090, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e490664b-02d7-4766-b1d4-39d0b819c8ad": {"__data__": {"id_": "e490664b-02d7-4766-b1d4-39d0b819c8ad", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7a7dc66d-5ddc-45f3-9bc3-5c582755c11e", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "a60b591ddc3968cdf2f36013b966adeb764f44689af1c937a03c3310accd1ee0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "865f3223-13b0-43be-ac41-c471c0280f3c", "node_type": "1", "metadata": {}, "hash": "e8c945cdbb0c0ae2b454c0e07b23dd8c3a73a196b3b19e159884ecf3efe1c08b", "class_name": "RelatedNodeInfo"}}, "text": "The plants were irrigated with 5 ml sterile \n\nwater on daily basis. The growth was observed for 15 \n\ndays and survival rate of the plants was evaluated.  \n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    30 \n\nData analysis \n\nData framing, table construction and bar graph design \n\nwas done using Microsoft Excel version 2013 and the \n\nanalysis of variance (ANOVA) followed by post-hoc \n\nanalysis by Tukey HSD for the mean comparison at 95% \n\nconfidence interval was done using R Studio V.2022.10.1. \n\nResults and Discussion \nThe variation in growth regulators used for propagating \n\npotato nodal propagation resulted in different responses \n\nin plant growth. However, the application of GA3 at \n\nvarious concentrations in agar media did not yield \n\nconclusive results compared to the control group without \n\nany growth regulators. None of the observed attributes \n\nshowed any improvement compared to the negative \n\ncontrol when GA3 was applied in concentrations ranging \n\nfrom 0 to 2 mg/ml in agar-based media (ABM), with a \n\n95% confidence interval. Nonetheless, the application of \n\nGA3 was found to be significantly effective when using \n\nClarigel-based media (CBM). \n\nFor the Janak Dev variety, the CBM was found to exhibit \n\ngreater root length (8.67 \u00b1 1.15 cm), quantity of root hairs \n\n(4.33 \u00b1 0.58), leaf size (33.67 \u00b1 5.86 mm2), and dry biomass \n\ncontent of the plants (12.48 \u00b1 0.88 mg) than the negative \n\ncontrol of ABM. However, there were only minor \n\ndifferences in the two varieties' node counts and \n\ninternode lengths. With a value of 10.03 \u00b1 0.31 cm, root \n\nlength for the cardinal variety was determined to be \n\nsuperior in ABM to CBM. The number of nodes and the \n\nnumber of nodes did not differ noticeably between the \n\ntwo circumstances. All other characteristics, including \n\nshoot length, number of root hairs, leaf size, and dry \n\nbiomass, respectively being 6 \u00b1 1 cm, 5.33 \u00b1 2.31, 23.33 \u00b1 \n\n8.02 mm2, and 11.84 \u00b1 2.69 mg, were significantly better \n\nexpressed in CBM than in ABM, both of which lacked \n\ngrowth regulator additions. \n\nWhen supplemented with 0.25 mg/ml GA3 to both of \n\nthese two basal media, CBM was better than ABM for \n\nboth cardinal and Janak Dev varieties in terms of all the \n\nattributes under investigation. The results are displayed \n\nin Table 1 and Table 2. \n\nFurther, comparing the performance of different \n\nconcentrations of CW (varying from 10 to 200 mg/L) with \n\nthat of 0.25 mg/L GA3 in CBM, addition of CW up to 200 \n\nmL/L expressed significantly higher values for root \n\nlength, number of root hairs, plant dry biomass and \n\nnumber of nodes for both varieties. Combination of 0.25 \n\nmg/ml GA3 and 10 mL/L CW was statistically found to \n\nbe superior for the enhancement of shoot length and leaf \n\nsizes for both varieties. GA3 resulted in longer internode \n\nlength than CW treatment in CBM for both varieties. The \n\nsurvival efficiency of the screen house acclimatized \n\nplantlets ranged in between 85 to 95% for both cultivars \n\nfrom all media systems used. Plants from Clarigel media \n\nsupplemented with GA3 had survival rate of 95% (19 \n\nsurvivors out of 20 transplanted plants) followed by the \n\nplants from CW supplied Clarigel media with the \n\nsurvival rate of 90%. Plants grown in AGM with GA3 had \n\nthe least survival rate of all (85%).  \n\nThe study investigated the effects of growth regulators \n\nand media on potato nodal propagation. GA3 in ABM \n\nshowed no conclusive results, but was effective in CBM. \n\nCBM outperformed ABM in root length, root hairs, leaf \n\nsize, and dry biomass for Janak Dev variety. ABM had \n\nbetter root length for cardinal variety.", "start_char_idx": 15023, "end_char_idx": 18688, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "865f3223-13b0-43be-ac41-c471c0280f3c": {"__data__": {"id_": "865f3223-13b0-43be-ac41-c471c0280f3c", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e490664b-02d7-4766-b1d4-39d0b819c8ad", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "4217ccc17d954562b204db4161246832a145d9b567183155813912dbac5c6ad1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c71ea141-936b-4596-90f5-002892704315", "node_type": "1", "metadata": {}, "hash": "1aafb61ab467ecd458b440d2905e0d4c2c3f376f521980ae0273a7a20ef0bfcd", "class_name": "RelatedNodeInfo"}}, "text": "ABM had \n\nbetter root length for cardinal variety. CBM with GA3 \n\nshowed superior performance for both varieties.  The \n\nability of Clarigel, a bacterial polysaccharide to gel more \n\nconsistently than agar, a polysaccharide from seaweeds \n\nand its physical smoothness and softness when \n\ncompared to agar aid in the uniform growth of in vitro \n\nplants [19]. Study of Clarigel\u2019s advantages over agar in \n\nthe tissue culture of potato remains limited, it\u2019s better \n\nwater retention qualities than agar, can provide a more \n\nconstant and suitable moisture level for the tissue culture. \n\nWater availability has a significant impact on plant \n\nmetabolism and nutrient absorption. However, in some, \n\ngelrite have been reported to show hyperhydration \n\nwhich in case of some plants like those with hard woods \n\nwas proven to be a drawback of this very nature of gelrite \n\n[20]. The inertness and purity of Clarigel provide more \n\naccurate results, especially considering that the MS basal \n\nmedia is already defined based on salts. In contrast, agar \n\ncontains various impurities, including salts of metals and \n\nsulfur. [21].  \n\nThe results presented here represent the mean values \u00b1 \n\nstandard deviation of three separate observations. The \n\nletters superscripted with the numerical values denote \n\nthe groupings obtained from the Tukey HSD test with an \n\nalpha of 0.05 conducted after ANOVA. Values sharing \n\nthe same letter are not significantly different at the 95% \n\nconfidence level. \n\nThe results presented here represent the mean values \u00b1 \n\nstandard deviation of three separate observations. The \n\nletters superscripted with the numerical values denote \n\nthe groupings obtained from the Tukey HSD test with an \n\nalpha of 0.05 conducted after ANOVA. Values sharing \n\nthe same letter are not significantly different at the 95% \n\nconfidence level. \n\n  \n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    31 \n\nTable 1: Variation of GA3 concentration using Agar based media (ABM) \n\nVariety \n\nGA3 \nconcent-\n\nration \n(mg/ml) \n\nShoot \nlength \n(cm) \n\nRoot length \n(cm) \n\nNumber of \nroot hairs \n\nNumber of \nnodes \n\nInternode \nlength (cm) \n\nDry biomass \n(mg) \n\nBiomass (%) \n\nJanak \nDev \n\n0 8.07a \u00b1 0.42 4.47a \u00b1 0.93 3.67a \u00b1 1.15 8.33ab \u00b1 1.53 0.99b \u00b1 0.19 9.06a \u00b1 1.76 8.58ab \u00b1 0.85 \n0.1 8.57a \u00b1 1.38 5.33a \u00b1 0.15 2ab \u00b1 0 10a \u00b1 1 0.86b \u00b1 0.16 10.05a \u00b1 0.82 9.77ab \u00b1 1.92 \n\n0.25 5.8a \u00b1 0.87 4.37a \u00b1 1.89 1b \u00b1 0 6.67ab \u00b1 2.08 0.92b \u00b1 0.28 10.02a \u00b1 0.61 11.31a \u00b1 0.78 \n0.5 7.73a \u00b1 1.42 6.73a \u00b1 0.06 1.67ab \u00b1 1.15 7.67ab \u00b1 0.58  1.01b \u00b1 0.12 9.34a \u00b1 1.22 8.96ab \u00b1 0.42 \n1 6.37a \u00b1 1.59 4.4a \u00b1 1.39 1.67ab \u00b1 0.58 6ab \u00b1 1 1.06ab \u00b1 0.15 8.46a \u00b1 2.44 8.35b \u00b1 1.10 \n2 8.53a \u00b1 3.06 3.8a \u00b1 0.98 2ab \u00b1 1 5.33b \u00b1 2.08 1.67a \u00b1 0.41 9.37a \u00b1 1.58 9.42b \u00b1 0.", "start_char_idx": 18638, "end_char_idx": 21411, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c71ea141-936b-4596-90f5-002892704315": {"__data__": {"id_": "c71ea141-936b-4596-90f5-002892704315", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "865f3223-13b0-43be-ac41-c471c0280f3c", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "0c071fc09fe98004052bd1f10aa9d08e2d700e23242a79d07f6c31bd665a9cd3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1e505253-aab1-48ee-b12f-68728be8f626", "node_type": "1", "metadata": {}, "hash": "9ec90696440d5aeaedea000da2198781b4b45a974371f6e29931dc34fd371092", "class_name": "RelatedNodeInfo"}}, "text": "41 9.37a \u00b1 1.58 9.42b \u00b1 0.33 \n\nCardinal \n\n0 5.5b \u00b1 0.46 10.03a \u00b1 0.31 2.67a \u00b1 0.58 7.67a \u00b11.53 0.73b \u00b1 0.10 10.46a \u00b1 1.57 9.84a \u00b1 0.68 \n0.1 7.93a \u00b1 0.86 9.57a \u00b1 0.50 2.33a \u00b1 0.58 6.67ab \u00b1 1.15 1.31ab \u00b1 0.38 11.56a \u00b1 3.08 10.48a \u00b1 2.01 \n\n0.25 7.47a \u00b1 0.67 8.47a \u00b1 1.01 2.33a \u00b1 0.58 4.33bc \u00b1 1.15 1.18ab \u00b1 0.03 12.33a \u00b1 3.06 10.39a \u00b1 2.03 \n0.5 8.27a \u00b11.17 8.83a \u00b1 0.75 2a \u00b1 0 7.33a \u00b1 1.15 1.14ab \u00b1 0.14 14.99a \u00b1 3.25 12.44a \u00b1 2.87 \n1 6.57ab \u00b1 .32 4.6b \u00b1 1.11 2.33a \u00b1 0.58 3.67c \u00b1 0.58 1.62a \u00b1 0.57 9.69a \u00b1 1.43 9.04a \u00b1 0.70 \n2 6.8ab \u00b1 0.1 8.07a \u00b1 1.34 2.66a \u00b1 0.58 6.33abc \u00b1 1.58 1.89a \u00b1 0.33 13.59a \u00b1 2.10 11.20a \u00b1 0.77 \n\n \n\nCW in CBM enhanced root length, root hairs, biomass, \n\nand nodes compared to the most widely used \n\nconcentration of GA3 in potato tissue culture (0.25 \n\nmg/L). GA3 and CW combination improved shoot \n\nlength and leaf size as shown in Figure 2. These findings \n\nindicate that the choice of growth regulator and media \n\ntype significantly influenced the growth and \n\ndevelopment of potato plants.  \n\nCW is a natural, organic source of growth-promoting \n\nelements such vitamins, minerals, amino acids, and \n\nphytohormone [22]. \n\nTable 2: Coconut water (CW) versus GA3 in Clarigel-based media (CBM) \n\nVariety Treatments \nShoot \nlength \n(cm) \n\nRoot length \n(cm) \n\nNumber of \nroot hairs \n\nNumber of \nnodes \n\nLeaf size \n(mm2) \n\nInternode \nlength (cm) \n\nDry biomass \n(mg) \n\nJanak \nDev \n\n0 7e \u00b1 1 8.67a \u00b1 1.15 4.33c \u00b1 0.58 7.67b \u00b1 0.58 33.67e \u00b1 5.86 0.92c \u00b1 0.2 12.48e \u00b1 0.88 \n\nCW 10 mL/L \n7.83e \u00b1 \n\n0.29 \n8.33a \u00b1 2.31 5.67c \u00b11.15 8.67b \u00b1 0.53  \n\n52.33d \u00b1 \n4.51 \n\n0.93c \u00b1 0.19 \n15.25de \u00b1 \n\n1.27 \n\nCW 20 mL/L 9.5de \u00b1 0.5 5.67a \u00b1 1.15 7c \u00b1 2 9.33ab \u00b1 0.58 \n54.33d \u00b1 \n\n2.52 \n1.02bc \u00b1 0.11 26.38c \u00b1 0.67 \n\nCW 50 mL/L 12cd \u00b1 1 6.83a \u00b1 1.26 \n7.33bc \u00b1 \n\n2.08 \n9.67ab \u00b1 0.58 \n\n55.67cd \u00b1 \n5.03 \n\n1.25bc \u00b1 0.18 \n29.52bc \u00b1 \n\n2.82 \n\nCW 100 mL/L \n13.33abc \u00b1 \n\n1.15 \n5.", "start_char_idx": 21385, "end_char_idx": 23255, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1e505253-aab1-48ee-b12f-68728be8f626": {"__data__": {"id_": "1e505253-aab1-48ee-b12f-68728be8f626", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c71ea141-936b-4596-90f5-002892704315", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "8151287fe84312bd9d30cb35cdd87ecb19be65b9d413435c9c5d1238a0bc1168", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7aefed6b-8f54-4a8a-8dcf-37d105dd592a", "node_type": "1", "metadata": {}, "hash": "5aa00a4e4becd748364072ec79f0482bbc0f2bfb16d14a0057d78a38a3031f82", "class_name": "RelatedNodeInfo"}}, "text": "82 \n\nCW 100 mL/L \n13.33abc \u00b1 \n\n1.15 \n5.5a \u00b1 0.5 \n\n11.67ab \u00b1 \n\n2.52 \n9.67ab \u00b1 0.58 76.67b \u00b1 3.79 1.39bc \u00b1 0.18 \n\n32.63ab \u00b1 \n1.72 \n\nCW 200 mL/L 15ab \u00b1 1 8.33a \u00b1 2.31 \n13.67a \u00b1 \n\n1.53 \n11a \u00b1 1  \n\n82.67ab \u00b1 \n7.64 \n\n1.37bc \u00b1 0.18 36.42a \u00b11.83 \n\nGA3 0.25 mg/L \n15.33a \u00b1 \n\n1.15 \n5.67a \u00b1 2.08 4.33c \u00b1 0.58 7.67b \u00b1 0.58 \n\n71.33bc \u00b1 \n2.52 \n\n2.01a \u00b10.27 \n14.13de \u00b1 \n\n0.83 \n\nCW 10 mL/L + \nGA3 0.25 mg/L \n\n12.33bcd \u00b1 \n1.53 \n\n6.67a \u00b1 0.58 \n7.67bc \u00b1 \n\n1.53 \n8b \u00b1 0 97a \u00b1 10.54 1.54ab \u00b1 0.19 18.44d \u00b1 2.40 \n\nCardinal \n\n0 6e \u00b1 1 7.67a \u00b1 1.15 5.33cd \u00b1 2.31 7.67 b \u00b1 0.58 \n23.33d \u00b1 \n\n8.02 \n0.78b \u00b1 0.08 11.84e \u00b1 2.69 \n\nCW 10 mL/L \n7.83de \u00b1 \n\n0.29 \n7.67a \u00b1 2.08 \n\n6.33bcd \u00b1 \n1.53 \n\n8.67ab \u00b1 1.53 50 c\u00b1 7.21 1.08b\u00b10.45 \n14.69de \u00b1 \n\n1.16 \nCW 20 mL/L 9.5cd \u00b1 0.5 5.67a \u00b1 1.15 7bc \u00b1 1 9.33ab \u00b1 0.58 55c \u00b1 6.24 1.02b \u00b1 0.11 22.78bc \u00b1 1.16 \n\nCW 50 mL/L 12bc \u00b1 1 6.83a \u00b1 1.26 \n8.67abc \u00b1 \n\n2.52 \n9.67ab \u00b1 0.58 \n\n65.33bc \u00b1 \n12.1 \n\n1.25b \u00b1 0.18 27.77b \u00b1 2.25 \n\nCW 100 mL/L \n13.33ab \u00b1 \n\n1.15 \n5.5a \u00b1 0.5 10 ab \u00b1 1 9.33ab \u00b1 0.58 86 ab \u00b1 4 1.43ab \u00b1 0.21 33.46a \u00b1 1.26 \n\nCW 200 mL/L 15a \u00b1 1 8a \u00b1 1.73 11.67a \u00b1 0.58 10.67a\u00b1 1.53 \n97.67 a \u00b1 \n\n6.51 \n1.42 ab \u00b1 0.25 38.46a \u00b1 1.57 \n\nGA3 0.25 mg/L 15a \u00b1 1.73 5.67a \u00b1 2.08 2.33d \u00b1 0.58 7.33b \u00b1 0.58 \n56.33c \u00b1 \n\n10.21 \n1.97a\u00b10.27 \n\n15.04de \u00b1 \n1.69 \n\nCW 10 mL/L + \nGA3 0.25 mg/L \n\n11bc \u00b1 1 6.67a \u00b1 0.58 9abc \u00b1 1 8ab \u00b1 0 \n107.67a \u00b1 \n\n5.86 \n1.37ab \u00b1 0.13 19.7cd \u00b1 3.19", "start_char_idx": 23216, "end_char_idx": 24589, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7aefed6b-8f54-4a8a-8dcf-37d105dd592a": {"__data__": {"id_": "7aefed6b-8f54-4a8a-8dcf-37d105dd592a", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1e505253-aab1-48ee-b12f-68728be8f626", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "a3da39539736cd3c77907777bf80aa3b6b8a75b1d6053812d31065f4a34ae249", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6cae94af-29e9-493b-a81f-0fb4c1e8cd84", "node_type": "1", "metadata": {}, "hash": "baab3e45a7c1759e52a73d0811032bcbaefa7fe831f2395b07b5a5baf2e06b26", "class_name": "RelatedNodeInfo"}}, "text": "37ab \u00b1 0.13 19.7cd \u00b1 3.19 \n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    32 \n\n  \n\na\n\nab\nab\n\na\n\na\n\nabc\nab\n\nc\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\nGG 200C GG G GG (G+C) Ag G\n\nR\no\n\no\nt \n\nle\nn\ng\nth\n\n (\nc\nm\n\n)\n\nCondoitionsb\nCardinal Janak Dev\n\na\n\nc\n\nbc\n\nc\n\na\n\nbc\n\nc\n\nd\n\n0\n\n5\n\n10\n\n15\n\n20\n\n25\n\n30\n\n35\n\n40\n\n45\n\nGG 200C GG G GG (G+C) Ag G\n\nD\nry\n\n b\nio\n\nm\na\nss\n\n (\nm\n\ng\n)\n\nCondoitions\nd\n\nCardinal Janak Dev\n\na a\n\nb\n\nc\n\na a\n\nab\n\nc\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\n18\n\nGG 200C GG G GG (G+C) Ag G\n\nS\nh\no\n\no\nt \n\nle\nn\ng\nth\n\n (\nc\nm\n\n)\n\nConditions\na\n\nCardinal Janak Dev\n\na\n\nc\n\nbc\n\nc\n\na\n\ncd\n\nbc\n\nd\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\nGG 200C GG G GG (G+C) Ag G\n\nN\nu\nm\n\nb\ne\nr \n\no\nf \n\nro\no\n\nt \nh\na\nir\n\ns\n\nCondoitionsc\nCardinal Janak Dev\n\na\n\nc\nbc\n\nc\n\na\n\nb\n\nb\n\nc\n\n0\n\n2\n\n4\n\n6\n\n8\n\n10\n\n12\n\n14\n\nGG 200C GG G GG (G+C) Ag G\n\nN\nu\nm\n\nb\ne\nr \n\no\nf \n\nn\no\n\nd\ne\ns\n\nCondoitionse\n\nCardinal Janak Dev\n\na\n\nb\n\na\n\nc\n\nab\n\nb\n\na\n\nc\n\n0\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\nGG 200C GG G GG (G+C) Ag G\n\nL\ne\na\nf \n\nsi\nz\ne\n (\n\nm\nm\n\n2\n)\n\nCondoitionsf\nCardinal Janak Dev\n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    33 \n\n   \nFigure 2: Comparison of shoot length (a), root length (b), \n\nnumber of root hairs (c), dry biomass (d), number of nodes per \n\nplant (e), leaf size (f) and average internode length (g) of \n\ncardinal and Janak dev varieties of potato plantlets in tissue \n\nculture condition grown in: gellan gum/Clarigel based media \n\nwith 200 mL/L coconut water [GG 200C], gellan gum/Clarigel \n\nbased media with 0.25 mg/L GA3 [GG G], gellan gum/Clarigel \n\nbased media with combination of 0.25 mg/L GA3 and 10 mL/L \n\ncoconut water [GG(G+C)] and agarbased media with 0.25 \n\nmg/L GA3 (Ag G). \n\nCompared to synthetic phytohormones like GA3, it \n\ncomprises a complex blend of substances that can offer a \n\nmore complete and balanced nutrient profile for plant \n\ngrowth. Auxins, cytokinins, and gibberellins, among \n\nother growth-promoting compounds found in CW, can \n\njointly affect different aspects of plant growth and \n\ndevelopment [23]. This encompassing effect boost a \n\nvariety of tissue culture performance factors, including \n\nroot length, root hairs, biomass, and node development \n\n[24]. On root initiation and development, CW has \n\nreportedly been shown to have remarkable effects. It \n\nincludes auxins, especially indole-3-acetic acid (IAA), \n\nwhich is essential for promoting root growth and \n\nexpanding the quantity of root hairs [24].", "start_char_idx": 24564, "end_char_idx": 27030, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6cae94af-29e9-493b-a81f-0fb4c1e8cd84": {"__data__": {"id_": "6cae94af-29e9-493b-a81f-0fb4c1e8cd84", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7aefed6b-8f54-4a8a-8dcf-37d105dd592a", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "64cd9ff1ffc3a6a4a93da8c48a0b2dca0784d19c313e94cefdf2682aaa9d63bc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d9b71749-16d6-4791-9ad5-419324c88ee6", "node_type": "1", "metadata": {}, "hash": "c5c18b1109280b8fef06f375548d6cb33d5d260b12999edc96efefa3b0f81d04", "class_name": "RelatedNodeInfo"}}, "text": "In this very study, the addition of CW at certain \n\nconcentrations showed positive effects on various \n\nattributes in CBM. These results provide valuable \n\ninsights for optimizing potato nodal propagation \n\ntechniques and selecting appropriate growth regulators \n\nand media combinations to enhance plant growth and \n\nbiomass production. Based on the findings of this study, \n\nit is recommended that adding CW supplements to \n\nClarigel or gelrite-based media in the range of 50 to 200 \n\nmg/ml can be helpful for improving a variety of traits in \n\nplant tissue culture. Additionally, 10 mL/L of CW and \n\n0.25 mg/L of GA3 in Clarigel or gelrite-based media have \n\ndemonstrated encouraging effects when combined. \n\nConclusion \nIn comparison to GA3 in agar-based media, the use of \n\ncoconut water (CW) in Clarigel based medium greatly \n\nimproves the health and development of in vitro potato \n\nplants. CW has the potential to be a safe and efficient \n\ngrowth-enhancing agent for potato nodal propagation \n\ndue to its superior efficacy in promoting root length, root \n\nhairs, leaf size, dry biomass, shoot length, and root \n\ngrowth. These results underline how crucial it is to \n\nchoose growth regulators and media carefully in order to \n\nachieve the best plant quality and multiplication in \n\npotato plantlet in in vitro conditions. Exploring the \n\nunderlying principles and possible uses of CW in \n\nenhancing potato propagation methods call for more \n\ninvestigation. \n\nAcknowledgement \nOur sincere gratitude to the Kathmandu University-\n\nIntegrated Rural Development Program/Nepal \n\nTechnology Innovation Center (KU-IRDP/NTIC) for \n\ngrant support and Khadyanna Tatha Biu Aaalu Alaichi \n\nTatha Falful Nursery Udyog, Panauti-3, Kavre, for \n\nproviding us with the potato materials and screen house \n\nas workspace. \n\nReference \n1.  Gamborg OL. Plant tissue culture. biotechnology. milestones. In \n\nVitro Cellular & Developmental Biology-Plant. 2002 Mar;38:84-92. \nhttps://doi.org/10.1079/IVP2001281 \n\n2.  Hussain T. Potatoes: ensuring food for the future. Adv Plants Agric \nRes. 2016;3(6):178-82. \n\n3.  Gong H, Igiraneza C, Dusengemungu L. Major in vitro techniques \nfor potato virus elimination and post eradication detection \nmethods. A review. American journal of potato research. 2019 Aug \n15;96:379-89. https://doi.org/10.1007/s12230-019-09720-z \n\n4.  Shoala T, Eid KE, El-Fiki IA. Impact of chemotherapy and \nthermotherapy treatments on the presence of potato viruses PVY, \nPVX and PLRV in tissue-cultured shoot tip meristem. Journal of \nPlant Protection and Pathology. 2019 Dec 1;10(12):581-5. \n\n5.  MoAl-2018/2019. Statistical Information on Nepalese Agriculture \n(2018/2019). 2019.  \n\n6.  Kaur A, Kumar A. The effect of gelling agent, medium pH and silver \nnitrate on adventitious shoot regeneration in Solanum tuberosum. \nbioRxiv. 2020 Jan 3:2020-01. \n\n7.  Repalli SK, Geda CK, Pradhan NS, Rao GJ. Influence of Additional \nNutrients and Gelling Agents on in Vitro Response of Selected \nIndica Rice Varieties. International Journal of Biology. 2021;11(4):1-\n26. \n\n 8.  Murashige T, Skoog F. A revised medium for rapid growth and bio \nassays with tobacco tissue cultures. Physiologia plantarum. 1962 \nJul;15(3):473-97. https://doi.org/10.1111/j.1399-\n3054.1962.tb08052.x  \n\n9.  Phillips GC, Garda M. Plant tissue culture media and practices: an \noverview. In Vitro Cellular & Developmental Biology-Plant.", "start_char_idx": 27034, "end_char_idx": 30438, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d9b71749-16d6-4791-9ad5-419324c88ee6": {"__data__": {"id_": "d9b71749-16d6-4791-9ad5-419324c88ee6", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6cae94af-29e9-493b-a81f-0fb4c1e8cd84", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "30ce6c3b03cf33d8c2e0f75f03c234b3a3902bdfdce70acb9b4ca4a0a785a870", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "be6e5547-bc39-44a6-b762-84de0ef63e85", "node_type": "1", "metadata": {}, "hash": "2b5d4eb56c1d7cdef0b647e40fb6cae51475757462e12bfb3f708cfcb529c22c", "class_name": "RelatedNodeInfo"}}, "text": "In Vitro Cellular & Developmental Biology-Plant. 2019 \nJun 15;55:242-57. https://doi.org/10.1007/s11627-019-09983-5 \n\n 10.  Mehmood A, Shah AH, Sajid M, Ahmad H. Investigation of \nGA3 effect on in vitro micropropagation of potato varieties. \nInternational Journal of Agronomical and Agricultural Research. \n2016;9(5):21-30. \n\n 11.  Sembiring R, Hayati M, Kesumawti E. Potato tuber (Solanum \ntuberosum L.) formation due to the application of different \nconcentrations of coconut water in in-vitro. InIOP Conference \n\nab a\n\nb\n\nb\n\nc\n\na\n\nab\n\nbc\n\n0\n\n0.2\n\n0.4\n\n0.6\n\n0.8\n\n1\n\n1.2\n\n1.4\n\n1.6\n\n1.8\n\n2\n\nGG 200C GG G GG (G+C) Ag G\n\nIn\nte\n\nrn\no\n\nd\ne\n l\n\ne\nn\ng\nth\n\n (\nc\nm\n\n)\n\nCondoitions\ng Cardinal Janak Dev\n\n\n\nNepal J Biotechnol. 2 0 2 3  J u l y ;  1 1  (1):27-34     Kafle et al.  \n\n\u00a9NJB, BSN    34 \n\nSeries: Earth and Environmental Science 2021 Mar 1 (Vol. 711, No. \n1, p. 012021). \n\n12.  Hafsan H, Mustami MK, Masriany M, Aziz IR, Mustakim M. The \nUtilization of Coconut Water Waste as a Growth Media of the In-\nVitro Potato Cutting. Scientiae Educatia: Jurnal Pendidikan Sains. \n2018 Dec 31;7(2):108-16. \n\n \n13.  Bhuiyan FR. In vitro meristem culture and regeneration of three \n\npotato varieties of Bangladesh. Research in Biotechnology. 2013 Jun \n24;4(3). \n\n14.  Cassells AC, Long RD. The elimination of potato viruses X, Y, S and \nM in meristem and explant cultures of potato in the presence of \nVirazole. Potato Research. 1982 Jun;25:165-73. \nhttps://doi.org/10.1007/BF02359803 \n\n15.  Sarkar D, Naik PS. Phloroglucinol enhances growth and rate of \naxillary shoot proliferation in potato shoot tip cultures in vitro. \nPlant Cell Tissue Organ Cult. 2000;60(2):139\u201349.  \n\n16.  Da Rocha PS, de Oliveira RP, Scivittaro WB. New light sources for \nin-vitro potato micropropagation. Orignal Article Biosci J. \n2015;(5):1312\u20138.  \n\n17.  Murshed R, Najla S, Albiski F, Kassem I, Jbour M, Al-Said H. Using \ngrowth parameters for in-vitro screening of potato varieties tolerant \nto salt stress. 2018 Feb 20.  \n\n18.  Sakha BM, Rai GP, Dhital SP, Nepal RB. Disease-free Pre-Basic Seed \nPotato Production through Tissue Culture in Nepal. Nepal Agric \nRes J. 2014;8(November 2014):7\u201313.  \n\n19.  Mohamed GM, Amer AM, Osman NH, Sedikc MZ, Hussein MH. \nEffects of different gelling agents on the different stages of rice \nregeneration in two rice cultivars. Saudi Journal of Biological \nSciences. 2021 Oct 1;28(10):5738-44. \n\n20.  Zimmerman TW, Cobb BG. Vitrification and soluble carbohydrate \nlevels in Petunia leaves as influenced by media Gelrite and sucrose \nconcentrations. Plant Cell Reports. 1989 Jun;8:358-60. \nhttps://doi.org/10.1007/BF00716673 \n\n21.", "start_char_idx": 30390, "end_char_idx": 33024, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "be6e5547-bc39-44a6-b762-84de0ef63e85": {"__data__": {"id_": "be6e5547-bc39-44a6-b762-84de0ef63e85", "embedding": null, "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-274", "node_type": "4", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "152a4c5023c4dfb8a61e1c6195f0e5a0b67eb95487c0d7de8c8df89e58985afb", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d9b71749-16d6-4791-9ad5-419324c88ee6", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "e1aedeeebbc9faacc06b944d8b9c4caa1e4325108ab4a53644e9b7a9a59f02d4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "27695f2d-bcc3-4bf4-bf59-3c8347937c2b", "node_type": "1", "metadata": {}, "hash": "b04062e54cac813896d4e94599f0e8c15f49d7ea308428befe46a989d4645f64", "class_name": "RelatedNodeInfo"}}, "text": "Scherer PA, M\u00fcller E, Lippert H, Wolff G. Multielement analysis of \nagar and gelrite impurities investigated by inductively coupled \nplasma emission spectrometry as well as physical properties of \ntissue culture media prepared with agar or the gellan gum gelrite. \nInInternational Symposium on Propagation of Ornamental Plants \n226 1987 Aug 23 (pp. 655-658). \nhttps://doi/10.17660/ActaHortic.1988.226.91 \n\n22.  Yong JW, Ge L, Ng YF, Tan SN. The chemical composition and \nbiological properties of coconut (Cocos nucifera L.) water. \nMolecules. 2009 Dec 9;14(12):5144-64. \nhttps://doi.org/10.3390/molecules14125144 \n\n23.  Hafsan H, Mustami MK, Masriany M, Aziz IR, Mustakim M. The \nUtilization of Coconut Water Waste As A Growth Media of The in \nvitro Potato Cutting. Sci Educ. 2019 Jan 2;7(2):108.  \n\n24.  Aishwarya PP, Seenivasan N, Naik DS. Coconut water as a root \nhormone: Biological and chemical composition and applications. \nMagnesium (mg/100mL). 2022;22(20.87):31-65. \n\n \n\nhttps://doi.org/10.17660/ActaHortic.1988.226.91", "start_char_idx": 33026, "end_char_idx": 34053, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "27695f2d-bcc3-4bf4-bf59-3c8347937c2b": {"__data__": {"id_": "27695f2d-bcc3-4bf4-bf59-3c8347937c2b", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "be6e5547-bc39-44a6-b762-84de0ef63e85", "node_type": "1", "metadata": {"identifier": "njb-274", "author": "NJB, Nepal Journal of Biotechnology", "title": "Effect of Coconut Water and GA3 Concentrations on in vitro Clonal Propagation of Potato Cultivars from Nepal", "date": "2023-07-31", "file": "njb-274.pdf"}, "hash": "42d7cceba85dab6ca8eff4b4f7f8b0a121e451ff783bf52d022ee5090bb0e495", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d9435550-e3b6-42e3-9ee8-e407eb4422ff", "node_type": "1", "metadata": {}, "hash": "105d9e1bb9f892c6cc568a851e93f70e7ab601f459a9d71e578b751a3db04c46", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 13-18   ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   13                    Nepjol.info/index.php/njb \n\n \n\nCharacterization of Nepalese Barley Gene Pool for Leaf Rust \nResistance \n\nResham Babu Amgai1*, Sumitra Pantha2, Madan Raj Bhatta3 \n1Biotechnology Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. \n\n2Agriculture Botany Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. \n3National Plant Genetic Resource Centre, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. \n\nAbstract \nBarley (Hordeum vulagare L) is the major crop for the people living in the high hills and mountainous \n\nregion of Nepal. Leaf rust (caused by Puccinia hordei) is one of the major production threats for barley \n\ncultivation. A lot of variation can be observed on Nepalese barley accessions with respect to leaf rust \n\nresistance characteristics. Two hundred and forty one barley accessions were screened for leaf rust \n\nresistance characteristics on heading stage at Khumaltar, Lalitpur, Nepal. Among them, one hundred \n\nand nine Nepalese barley accessions showing promising for disease resistance were screened using \n\nsix SSR markers linked to leaf rust resistance genes. Bonus and Local Jau was used as the resistant and \n\nsusceptible check respectively. Leaf rust resistance genes Rph1, Rph2, Rph3, Rph7, QBLR-P and QTL on \n\nchromosome 5HS were detected on Nepalese barley accessions using respective SSR markers. Eight \n\nNepalese barley accessions showed presence of three and more leaf rust resistant genes. The poor \n\nrelationship between the field disease resistance and molecular markers linked with specific leaf rust \n\nresistance gene proved that Nepalese barley gene pool contains other leaf resistance genes. \n\nKeywords: leaf rust, Puccinia hordei, resistant gene, Nepalese barley, Simple Sequence Repeats (SSR) \n\n*Corresponding Author \n\nEmail: reshamamgain@yahoo.com \n\nIntroduction \nBarley (Hordeum vulgare L.) occupies total area of \n\n29598 hectare and total production 33782 metric \n\nton with average productivity of 1.141 metric \n\nton/ha in Nepal [1]. The maximum area of the \n\ncrop lies in the mid-western development region. \n\nOut of the total barley area, more than 50 % is in \n\nthe hill region, while 40% is in the mountain \n\nregion [2]. However, its production and \n\nproductivity is declining due to diseases and \n\nunpredictable climatic condition of the mountain \n\narea [3]. Leaf rust (caused by Puccinia hordei) is one \n\nof the major problematic diseases for barley \n\nproduction in Nepal [4]. Therefore, rust resistant \n\nbarley varieties are another need of mountain \n\nfarmers. \n\nNepal harbours hundreds of the barley landraces. \n\nHigh level of genetic variation can be observed in \n\nNepalese barley [5]. Similarly, a lot of variation \n\nwas observed among the Jumla collection of \n\nNepalese barley for many yield attributing \n\ncharacters [6]. Many of these landraces possess \n\none or more characteristics for abiotic and biotic \n\nstress tolerance [2,3]. Variation on rust resistance \n\ncharacteristics is very important for rust resistance \n\nbreeding program. \n\nTherefore, use of these germplasm for rust \n\nresistance gene pyramiding is highly beneficial to \n\nNepalese farmers. Identification of particular rust \n\nresistance gene and its incorporation is the only \n\noption for the development of barley varieties for \n\nleaf rust rust resistance. Use of the molecular \n\nmarker for this purpose is highly sought for this.", "start_char_idx": 47, "end_char_idx": 3665, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d9435550-e3b6-42e3-9ee8-e407eb4422ff": {"__data__": {"id_": "d9435550-e3b6-42e3-9ee8-e407eb4422ff", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "27695f2d-bcc3-4bf4-bf59-3c8347937c2b", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "71d9b2c9761a6a3016c79c72dc798c4302c28ce1ae81b790c4878a238180babf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "37061136-2fd0-4093-aa84-c6f849896a6c", "node_type": "1", "metadata": {}, "hash": "fbb4ca60fdc3ba121bc18f55982e3bdb8b93741219ae3eabb63747a123924369", "class_name": "RelatedNodeInfo"}}, "text": "Use of the molecular \n\nmarker for this purpose is highly sought for this. \n\nMaterial and Methods \nGermplasm Collection \n\nOne hundred and fifty five Nepalese barley \n\naccessions (NPGR No.s) collected from different \n\nparts of Nepal were obtained from National Plant \n\nGenetic Resources Centre (NPGRC); and forty \n\nseven Jumla collection (JC# series), two local \n\ncollection (Acc# series) and thirty six barley \n\nbreeding lines (NB, B, GR and Xveola series) were \n\ncollected from Hill Crops Research Program \n\n(HCRP), Dolakha of Nepal Agricultural Research \n\nCouncil (NARC) (Table 1). Similarly, one hundred \n\nand nine Nepalese barley accessions were selected \n\nbased on their disease resistance data (Table 2) for \n\n  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:13-18   Amgai et al.  \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   14                    Nepjol.info/index.php/njb \n\n \n\nTable 1: Nepalese barley accessions showing variation on leaf rust resistance characteristics.  \n\nGenotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust \n\n1522 \n \n\n2060 \n \n\n2496 \n \n\n7436  JC#14 \n \n\n1535 \n \n\n2062 \n \n\n2505 \n \n\n7441  JC#15 \n \n\n1537 \n \n\n2064 \n \n\n2506 \n \n\n8252  JC#16 \n \n\n1538 \n \n\n2065 \n \n\n2507 \n \n\n9436  JC#17 \n \n\n1540 \n \n\n2066 \n \n\n2508 \n \n\n9963  JC#18 \n \n\n1543 \n \n\n2069 \n \n\n2511 \n \n\n12069  JC#19 80s \n\n1544 \n \n\n2072 \n \n\n2512 \n \n\n12538  JC#21 40s \n\n1545 \n \n\n2073 \n \n\n2513 40s 20774  JC#22 \n \n\n1546 \n \n\n2074 \n \n\n2514 \n \n\n22463  JC#23 \n \n\n1547 \n \n\n2075 \n \n\n2515 \n \n\n112-14  JC#24 \n \n\n1550 \n \n\n2078 \n \n\n2518 \n \n\nAcc#1545  JC#25 \n \n\n1574 \n \n\n2079 \n \n\n2520 \n \n\nAcc#1574  JC#26 60s \n\n1575 \n \n\n2080 \n \n\n2523 \n \n\nArupos/oy-\nB-oy \n\n JC#27 \n \n\n1576 \n \n\n2081 \n \n\n2525 \n \n\nB86019-1-0  JC#28 \n \n\n1589 \n \n\n2082 \n \n\n2527 \n \n\nB86019-1K2  JC#29 \n \n\n1664 \n \n\n2083 \n \n\n2530 \n \n\nB86023-1K ts JC#31 \n \n\n1999 \n \n\n2084 \n \n\n2532 \n \n\nB86065-1-4  JC#32 80s \n\n2000 \n \n\n2086 \n \n\n2533 \n \n\nB86099-1K  JC#33 80s \n\n2001 \n \n\n2087 \n \n\n2534 tms B86099-2-1  JC#34 20s \n\n2002 \n \n\n2088 \n \n\n2539 \n \n\nB86099-2K  JC#35 \n \n\n2003 \n \n\n2089 \n \n\n2542 \n \n\nB86115  JC#36 60s \n\n2004 \n \n\n2090 \n \n\n5177 \n \n\nB86122-1-5  JC#37 90s \n\n2005 \n \n\n2181 \n \n\n5617 \n \n\nB86122-1-5-\n0K3 \n\n JC#38 ts \n\n2008 \n \n\n2244 \n \n\n6035 \n \n\nB86122-1K  JC#39 \n \n\n2009 \n \n\n2447 \n \n\n6036 \n \n\nB86152-2  JC#41 \n \n\n2010 \n \n\n2453 \n \n\n6038 \n \n\nB86152-2-2-0-\n0K \n\n JC#42 60s \n\n2011 \n \n\n2454 \n \n\n6041 \n \n\nB86157-1-1-5-\n0-0K3 \n\n JC#43 20s \n\n2013 \n \n\n2456 \n \n\n6044 \n \n\nB86615-1-4  JC#44 40ms", "start_char_idx": 3592, "end_char_idx": 6052, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "37061136-2fd0-4093-aa84-c6f849896a6c": {"__data__": {"id_": "37061136-2fd0-4093-aa84-c6f849896a6c", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d9435550-e3b6-42e3-9ee8-e407eb4422ff", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "26875201db7790863b61d81ebd07482bd34f2b18989e4149d3b093bad01a352e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "47c54904-1f62-4d94-9584-a9980157a196", "node_type": "1", "metadata": {}, "hash": "50e77ad79fb51553c3fd13ceda39d4e0d6935d889df34f483c0e6e2c7f1f3a59", "class_name": "RelatedNodeInfo"}}, "text": "6044 \n \n\nB86615-1-4  JC#44 40ms \n\n2014 \n \n\n2457 \n \n\n6045 \n \n\nB90K-007-1  JC#45 40s \n\n2016 \n \n\n2458 \n \n\n6055 \n \n\nB90K-01-2K  JC#48 40s \n\n2018 \n \n\n2459 \n \n\n6063 80s B90K-014-1 ts JC#49 \n \n\n2023 \n \n\n2461 \n \n\n6235 \n \n\nB90K-024-1  JC#50 \n \n\n2024 \n \n\n2464 \n \n\n6289 \n \n\nB90K-038  JC#51 60s \n\n2027 \n \n\n2465 \n \n\n6292 \n \n\nB90K-090  Jumla coll \n \n\n2029 \n \n\n2467 \n \n\n6293 \n \n\nBonus  \nLG-\n\n51/Xve  \n\n2033 \n \n\n2468 \n \n\n6304 \n \n\nCOQ/KI/Pes\ncii \n\n Local Jau \n \n\n2035 \n \n\n2469 \n \n\n6309 \n \n\nGR-25-85  Maticos \n \n\n2037 \n \n\n2470 \n \n\n6310 \n \n\nJC#01  \nNB-1003-\n\n37  \n\n2040 \n \n\n2471 \n \n\n6311 \n \n\nJC#02  \nNB-1003-\n37/1034  \n\n2042 \n \n\n2472 \n \n\n6315 \n \n\nJC#03  \nNB-1003-\n37/1038  \n\n2043 90ms 2473 \n \n\n6316 \n \n\nJC#04  \nNB-1003-\n37/1214  \n\n2046 \n \n\n2482 \n \n\n6319 \n \n\nJC#05  \nNB-\n\n1207/CI  \n\n2048 \n \n\n2483 \n \n\n6320 \n \n\nJC#06 20s Xveola-12 \n \n\n2049 \n \n\n2485 \n \n\n6326 \n \n\nJC#07 60s XVeola-13 \n \n\n2050 \n \n\n2486 \n \n\n6327 \n \n\nJC#08 10s XVeola-28 \n \n\n2051 \n \n\n2487 \n \n\n6334 \n \n\nJC#09 30s Xveola-38 \n \n\n2056 \n \n\n2488 \n \n\n6342 \n \n\nJC#11 20s Xveola-43 \n \n\n2057 \n \n\n2491 \n \n\n6447 \n \n\nJC#12  \n  \n\n2058 \n \n\n2494 40s 6557 \n \n\nJC#13  \n  \n\nNote: r-resistance, mr-moderately resistance, tms-trace moderately susceptible, tmr-trace moderately resistance, ts-trace susceptible, s-\nsusceptible; blank=no disease i.e. 0 score", "start_char_idx": 6021, "end_char_idx": 7297, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "47c54904-1f62-4d94-9584-a9980157a196": {"__data__": {"id_": "47c54904-1f62-4d94-9584-a9980157a196", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "37061136-2fd0-4093-aa84-c6f849896a6c", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "0cbb54dac8f222df3e412297827706544e9d98dfe8292f83fa85c8010baebfb2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b04235e4-4a58-469a-96a6-d92ed1265976", "node_type": "1", "metadata": {}, "hash": "ff2cd1b890f3dd776fc86243ad857c3af31092adf850b3494b6e28b5b5f4f236", "class_name": "RelatedNodeInfo"}}, "text": "s-\nsusceptible; blank=no disease i.e. 0 score \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:13-18   Amgai et al.  \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   15                    Nepjol.info/index.php/njb \n\n \n\n  \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n  \n\nTable 2: Nepalese barley accessions used for screening leaf rust resistance using simple sequence repeats marker \n\n1535 2002 2046 2081 2458 2483 6036 6327 8242 JC#18 \n\n1537 2003 2051 2082 2459 2489 6293 6340 8244 JC#46 \n\n1538 2004 2062 2084 2461 2494 6304 6342 8245 JC#49 \n\n1540 2008 2064 2086 2464 2496 6309 6343 8246 Local Jau \n\n1544 2009 2069 2087 2467 2505 6310 6344 8252 Maticos \n\n1545 2010 2072 2088 2468 2506 6314 6350 9436 NB-1003-37 \n\n1572 2014 2073 2089 2470 2508 6315 6557 9963 NB-1003-37/1038 \n\n1574 2023 2074 2090 2471 2515 6316 7436 \nArupos/oy-\n\nB-oy \nSolu Uwa \n\n1575 2026 2075 2441 2478 2537 6319 7441 \nB86122-1-5-\n\n0K3 \nXveola-12 \n\n1594 2033 2078 2456 2480 2541 6320 8239 Bonus XVeola-13 \n\n1601 2043 2080 2457 2482 2542 6326 8240 GR-25-85 Xveola-43 \n\n2000 \n         \n\nNote: Name as numbers without any alphabet denote the Nepalese Plant Genetic Resource (NPGR) number. \nTable 3: SSR markers used to identify presence of leaf rust resistance gene in Nepalese barley gene pool \n\nMarker \nName \n\nSequence-F \n[5'\u2026 \u2026 3'] \n\nSequence-R \n[5'\u2026 \u2026 3'] \n\nAnnealing \nTemperature \n\nStripe rust \nresistance \n\ngene \n\nPCR \nproduct \n\nsize \n\nChromosome \nNo. \n\nRef \n\nAY642926-\nCA11 \n\nCCAAAAAC\nAATTGAGAA\nAACCCTA \n\nCCTCCC \nTGAGAG \nACCTCCTAT T \n\n58 Rph7 183 3H [12] \n\nBmac096 \nGCTATGGCG\nTACTATGTA\nTGGTTG \n\nTCACGATGA\nGGTATGATCA\nAAGA \n\n58 Rph2 173 5HS [13] \n\nBmag0225 \nAACACA CA \nAAATATTAC\nAT CA \n\nCGAGTAGTTC\nCC ATG TGA C \n\n58 Rph7 162 3H [14] \n\nBmag337 \nACAAAGAG\nGGAGTAGTA\nCGC \n\nGACCCATGAT\nATATGAAGA\nTCA \n\n55 QTL 129-150 5HS [13] \n\nEBmac075\n5 \n\nAGCCTTGTG\nTATCAGGAC\nA \n\nCTGCTGGTGT\nTCTCTAAAAG\nT \n\n55 Rhp3 Rhp19 144-155 7HL [15] \n\nRis44 \nACGGATCTA\nCTTTAGCTA\nGCA \n\nAAACAACCC\nCACACAATC \n\n58 QBLR-P 105-110* 7HL [16] \n\nNote: *=product size determined based on field data for disease resistance", "start_char_idx": 7252, "end_char_idx": 9293, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b04235e4-4a58-469a-96a6-d92ed1265976": {"__data__": {"id_": "b04235e4-4a58-469a-96a6-d92ed1265976", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "47c54904-1f62-4d94-9584-a9980157a196", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "a3d7cf9276e3a94d669ed1bfe7add0f33960dabbaff6f1fe675bc2eb7cd316e6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6738509c-c2fc-48e6-85b0-a17354a66159", "node_type": "1", "metadata": {}, "hash": "67565ea074ba8a40e3b7717203507b95f520f657f682a3e97bccf125658c73fa", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:13-18   Amgai et al.  \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   16                    Nepjol.info/index.php/njb \n\n \n\nTable 4: Nepalese barley germplasm with different leaf rust resistant gene identified using different \n\nmolecular markers \n\nRph7 \n\n(AY642926-\n\nCA11) \n\nRph2 \n\n(Bmac096) \n\nRph3 & Rph1 \n\n(EBmac0755) \nRph7 (Bmag0225) \n\nQTL on 5HS \n\n(Bmag0337) \n\nQBLR-P \n\n(Ris44) \n\n2480 GR-25-85 1538 GR-25-85 1540 GR-25-85 \n\n2062 2470 2008 1545 1601 1544 \n\n2483 2471 2033 1574 2010 1545 \n\n2541 2515 2062 2010 2023 1574 \n\nSolu Uwa 6036 2478 2080 2033 1575 \n\n \n1537 2480 2505 2043 1601 \n\n \n2074 2483 2508 2051 2002 \n\n \n2082 2489 6304 2478 2003 \n\n \n2506 2515 6310 2480 2004 \n\n \nNB-1003-37 2541 6315 2482 2072 \n\n \n\nNB-1003-\n\n37/1038 \n6342 6343 2489 2073 \n\n \nXveola-43 8252 6350 2494 2075 \n\n  \n9963 2000 2505 2078 \n\n  \n1594 2026 2515 2080 \n\n  \n2506 6309 2537 2081 \n\n  \n2537 Arupos/oy-B-oy 2541 2084 \n\n  \n6557 Bonus 6036 2086 \n\n  \nJC#46 JC#49 6340 2088 \n\n  \nJC#49 Local Jau 6342 2089 \n\n  \nNB-1003-37/1038 Maticos 6344 2090 \n\n  \nSolu Uwa NB-1003-37 8246 2456 \n\n  \nXveola-43 XVeola-13 8252 2457 \n\n    \n9963 2459 \n\n    \n1537 2461 \n\n    \n2000 2464 \n\n    \n2014 2467 \n\n    \n2046 2468 \n\n    \n2074 2470 \n\n    \n2082 6304 \n\n    \n2506 6310 \n\n    \n2542 6314 \n\n    \n6557 6315 \n\n    \n7436 6316 \n\n    \n8239 6319 \n\n    \n8245 6326 \n\n    \nB86122-1-5-0K3 6327 \n\n    \nJC#49 1535 \n\n    \nLocal Jau 2009 \n\n    \nNB-1003-37 2064 \n\n    \nNB-1003-37/1038 2069 \n\n    \nSolu Uwa 2458 \n\n    \nXveola-43 2471 \n\n     \n6293 \n\n     \n6309 \n\n     \n6320 \n\n     \nBonus \n\n     \nNB-1003-37 \n\nNote: combination on parenthesis is respective SSR markers used to detect particular gene. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1:13-18   Amgai et al.  \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   17                    Nepjol.info/index.php/njb \n\n \n\nTable 5: Nepalese barley germplasm having three and more leaf rust resistance gene detected by molecular \n\nmarkers \n\nGenotype Rph3 & Rph1 QTL on 5HS Rph2 QBLR-P Rph7 \n\nGR-25-85 0 0 1 1 1 \n\nNB-1003-37 0 1 1 1 1 \n\nNPGR No. 2506 1 1 1 0 0 \n\nXveola-43 1 1 1 0 0 \n\nNB-1003-37/1038 1 1 1 0 0 \n\nNPGR No.", "start_char_idx": 9298, "end_char_idx": 11479, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6738509c-c2fc-48e6-85b0-a17354a66159": {"__data__": {"id_": "6738509c-c2fc-48e6-85b0-a17354a66159", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b04235e4-4a58-469a-96a6-d92ed1265976", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "93e611429fd0f6cf632c468c2edfab9674c2ec760c314e70d39bdbe5961cc4c8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "53640971-06d6-43d1-ae5e-708cfd36d125", "node_type": "1", "metadata": {}, "hash": "45f3121d31a5dfca9c8f2fdde78c92ff6e34150010b5c085bb91a2949bc5b838", "class_name": "RelatedNodeInfo"}}, "text": "2515 1 1 1 0 0 \n\nJC#49 1 1 0 0 1 \n\nSolu Uwa 1 1 0 0 1 \n\nNote: 1=present, 0=absent \n\n \n\nmolecular marker screening. Bonus and Local Jau \n\nwere used as resistant and susceptible check \n\nrespectively. \n\nLeaf Rust Evaluation at Field \n\nBarley lines were screened for leaf rust at heading \n\nstage at Khumaltar, Lalitpur, Nepal during normal \n\nbarley growing season. Bonus (origin Sweden) \n\nand Local Jau (Nepalese landrace) was used as \n\nresistance and susceptible check respectively. Two \n\nrows (spacing 20cm) of 1.5m long per accession \n\nwere sown. The resistance and susceptible checks \n\nwere repeated after every 15 test lines. Two \n\nspreader rows of Local Jau were sown around the \n\ndisease screening plots. Disease scoring was \n\nconducted according to the modified Cobb scale \n\n[7]. \n\nMolecular Marker  \n\nSix SSR markers were selected for screening leaf \n\nrust resistance gene (Table 3). Molecular markers \n\nare selected based on their linkage with particular \n\nleaf rust resistance gene.  \n\nDNA extraction, PCR reaction and data analysis \n\nGenomic DNA of barley accessions was prepared \n\nusing modified CTAB method as described by Sul \n\nand Korban [8]. Each PCR reaction was conducted \n\nwith100ng of genomic DNA, 1 \u00b5M of each primer \n\nand 7.5 \u00b5l of 2x GoTaqGreen PCR Master Mix \n\n(Promega Corporation, Madison, WI, USA). PCR \n\nmixture was amplified in MJ Research PTC-100TM \n\nProgrammable Thermal Controller (MJ Research, \n\nInc, Watertown, MA, USA) with the following \n\ntemperature regimes: initial denaturation for 2 \n\nmin at 95oC followed by 30 cycles of 95oC for 30 \n\nsec, annealing as per primer for 1 min, extension at \n\n72oC for 2 min and final extension at 72oC for 7 \n\nmin followed by holding at 4oC as described on \n\nTable 3 and Scottish Crop Research Institute [9]. \n\nAmplified PCR products were separated in 2% \n\nanalytical grade agarose gel at 100V for 1H. Gels \n\nwere stained with 0.1 \u00b5g/ml ethidium bromide \n\n(Promega Corporation, Madison, WI, USA) and \n\nthen visualized under UV trans illuminator gel \n\ndocumentation system (Wilber Lourmat, Marne-\n\nLa-Valleen, France) using 1 \u00b5g guide size DNA \n\nladder (Genetix, Biotech Asia Pvt. Ltd.). The \n\npresence and absence of particular band size was \n\nscored for screening disease resistance genes. \n\nResults and Discussion \nA lot of variation was observed in Nepalese barley \n\ngermplasm for leaf rust resistance characteristics \n\n(Table 1). Leaf rust resistance gene Rph1, Rph2, \n\nRph3, Rph7, QBLR-P and QTL on 5HS was \n\ndetected on Nepalese barley accessions using \n\nrespective SSR markers. Twelve landraces showed \n\npresence of Rph2, 22 accessions showed presence \n\nof Rph1 and Rph3, 27 accessions showed presence \n\nof Rph7, forty two accessions showed presence of \n\nQTL on chromosome 5HS and 47 accessions \n\npossessed leaf rust resistant QTL QBLR-P (Table \n\n4). Similarly, eight Nepalese barley accessions \n\nshowed presence of three and more leaf rust \n\nresistant gene (Table 5). \n\nNepalese barley germplasm showed good \n\nresistance with leaf rust which may be due to the \n\npresence of leaf rust resistance major genes and \n\nquantitative trait loci (QTLs) as detected by \n\ndifferent SSR markers. Tyryshkin [10] and \n\nHenderson [11] also concluded that Nepalese \n\nbarley has good resistance against the leaf rust. \n\nThe released hulless barley variety \u201cSolu Uwa\u201d \n\nshow good resistance with leaf rust and have \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No.", "start_char_idx": 11480, "end_char_idx": 14919, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "53640971-06d6-43d1-ae5e-708cfd36d125": {"__data__": {"id_": "53640971-06d6-43d1-ae5e-708cfd36d125", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6738509c-c2fc-48e6-85b0-a17354a66159", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "ce8486a7a0ce40f374bf61565d5190a4f58592988bdd97caf484d68ff0c3f78c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0a592ab8-5363-4168-844e-002e79aa80ce", "node_type": "1", "metadata": {}, "hash": "36604d86df687c88a08f9e658064b5eb67f754f397c187e177fb8067e10389ea", "class_name": "RelatedNodeInfo"}}, "text": "2 0 1 6  Vol. 4, No. 1:13-18   Amgai et al.  \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   18                    Nepjol.info/index.php/njb \n\n \n\nQTLs (Table 4) and major genes including Rph7 \n\n(Figure 1). Similarly, adult plant resistance for leaf \n\nrust was also observed by Tyryshkin [10] for \n\nNepalese barley NB-3002 while screening world \n\ncollection of barley for leaf rust and proved to \n\nhave one dominant gene. This gene may be Rph7. \n\nSimilarly, the poor relationship between the field \n\ndisease resistance and molecular markers linked \n\nwith specific leaf rust resistance gene proved that \n\nNepalese barley gene pool contains new leaf \n\nresistance genes that cannot be defined by the \n\ntested molecular markers. \n\nConclusion \nMany Nepalese barley landraces showed field \n\nresistance with leaf rust, however, some \n\npromising lines still lacks any major resistant \n\ngenes as defined by molecular markers and need \n\nto be incorporated from other lines to address \n\nfuture unwanted leaf rust spread. Barley \n\ngenotypes with more than three resistant genes \n\ncould be the choice of donor parents for leaf rust \n\nresistant breeding through molecular marker \n\nassisted selection in Nepal.  \n\nAcknowledgement \nThis work was conducted under Global \n\nBiodiversity Trust Grant No. GS10027. \n\nReferences \n1. GoN: Statistical information on Nepalese \n\nagriculture 2012/13. Ministry of Agriculture \nDevelopment. Agri-Business Promotion and \nStatistics Division, Kathmandu, Nepal, 2013. \n\n2. Upreti RP: Status of food barley in Nepal. In-S. \nGrando and H. Gormez Macpherson (eds.), \nFood Barley: Importance, Uses and Local \nKnowledge. Proceedings of the International \nWorkshop on Food Barley Improvement, 14-17 \nJanuary 2002, Hammamet, Tunisia. ICARDA, \nAleppo, Syria, 2005: 99-114. \n\n3. HCRP: Barley Research Reports 2007/08. Hill \nCrops Research Program, Dolakha, Nepal, 2008. \n\n4. Prasad RC, Karki CB, Sharma S: Pathological \nReport on Barley. In-Hill Crops Proceedings. \nNational Hill Crops Research Program, Kabre \nDolakha, Nepal, 1993: 52-78. \n\n5. Bajracharya J, Tiwari PR, Shakya DM, Baniya \nBK, Sthapit BR: Genetic variation in barley \nlandraces (Hordeum vulgare L.) of Jumla ecosite \n\nrevealed by isozyme analysis. In BR Sthapit, A \nSubedi, MP Upadhaya and BK Baniya, (eds.), \nProceedings of a National Workshop on \nStrengthening the Scientific Basis of In Situ \nConservation of Agricultural Biodiversity \nduring 24-26 April 2001at Lumle, Kaski, Nepal. \nNARC/LIBIRD/IPGRI, 2001. \n\n6. Gupta SR, Upadhyay MP, Shah US: Agro-\nmorphological Variability Study of Barley \n(Hordeum vulgare L.) landraces in Jumla, Nepal. \nNepal Agric. Res. J. 2009, 9:1-11. \n\n7. Peterson RF, Campbell AB, Hannah AE: A \ndiagrammatic scale for estimating rust intensity \nof leaves and stem of cereals. Can J Res Sci 1948, \n26:496-500. \n\n8. Sul IW, Korban SS: A highly efficient method \nfor isolating genomic DNA from plant tissues. \nPlant Tiss. Cult. Biotech. 1996, 2: 113-116. \n\n9. Scottish Crop Research Institute: Barley SSRs \n1.0. http://bioinf.scri.ac.uk/ssr/barley_s.html \n(accessed 24 April 2016). 2005. \n\n10.", "start_char_idx": 14899, "end_char_idx": 17993, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0a592ab8-5363-4168-844e-002e79aa80ce": {"__data__": {"id_": "0a592ab8-5363-4168-844e-002e79aa80ce", "embedding": null, "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-28", "node_type": "4", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "b918553898323ba539882fd8c5692aae31ec3245e4257e03b5be2d6c79bc20ff", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "53640971-06d6-43d1-ae5e-708cfd36d125", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "50c1103ae363a82ae47753dcef6402cccd400e62db509fefdbcd7275600472d2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2fe4edb4-338d-4d47-9fd6-003363e7de4f", "node_type": "1", "metadata": {}, "hash": "fdc93752e12e28e27dd0089f0c0c5c377981ddaa9a917bc264b3fd3c5c3dc0c1", "class_name": "RelatedNodeInfo"}}, "text": "2005. \n\n10. Tyryshkin, LG: Genetic control of effective leaf \nrust resistance in collection accessions of barley \nHordeum vulgare L. Russ J Genet. 2009, 45 (3): \n376-378. \n\n11. Henderson MT: Studies of sources of resistance \nand inheritance of reaction to leaf rust Puccinia \nanomala Rostr. in barley. Ph.D. Thesis, \nUniversity of Minnesota, Minneapolis. 1945. \n\n12. Mammadov JA, Brooks WS, Griffey CA, Saghai \nMaroof MA: Validating molecular markers for \nbarley leaf rust resistance genes Rph5 and \nRph7. Plant Breeding 2007, 126: 458-463. \n\n13. Karakousis A, Barr AR, Chalmers KJ, Ablett GA, \nHolton TA, Henry RJ, Lim P, Langridge P: \nPotential of SSR markers for plant breeding \nand variety identification in Australian barley \ngermplasm. Aust J. Agri. Res. 2003, 54: 1197-1210. \n\n14. Czembor PC, Pietrusinska A, Czembor HJ: \nMapping new resistance gene to Puccinia \nhordei Otth. in barley. Abstract In: J.L. Molina-\nCano, P. Christou, A. Graner, K. Hammer, N. \nJouve, B. Keller, J.M. Lasa, W. Powell, C. Royo, P \nShewry and A.M. Stanca (eds.), Cereal science \nand technology for feeding ten billion people: \ngenomics era and beyond. Options \nMediterraneennes, Series A, 2008, No.81:151p. \n\n15. Park RF, Poulsen D, Barr AR, Cakir M, Moody \nDB, Raman H, Read BJ: Mapping genes for \nresistance to Puccinia hordei in barley. Aust J \nAgri. Res. 2003, 54: 1323-1333. \n\n16. Rossi C, Cuesta-Marcos A, Vales I, Gomez-\nPando L, Orjeda G, Wise R, Sato K, Hori K, \nCapettini F, Vivar H, Chen X, Hayes P: Mapping \nmultiple disease resistance genes using a \nbarley mapping population evaluated in Peru, \nMexico, and the USA. Mol Breed. 2006, 18: 355 \u2013 \n366.", "start_char_idx": 17982, "end_char_idx": 19629, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2fe4edb4-338d-4d47-9fd6-003363e7de4f": {"__data__": {"id_": "2fe4edb4-338d-4d47-9fd6-003363e7de4f", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0a592ab8-5363-4168-844e-002e79aa80ce", "node_type": "1", "metadata": {"identifier": "njb-28", "author": "Amgai, Resham Babu; Pantha, Sumitra; Bhatta, Madan Raj", "title": "Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance", "date": "2016-12-31", "file": "njb-28.pdf"}, "hash": "606dca1afba932461a0ee96ac100e1ea7170c25a34db3537aee602cc2092649f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e0303e9f-ac0a-4011-85ac-3588e668d133", "node_type": "1", "metadata": {}, "hash": "8c8d1702bb7feed84c79649093f086b7f996770caee9a4bb9fe7ffc1bf42691f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13   ISSN 2091-1130(Print)/ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    1     Nepjol.info/index.php/njb\n\n         \n\nAssessing the Role of Potential Biomarkers in Antimony \nSusceptible and Resistant Clinical Isolates of L. donovani from \n\nIndia  \nMahendra Maharjan1,2*, Swati Mandal1, Rentala Madhubala1 \n\n1School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India. \n2 Central Department of Zoology, Tribhuvan University, Kathmandu, Nepal. \n\n \n\nAbstract \nFailure of antimonial drugs, the mainstay therapy for leishmaniasis has become an escalating \n\nproblem in the treatment of Indian leishmaniasis. Using 14 clinical isolates from both visceral \n\n(VL) and post-kala-azar dermal leismaniasis (PKDL) patients, we have examined the role of \n\nATP-binding cassette transporter (ABC transporter) gene, multidrug resistant protein A \n\n(MRPA) and two building blocks of the major thiol, trypanothione namely, ornithine \n\ndecarboxylase gene (ODC) (a rate limiting enzyme in the polyamine biosynthesis) and \u03b3- \n\nglutamylcysteine synthetase (\u03b3-GCS) (a rate limiting enzyme in glutathione biosynthesis) in \n\nantimony resistance. Amplification of these three genes was observed in some but not all \n\nclinical isolates. Increased expression of the three RNAs as determined by real-time PCR was \n\nobserved in all SAG-R clinical isolates. Significant increase in cysteine and glutathione levels \n\nwas observed in the resistant isolates. Our studies report the underlying mechanism of \n\nantimony resistance in the clinical isolates. \n\nKey words: ABC transporter, ornithine decarboxylase, \u03b3- glutamylcysteine synthetase, \nantimony resistance. \n\n*Corresponding Author \nEmail: maharjan.m@gmail.com \n\nIntroduction \nThe protozoan parasite Leishmania is the \n\ncausative agent of kala-azar and is responsible \n\nfor a variety of clinical manifestations. It causes a \n\nwide spectrum of diseases ranging from the \n\nsimple self healing cutaneous form to the \n\ndebilitating visceral form. Visceral leishmaniasis \n\n(VL) is caused by L. donovani in the Indian sub-\n\ncontinent. Pentavalent antimonials (SbV) are the \n\nfirst line of drug used in the treatment against all \n\nforms of leishmanial infections [1,2]. Resistance \n\nto this drug is becoming a major barrier in the \n\ntreatment of VL in many endemic regions \n\nparticularly in India [3]. Kala-azar transmission \n\nin India is thought to be anthroponotic and post-\n\nkala-azar dermal leismaniasis (PKDL) patients \n\nare considered to serve as a source for new \n\noutbreaks [4]. The post-kala-azar dermal \n\nleismaniasis (PKDL) is a sequel to VL in India \n\nand Sudan; the disease develops months to years \n\nafter the patient\u2019s recovery from VL [5]. \n\nThe mechanism of action of sodium antimony \n\ngluconate (SAG) remained an enigma for more \n\nthan 60 years of its effective use against all forms \n\nof leishmaniasis. It is generally agreed that \n\npentavalent form (SbV) is reduced to a more \n\ntoxic trivalent form (SbIII) which constitutes the \n\nactive form of the drug against the parasite [6]. \n\nMolecules possibly implicated in reduction of \n\nSbV to SbIII include host and parasite thiols and \n\ntwo newly discovered parasite enzymes thiol-\n\ndependent reductase (TDR1) and arsenate \n\nreductase (ACR2) [7-9]. A loss of drug activation \n\nis reported to lead to resistance [10].", "start_char_idx": 47, "end_char_idx": 3478, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e0303e9f-ac0a-4011-85ac-3588e668d133": {"__data__": {"id_": "e0303e9f-ac0a-4011-85ac-3588e668d133", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2fe4edb4-338d-4d47-9fd6-003363e7de4f", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "1f7d9645665f144d945536b26b9dcea6c7ad84a86abc44ab4b271526a5f559b3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5c693204-5d93-4076-9e01-6335fa6b21c8", "node_type": "1", "metadata": {}, "hash": "ffac71101c2242fa763c584f6e060bfb57aa954c95c4e80a6c0a8b07bbf8bec6", "class_name": "RelatedNodeInfo"}}, "text": "A loss of drug activation \n\nis reported to lead to resistance [10]. The route of \n\nentry of SbV into Leishmania cells is still \n\nunknown but SbIII has been reported to be \n\ntransported in Leishmania through \n\naquaglyceroporin (AQP1) [11,12]. Recent \n\nevidence has suggested that part of the mode of \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    2     Nepjol.info/index.php/njb\n\n         \n\naction of SbV could be in depleting the cells of its \n\nreduced thiols [13]. Trypanothione (TSH), a \n\nmajor reduced thiol of Leishmania is a N1, N8 \n\nbisglutathione spermidine conjugate. It is \n\nthought to bind to the active reduced form of the \n\nmetal. These metal-trypanothione conjugates are \n\neither sequestered into an intracellular organelle \n\nby the ABC transporter MRPA or extruded \n\noutside the cell by an efflux pump [13-16]. A \n\nnumber of candidate genes associated with \n\nincreased thiol concentration have been \n\ndescribed in Leishmania laboratory mutants.  \n\nResistance was induced in these mutants in vitro \n\nin the presence of antimony related metals such \n\nas arsenic or antimony [2,17,18].  \n\nHowever, till date it remains unclear as to \n\nwhether similar mechanisms can be extrapolated \n\nto clinical isolates from geographical zones with \n\na high incidence of primary antimony resistance. \n\nTo address this question, we have characterized \n\nboth the VL and the PKDL isolates from India \n\nand report that diverse mechanisms of resistance \n\nare operative in these isolates. This work aims at \n\ncharacterizing possible biomarkers for \n\nmonitoring antimonial resistant visceral \n\nleishmaniasis and post-kala-azar dermal \n\nleismaniasis in the field isolates. \n\nIn the present study, we report the role of thiols \n\nand also assessed the role of ABC transporter \n\n(MRPA), ornithine decarboxylase (ODC) and \u03b3-\n\nglutamylcysteine synthetase (\u03b3-GCS) genes as \n\npotential biomarkers for monitoring antimonial \n\nresistance in Indian leishmaniasis.  \n\nMaterials and Methods \nParasite and culture conditions \n\nPromastigotes of Leishmania donovani clones, \n\nAG83 (MHOM/IN/80/AG83), 2001, MC4, MC7, \n\nMC8 and  MC9 were isolated from patients with \n\nVL and strains, RK1, MS2, NR3A, RMP8 \n\n(HM/IN/RMP-8), RMP-19 (HM/IN/RMP-19), \n\nRMP142 (HM/IN/RMP-142), RMP155 \n\n(HM/IN/RMP-155) and  RMP240 \n\n(HM/IN/RMP-240) used in the present study \n\nwere isolated from patients with PKDL [19]. \n\nClinical isolates obtained from VL and PKDL \n\npatients who responded to SAG chemotherapy \n\nwere designated as SAG-S (SAG-sensitive) \n\nwhereas isolates from VL and PKDL patients \n\nwho did not respond to SAG were designated as \n\nSAG-R (SAG-resistant). SAG-sensitive strains, \n\nAG83-S, 2001-S, MC7-S, RK1-S, MS2-S and the \n\nnine SAG-R isolates, MC4-R MC8-R, MC9-R, \n\nNR3A-R, RMP8-R, RMP19-R, RMP142-R, \n\nRMP155-R and RMP240-R have been \n\ncharacterized earlier [20].  Clinical history of the \n\npatient infected with the strain RK1-S showed \n\nthat the interval between the cure of VL and the \n\nonset of PKDL was 2.5 years where as in the case \n\nof PKDL patients infected with the strains, MS2-\n\nS and NR3A-R, the interval between the cure of \n\nVL and the onset of PKDL was 7 and 11 years \n\nrespectively.", "start_char_idx": 3411, "end_char_idx": 6705, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5c693204-5d93-4076-9e01-6335fa6b21c8": {"__data__": {"id_": "5c693204-5d93-4076-9e01-6335fa6b21c8", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e0303e9f-ac0a-4011-85ac-3588e668d133", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "1f1257c7bfd5b902c9d6bca3d644d8abdb7063ffda10e57cc6c85db1f7654e98", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "80386506-da90-421c-8962-6f3188b3fd60", "node_type": "1", "metadata": {}, "hash": "9a9749acd74595c68c622cc53c0ff4e1020961273867d12d454e425c78109836", "class_name": "RelatedNodeInfo"}}, "text": "The interval between the cure of VL \n\nand the onset of PKDL for the remaining isolates \n\nis not known. The clinical isolates were \n\nmaintained in vitro in the absence of the drug \n\npressure. Promastigotes were routinely cultured \n\nat 220C in modified M-199 medium (Sigma, \n\nUSA) supplemented with 10% heat-inactivated \n\nfetal bovine serum (FBS; Gibco/BRL, Life \n\nTechnologies Scotland, UK) and 0.13 mg/mL \n\npenicillin and streptomycin. This study has the \n\napproval of the Institutional level ethics \n\ncommittee.  \n\nDNA and RNA manipulations \n\nChromosomes of the clinical isolates were \n\nseparated by pulse field gel electrophoresis \n\n(PFGE) in which low melting agarose blocks, \n\ncontaining embedded cells (108/ml log phase \n\npromastigotes) were electrophoresed in a \n\ncontour clamped homogeneous electric field \n\napparatus (CHEF DRIII, Bio-Rad) as reported \n\nearlier [20]. Mid log phase promastigotes (~2 x \n\n109 cells) of all the field isolates were used for \n\nisolation of genomic DNA. 5 \u00b5g of genomic \n\nDNA was digested with HindIII enzyme and \n\nsubjected to electrophoresis. Total RNA was \n\nisolated from promastigotes (2 x 108 cells) using \n\nRNAeasy Plus Mini Kit (Qiagen). Standard \n\nprotocols were followed for Southern \n\nhybridization [21]. DNA probes used in the \n\npresent study included a 400-bp MRPA \n\nfragment (released from plasmid PM12 that was \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    3     Nepjol.info/index.php/njb\n\n         \n\ndigested with BamHI and PstI), a 2.3-kb \u03b3GCS \n\nfragment - (derived from plasmid psp\u03b1hygro\u03b1-\n\n\u03b3GCS digested with HindIII and XbaI), a 2.0-kb \n\nODC-full length probe (derived from plasmid \n\npsp\u03b1hygro\u03b1-ODC digested with HindIII and \n\nXbaI) and a 1.6-kb 5\u2019-PTR1 probe derived from \n\nplasmid (psp72-Y-hygro-5\u2019-PTR1). \n\ncDNA synthesis and real time RT- PCR  \n\nTotal RNA was isolated from 108 Leishmania cells \n\nin the mid-log phase of growth using the \n\nRNeasy Plus Mini Kit (Qiagen) as described by \n\nthe manufacturer. Quality and quantity of the \n\nRNA were determined using the RNA 6000 \n\nNano Lab chip kit on the Bio-analyzer 2100 \n\n(Agilent Technologies). The sequences of the \n\nprimers for MRPA are forward 5\u2019-\n\nGCGCAGCCGTTTGTGCTTGTGG-3\u2019 and \n\nreverse 5\u2019-TTGCCGTACGTCGCGATGGTGC-3\u2019 \n\nand for the glyceraldehyde 3-phosphate \n\ndehydrogenase (GAPDH) control forward 5\u2019-\n\nGAAGTACACGGTGGAGGCTG and reverse 5\u2019-\n\nCGCTGATCACGACCTTCTTC primers. The \n\nsequences of the primers for ODC are forward \n\n5\u2019- GATGGTGCGCCCTTACTTTGC-3\u2019and \n\nreverse 5\u2019-TTCCATCTCCAGCGGGTTGTCC-3\u2019, \n\nand for the \u03b3GCS, forward 5\u2019-\n\nCATTGGCTGGCGCGTTGAGTTC-3\u2019 and \n\nreverse 5\u2019-ATGTGCGCGGCCCATATTCTCG -3\u2019 \n\nprimer.  Complementary DNAs from \n\npromastigotes were synthesized from 500 ng of \n\ntotal RNA using the AccuSuperscript High \n\nFidelity RT-PCR kit (Stratagene, La Jolla) and \n\nOligo (dT)18 primers following manufacturer\u2019s \n\ninstructions.", "start_char_idx": 6706, "end_char_idx": 9683, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "80386506-da90-421c-8962-6f3188b3fd60": {"__data__": {"id_": "80386506-da90-421c-8962-6f3188b3fd60", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5c693204-5d93-4076-9e01-6335fa6b21c8", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "9af9236962d6639ceceb60c1669f3b2967ff59f97f64c1eae6ef43c92d883939", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "74acbad7-1112-49ce-a098-7fc12a206154", "node_type": "1", "metadata": {}, "hash": "cea9ef5f4c51ae24541c9832a6cf3f1a824ba25761bff04babcab2e8e06df337", "class_name": "RelatedNodeInfo"}}, "text": "Real-time PCR was performed in \n\ntriplicate in 25 \uf06dl volumes using QuantiFast \n\nSYBR Green PCR Master Mix (Qiagen) in an \n\nApplied Biosystem 7500. Reactions were run \n\nusing the following thermal profile: initial \n\ndenaturation at 95\u00b0C for 5 min followed by 40 \n\ncycles with denaturation at 95\u00b0C for 30 s, \n\nannealing at 62\u00b0C for 1 min and extension at \n\n72\u00b0C for 20 s. The PCR was followed by a melt \n\ncurve analysis to ascertain that the expected \n\nproducts were amplified. The relative amount of \n\nPCR products generated from each primer set \n\nwas determined based on the threshold cycle \n\n(Ct) value and amplification efficiencies and was \n\nnormalized by dividing the values by the \n\nrelative amount of the GAPDH gene used as a \n\ncontrol. \n\nTransfection and overexpression of the MRPA \n\nand \u03b3-GCS gene \n\nEpisomal Leishmania expression vectors, pGL-\n\n\uf061NEO\uf061LUC containing luciferase encoding \n\nDNA and neomycin phosphotransferase \n\nselectable marker, psp\uf061hygro\uf061-\uf067GCS containing \n\ncoding sequence for heavy subunit of \uf067GCS with \n\nhygromycin phosphotransferase as selectable \n\nmarker and pGEM72f-\uf061neo\uf061-MRPA containing \n\nMRPA coding DNA and neomycin \n\nphosphotransferase as selectable marker were \n\nobtained as gifts from Prof. Marc Ouellette, \n\nCentre de Recherche en Infectiologie du Centre \n\nde Recherche du CHUL, Universite Laval, \n\nQuebec, Canada. Twenty \u00b5g of each construct \n\nwas transfected into L. donovani promastigotes \n\nby electroporation. Electroporation was done \n\nwith a single pulse with the following \n\nparameters 450 V, 500 \uf06dF (Bio-Rad). \n\nTransfectants were selected for resistance to \n\neither G418 (40 \u03bcg/ml) or hygromycin B (80 \n\n\u03bcg/ml) as described earlier [22]. \n\nChemosensitivity profiles of SAG-S and SAG-R \n\nstrains in an amastigote macrophage model \n\nStationary phase Leishmania promastigotes \n\nexpressing the luciferase gene (pGL-\n\n\uf061NEO\uf061LUC) and psp\uf061hygro\uf061-\uf067GCS containing \n\ncoding sequence for heavy subunit of \uf067GCS with \n\nhygromycin phosphotransferase as selectable \n\nmarker or pGEM72f-\uf061neo\uf061-MRPA containing \n\nMRPA coding DNA and neomycin \n\nphosphotransferase as selectable marker were \n\ninfected into J774A.1 macrophages. Macrophage \n\ncell line J774A.1 (American Type Culture \n\nCollection) was maintained at 37\uf0b0C in RPMI-\n\n1640 medium (Sigma) containing 10% heat \n\ninactivated fetal bovine serum. Briefly, J774A.1 \n\nmurine macrophages (1 x 105 cells/ petridish) \n\nwere infected with 1 x 106 promastigotes \n\n(expressing the luciferase gene (pGL-\n\n\uf061NEO\uf061LUC) in M199 media with 10% FBS. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    4     Nepjol.info/index.php/njb\n\n         \n\nAfter 3 h, the non-internalized parasites were \n\nwashed off and SAG was added at different \n\nconcentrations (10 \u2013 100 \uf06dg/ml). After 5 days of \n\ndrug exposure, plates containing adherent \n\nmacrophages were washed and luciferase \n\nactivity was determined [22]. The 50% inhibitory \n\nconcentration (IC50) was determined from the \n\ngraph representing different concentrations of \n\nSAG plotted against relative light units (RLU) \n\nproduced by luciferase expressing parasites.", "start_char_idx": 9684, "end_char_idx": 12883, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "74acbad7-1112-49ce-a098-7fc12a206154": {"__data__": {"id_": "74acbad7-1112-49ce-a098-7fc12a206154", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "80386506-da90-421c-8962-6f3188b3fd60", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "d05b690797f8fa01f37c201555464bc90abb6e496b0ae58339827b7598ff2e02", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "771b5dca-14b9-41f1-b5ed-9d9f20f7367d", "node_type": "1", "metadata": {}, "hash": "c503696717762fb0640f676e585cbcf2db1c981543087e5d01f2e0d824a95081", "class_name": "RelatedNodeInfo"}}, "text": "Thiol analysis \n\nThiols were derivatized with \n\nmonobromobimane and separated by high-\n\nperfomance liquid chromatography (HPLC) as \n\nreported earlier [18].  \n\nStatistical analysis  \n\nData was analyzed by the Student\u2019s t- test. The \n\ndata is represented as mean \u00b1 S.D. The results \n\nare representative of three independent \n\nexperiments. A p value of < 0.05 was considered \n\nstatistically significant. \n\nResults \nIntracellular thiol levels in SAG-S and SAG-R \n\nclinical isolates \n\nResistance to antimonials in clinical isolates is \n\nnot well defined. Understanding the mechanism \n\nof resistance to antimony in clinical isolates of L. \n\ndonovani will aid in the development of \n\nbiomarkers for antimony resistance and this in \n\nturn will enable the clinicians to monitor the \n\ntreatment of the patients. With this background \n\nin mind, we used the previously characterized \n\nSAG-S and SAG-R, VL and PKDL clinical \n\nisolates for the present study [20]. \n\nA total of fourteen field isolates were used to \n\nassess the putative role of the ABC transporter \n\nMRPA, ornithine decarboxylase and \u03b3-\n\nglutamylcysteine synthetase in antimony -\n\nsusceptible and -resistant clinical isolates of L. \n\ndonovani from India. As reported earlier, SAG-S \n\nisolates, AG83-S, 2001-S, MC7-S, RK1-S and \n\nMS2-S coming from SAG responsive patients \n\nhad IC50 values 6.2 \u00b1 1.8, 0.9 \u00b1 0.12,  8 \u00b1 3.3, 0.01 \u00b1 \n\n0.02 and 4.75 \u00b1 0.12 \u00b5M respectively whereas the \n\nSAG-R isolates, MC4-R, MC8-R, MC9-R, NR3A-\n\nR, RMP8-R, RMP19-R, RMP142-R, RMP155-R, \n\nRMP240-R coming from the SAG-unresponsive \n\npatients had IC50s that were \uf07e2 to >10 fold higher \n\nthan that of the sensitive isolate, AG83-S.(20) \n\nRK1-S was the most sensitive of all the isolates \n\nwith an IC50   of 0.01 \u00b1 0.02 \u00b5M. \n\n Intracellular thiol levels were quantified in the \n\nSAG-S and SAG-R isolates (Figure 1). SAG-R \n\nisolates maintained significantly higher levels of \n\ncysteine and glutathione as compared to the  \n\nSAG-S isolates. Cysteine levels in the SAG \n\nresistant strains MC4-R, MC8-R, MC9-R, NR3A-\n\nR, RMP8-R, RMP19-R, RMP142-R, RMP155-R \n\nand RMP240-R were  ~1.7, ~1.6, ~1.5, ~2.7, ~2.0, \n\n~1.8, ~1.8,  ~2.0 and ~1.7 -fold higher \n\nrespectively when compared to the SAG-S \n\nisolate, 2001-S (Figure 1A). \n\nSimilarly GSH levels showed significant increase \n\nin the SAG-R isolates, MC4-R (~2.7-fold), MC8-R  \n\n(~3.6 -fold), MC9-R (~3.0 -fold), NR3A-R (~2.0 -\n\nfold), RMP8-R (~3.2 -fold), RMP19-R (~2.5 -fold), \n\nRMP142-R (~3.1 -fold), RMP155-R (~2.4 -fold) \n\nand RMP240-R (~3.0 -fold) when compared to \n\nthe SAG sensitive isolate, 2001-S (Figure 1B). A \n\nSAG-S strain, RK1-S had glutathione levels that \n\nwere ~2.1 -fold higher when compared to the \n\nSAG sensitive isolate, 2001-S.  Interestingly, no \n\nsignificant difference was observed in the \n\ntrypanothione levels between the SAG-S and \n\nSAG-R isolates (Figure 1C). Similar observation \n\nwas made in our earlier study using a small set \n\nof clinical isolates [23].", "start_char_idx": 12887, "end_char_idx": 15848, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "771b5dca-14b9-41f1-b5ed-9d9f20f7367d": {"__data__": {"id_": "771b5dca-14b9-41f1-b5ed-9d9f20f7367d", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "74acbad7-1112-49ce-a098-7fc12a206154", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "4d7353904eca9b22111f2668f892ca91e9db36bd0d061171ba8d335bcc4f3014", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "96fdf5a8-4225-41e6-9823-a4cce31ae6e2", "node_type": "1", "metadata": {}, "hash": "a5d463be403689e9853971ce72e3522c241872989d2843250d1ff9b939234d11", "class_name": "RelatedNodeInfo"}}, "text": "Gene copy number and expression profiling of \n\nthe ABC transporter MRPA in SAG-S and SAG-R \n\nclinical isolates \n\nOur earlier work on the clinical kala azar L. \n\ndonovani isolates from India showed MRPA \n\noverexpression as an important SAG resistance \n\nfactor [23]. To further validate, the role of MRPA \n\ngene in antimony resistance phenotype, we \n\nchecked the amplification of MRPA gene by \n\nSouthern blot hybridization. Southern blot \n\nhybridization of total genomic DNA digested \n\nwith HindIII followed by hybridization with \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    5     Nepjol.info/index.php/njb\n\n         \n\n \n\n \n\n \n\n \n\nFigure 1: Intracellular levels of thiols, cysteine, \n\nglutathione and trypanothione in SAG-S and SAG-R \n\nL. donovani VL and PKDL clinical isolates.  A: Cysteine \n\nlevels B: Glutathione levels C: Trypanothione levels. \n\nThiols were derivatized with monobromobimane and \n\nseparated by high-perfomance liquid chromatography \n\n(HPLC). Each value is the mean \uf0b1 SD of triplicates \n\nfrom two independent experiments.  a indicates p \n\n<0.01; b indicates p < 0.001; c indicates p < 0.0004 \n\nwhen compared to 2001-S respectively \n\nMRPA specific probe was done. SAG-sensitive \n\nand SAG-resistant field isolates showed a single \n\nhybridizing fragment of 11-kb indicating that \n\nMRPA gene exist as a single copy gene in all the \n\nisolates (Figure 2A). Quantitation of the \n\nSouthern blot hybridization signal was done \n\nusing ImageQuant 5.2 (Molecular Dynamics) \n\nand the fold difference in DNA copy number of \n\nthe isolates with the AG83-S or 2001-S was \n\ncalculated. Amplification of MRPA gene was \n\nobserved in the resistant isolates, MC4-R, MC8-\n\nR, MC9-R, RMP19-R, RMP142-R, RMP155-R and", "start_char_idx": 15852, "end_char_idx": 17687, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "96fdf5a8-4225-41e6-9823-a4cce31ae6e2": {"__data__": {"id_": "96fdf5a8-4225-41e6-9823-a4cce31ae6e2", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "771b5dca-14b9-41f1-b5ed-9d9f20f7367d", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "e16a46aeddac15d373031638c2a6ef8db173bc1cdd4db971fd26b2d8a4fc131d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7aed0040-57fc-4a2a-b087-4ecac058dc3f", "node_type": "1", "metadata": {}, "hash": "e74c69821eb930c837001304cb7487230fdde7ff036f5c042fbdb0fd8d4574b6", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    6     Nepjol.info/index.php/njb\n\n         \n\n \n\nFigure 2: A: Southern blot analysis of MRPA and PTR1 \ngenes in SAG-S and SAG-R L. donovani VL and PKDL \nclinical isolates. Total genomic DNA of isolates were \ndigested with HindIII, electrophoresed, blotted and \nhybridized with a MRPA specific probe of 400-bp and \n6 kb 5\u2019-PTR1 specific probe. The size of the \nhybridizing bands was determined using HindIII  \n \n\n \n\n digested lambda DNA. The blot was rehybridized \nwith \u03b1-tubulin probe to monitor the amount of \ndigested DNA layered on the gel. Quantitation of \nSouthern blot was done by Image-quant software 5.2 \n(Molecular dynamics) and the fold difference in DNA \ncopy number of MRPA is presented below each blot. \nB: Southern blot analysis of the pulse field gel \nelectrophoresis of SAG-sensitive (SAG-S) and SAG-\nresistant (SAG-R) isolates of Leishmania donovani \nchromosomes. Agarose blocks containing \nchromosomal DNAs of promatigotes of L. donovani \nclinical isolates were prepared and subjected to pulsed \nfield gel electrophoresis for 24 hours run time and \nhybridized with a MRPA specific probe of 400-bp. C: \nReal time RT-PCR expression analysis of MRPA gene \nin L. donovani clinical isolates. MRPA RNA expression \nratios in promastigotes of SAG -resistant isolates are \nrelative to the SAG-sensitive isolate (2001-S). The \ngraph represents mean of three independent \nexperiments performed from three different RNA \npreparations. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    7     Nepjol.info/index.php/njb\n\n         \n\nFigure 3: A: Characterization of ODC gene in SAG-S and SAG-R isolates L. donovani VL and PKDL clinical isolates. \nGenomic DNAs were isolated and digested with HindIII and hybridized with a full-length ODC specific probe, \nderived from the L. donovani ODC gene. The sizes of the hybridizing bands were determined using HindIII digested \nlambda DNA marker. The blot was rehybridized with \u03b1-tubulin probe to monitor the amount of digested DNA \nlayered on the gel. B: Real time RT-PCR expression analysis of ODC in L. donovani clinical isolates. ODC RNA \nexpression ratios in the SAG- resistant isolates are relative to the SAG-sensitive isolate, 2001-S. Results are mean of \nthree independent experiments performed from three different RNA preparations.  \n\nRMP240-R (Figure 2A). No amplification was \n\nobserved in the resistant isolates, NR3A-R and \n\nRMP8-R (Figure 2A). The amplification observed \n\nin the resistant isolates was further analyzed by \n\nPFGE (Figure 2B). None of the resistant isolates \n\nshowed circular amplification as was observed \n\nin our earlier studies with limited number of \n\nisolates [23]. Interestingly, two sensitive PKDL \n\nisolates, RK1-S and MS2-S and one resistant \n\nPKDL isolate, NR3A-R had the presence of \n\nMRPA on two chromosomes as indicated by \n\ntheir characteristic migration in PFGE (Figure \n\n2B). We had earlier reported co-amplification of \n\npterin reductase gene (PTR1) with MRPA in the \n\nclinical isolates [23]. In the present study, co-\n\namplification of PTR1 gene with MRPA gene \n\nwas not observed in any of the clinical isolates as", "start_char_idx": 17692, "end_char_idx": 21087, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7aed0040-57fc-4a2a-b087-4ecac058dc3f": {"__data__": {"id_": "7aed0040-57fc-4a2a-b087-4ecac058dc3f", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "96fdf5a8-4225-41e6-9823-a4cce31ae6e2", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "8347f18bae2acac43db427c4490bf981319cece863075e1edaba563286a26c25", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9aa2e6c2-6a0f-4064-8b93-7cac57225b4c", "node_type": "1", "metadata": {}, "hash": "ea84975379101694fbd3c1aec22aca9d7f3289fd9161f625539fe6636fe0bc02", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    8     Nepjol.info/index.php/njb\n\n         \n\nFigure 4: A: Southern blot analysis of the \uf067-GCS gene in the SAG-sensitive and the SAG-resistant Leishmania donovani \nVL and PKDL field isolates. Total genomic DNA was isolated and digested with HindIII. The digested DNA was \n\nelectrophoresed, blotted and hybridized with \uf067-GCS probe. The sizes of the hybridizing bands were determined using \nHindIII digested \u03bb- DNA. The blot was rehybridized with \u03b1-tubulin probe to monitor the amount of digested DNA \n\nlayered on the gel. B: Real time RT-PCR expression analysis of \uf067-GCS gene in L. donovani clinical isolates.  \uf067-GCS gene \nexpression ratios in the SAG-resistant isolates are relative to the SAG- sensitive isolate, 2001-S. Results are mean of \nthree independent experiments performed from three different RNA preparations.\n\ndetermined by Southern-blot analysis using \n\nPTR1-specific probe (Figure 2A). \n\nComparison of MRPA gene expression in SAG-S \n\nversus SAG-R field isolates was done to verify if \n\nthere is any correlation between MRPA gene \n\nexpression and SAG susceptibility profile of the \n\nclinical isolates. Total RNA from promastigotes \n\nof the clinical isolates was isolated and \n\ncomplementary DNAs were synthesized. Real-\n\ntime -PCR using QuantiFast SYBR Green PCR \n\nMaster Mix (Qiagen) with MRPA (gene specific) \n\nand GAPDH (internal control) primers was  \n\nperformed. Up-regulation of MRPA expression \n\nwas observed in the resistant clinical isolates. \n\nMRPA expression in the resistant strains, MC4-\n\nR, MC8-R, MC9-R, NR3A-R, RMP8-R, RMP19-R, \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    9     Nepjol.info/index.php/njb\n\n         \n\nRMP142-R, RMP155-R and RMP240-R was ~4-, \n\n~4.2-, ~9.6-, ~7.2-, ~6.1-, ~6.8-, ~6.0-, ~7.5-  and \n\n~4.4- fold more respectively when compared to \n\nthe expression in the sensitive isolate, 2001-S  \n\n(Figure 2C).  \n\nGene copy number and expression profiling of \n\nthe ornithine decarboxylase (ODC) gene in \n\nSAG-S and SAG-R clinical isolates. \n\nOrnithine decarboxylase (ODC) is the rate-\n\nlimiting enzyme of the polyamine biosynthetic \n\npathway. In addition to MRPA, overexpression \n\nof the ODC gene has been reported in the \n\nantimony resistant mutants [23-25]. These \n\nobservations prompted us to determine if \n\namplification of the ODC gene occured in our  \n\nclinical isolates. Southern-blot analysis of the \n\ntotal genomic DNA digested with HindIII and \n\nhybridized with the ODC specific probe was \n\ndone. SAG-S and SAG-R isolates showed a \n\nsingle copy of the ODC gene (Figure 3A) with \n\nthe exception of three PKDL isolates, RK1-S, \n\nMS2-S and NR3A-R. Interestingly, two copies of \n\nthe ODC gene were observed in these three \n\nPKDL isolates (Figure 3A). \n\nComparison of ODC gene expression in SAG-S \n\nversus SAG-R field isolates was done to verify if \n\nthere is any correlation between the gene \n\n expression and SAG susceptibility profile of the \n\nclinical isolates. Real-time PCR with the ODC \n\n(gene specific) and the GAPDH (internal control) \n\nprimers was performed. Up-regulation of the \n\nODC expression was observed in the resistant \n\nclinical isolates.", "start_char_idx": 21093, "end_char_idx": 24494, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9aa2e6c2-6a0f-4064-8b93-7cac57225b4c": {"__data__": {"id_": "9aa2e6c2-6a0f-4064-8b93-7cac57225b4c", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7aed0040-57fc-4a2a-b087-4ecac058dc3f", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "28a92152709e3b502694aceb2bd0fa9c7ab72bf476ac35c0eaccdeedc7f95dcc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "de6932c8-b273-4f8c-98c8-ed4d088c74c2", "node_type": "1", "metadata": {}, "hash": "3d51e531f486a4452a639a4275e4a579a8e198ad86913eb4556ff866a0113f80", "class_name": "RelatedNodeInfo"}}, "text": "Up-regulation of the \n\nODC expression was observed in the resistant \n\nclinical isolates. ODC expression in the resistant \n\nstrains, MC4-R, MC8-R, MC9-R, NR3A-R, RMP8-\n\nR, RMP19-R, RMP142-R, RMP155-R and \n\nRMP240-R was ~3.6-, ~7.4-, ~3.1-, ~2.7-, ~2.3-, \n\n~3.3-, ~3.5-, ~2.8-  and ~3.0- fold more \n\nrespectively when compared to the sensitive \n\nisolate, 2001-S  (Figure 3B). SAG-S isolate, RK1-S \n\nwas an exception since it was the only sensitive \n\nisolate that showed 2.9-fold up-regulation when \n\ncompared to the reference strain, 2001-S and also \n\nin comparison with all other SAG-S isolates \n\n(Figure 3B). No uniform correlation was \n\nobserved between gene amplification and ODC \n\ngene expression in these isolates. \n\nGene copy number and expression profiling of \n\nthe \u03b3-glutamylcysteine synthetase (\u03b3-GCS) gene \n\nin SAG-S and SAG-R clinical isolates  \n\nIn addition to MRPA and ODC, another locus \n\nthat has been reported to be amplified in the \n\nantimony-resistant isolates is the GSH1 gene \n\ncoding for the heavy subunit of \uf067-GCS. \uf067-GCS is \n\nthe rate limiting enzyme for GSH synthesis \n\n[14,23]. We performed Southern blot analysis of \n\nthe total genomic DNA digested with HindIII \n\nand hybridized with the \uf067-GCS specific probe. \n\nSouthern blot analysis of the \uf067-GCS gene showed \n\ntwo copies in a resistant isolate MC4-R but in all \n\nother isolates, \uf067-GCS probe hybridized to a single \n\nhybridizing fragment  of 10-kb (Figure 4A). \n\nSAG-S isolate, RK1-S was again an exception \n\nsince it showed amplification of the \uf067-GCS gene \n\n(Figure 4A). \n\nComparison of \uf067GCS gene expression in SAG-S \n\nversus SAG-R field isolates was done to verify if \n\nthere is any correlation between gene expression \n\nand SAG sensitivity profile of the clinical \n\nisolates. Real-time PCR using \uf067-GCS (gene \n\nspecific) and GAPDH (internal control) primers \n\nwas performed. Up-regulation of \uf067-GCS \n\nexpression was observed in the resistant clinical \n\nisolates. \uf067-GCS expression in the resistant strains, \n\nMC4-R, MC8-R, MC9-R, NR3A-R, RMP8-R, \n\nRMP19-R, RMP142-R, RMP155-R and RMP240-R \n\nwas 4.3-, 5.2-, 6.4-, 5.5-, 3.0-, 5.0-,  5.3-, 4.2-  and \n\n8.8- fold more respectively compared to the \n\nexpression in the sensitive isolate, 2001-S  \n\n(Figure 4B). In the present study though up-\n\nregulation of \uf067-GCS was observed in all SAG-R \n\nisolates, interestingly one SAG-S isolate, RK1-S \n\nshowed 5.5-fold up-regulation of \uf067GCS \n\nexpression when compared to a SAG-S isolate, \n\n2001-S (Figure 4B). L-butathione-sulfoxamine \n\n(BSO), an inhibitor of \uf067-GCS was used to \n\ncompare its effect on the SAG-S isolate, RK1-S \n\nand 2001-S. RK1-S promastigotes over-\n\nexpressing \uf067-GCS were ~5 fold more resistant to \n\nBSO when compared to the IC50  of \n\npromastigotes of the 2001-S, the IC50 being 6.5 \u00b1 \n\n0.5 mM and 1.3 \u00b1 0.3 mM respectively. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No.", "start_char_idx": 24406, "end_char_idx": 27275, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "de6932c8-b273-4f8c-98c8-ed4d088c74c2": {"__data__": {"id_": "de6932c8-b273-4f8c-98c8-ed4d088c74c2", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9aa2e6c2-6a0f-4064-8b93-7cac57225b4c", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "9a6fc98e6cf2ddf94d9ce765d4a829b29deed24b5e224d2340cc461e6d0f398c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4ab51173-4c01-441c-9745-428bc961d73b", "node_type": "1", "metadata": {}, "hash": "9d3701ebf83963473efbdb2b23aa31765c2476bfa5a7bf9877ab4cf2301f81aa", "class_name": "RelatedNodeInfo"}}, "text": "2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    10     Nepjol.info/index.php/njb\n\n         \n\n \n\n \nFigure 5: Over-expression of MRPA and \u03b3-GCS gene \nin a sensitive L. donovani isolate. A: Transfection of \nMRPA in SAG-sensitive isolate leads to resistance to \nantimony in amastigotes. AG83-S (-\u25a0-) was transfected \n\nwith pGL-\uf061NEO\uf061LUC, AG83-S + LdMRPA (-\u25a1-) was \n\ntransfected with pGEM72f-\uf061neo\uf061-MRPA + pGL-\n\n\uf061NEO\uf061LUC and sensitivity to SAG in the J774A.1 line \nwas measured as described in Materials and methods. \n\nEach data point represents the mean \uf0b1 SD of three \ndeterminations. Northern blot analysis (insets in A) of \nvector transfected controls and MRPA transfected \nlines was performed as described under methods \nsection B: Transfection of \u03b3-GCS gene in SAG-\nsensitive isolate leads to antimony resistance in \nintracellular amastigotes. AG83-S (-\u25a0-) transfected \n\nwith pGL-\uf061NEO\uf061LUC, AG83-S + \u03b3-GCS (-\u25a1-) \n\ntransfected with psp\uf061hygro\uf061-\uf067GCS + pGL-\n\n\uf061NEO\uf061LUC and sensitivity to SAG measured as \ndescribed in Materials and methods. Each data point \n\nrepresents the mean \uf0b1 SD of three determinations. \nNorthern blot analysis (insets in B) of vector \ntransfected controls and \u03b3-GCS transfected lines was \nperformed as described under methods section.  \n\nOverexpression of MRPA or \uf067-GCS in an \n\nantimony-sensitive isolate conferred increased \n\nexpression and resistance to antimony  \n\nTo determine whether overexpression of MRPA \n\nand \uf067-GCS conferred antimony resistance in a \n\nsensitive isolate, we transfected MRPA or \uf067-GCS \n\nconstructs into a SAG-S isolate, AG83-S. These \n\nMRPA and \uf067-GCS recombinant parasites were \n\nco-transfected with pGL-\uf061NEO\uf061LUC, encoding \n\nLUC. Intracellular amastigotes over-expressing \n\nMRPA (IC50, 32.3 \u00b1 6.4 \uf06dg/ml) were 3.6-fold \n\nmore resistant to SAG when compared to \n\namastigotes of the parent strain transfected with \n\nthe control vector (IC50, 9 \u00b1 0.5 \uf06dg/ml) (Figure \n\n5A).  Intracellular amastigotes over-expressing \uf067-\n\nGCS were 3.3-fold resistant to SAG when \n\ncompared to the sensitive L. donovani the IC50 \n\nvalue being (29.7 \u00b1 1.5 \uf06dg/ml) respectively \n\n(Figure 5B). In our previous studies we had \n\ndemonstrated that the ODC overexpressors \n\nexhibited significant resistance to Pentostam \n\ncompared to the wild type cells (26). Intracellular \n\namastigotes over-expressing ODC (IC50, > 80 \n\n\uf06dg/ml) were >8.8-fold more resistant to SAG \n\nwhen compared to amastigotes of the parent \n\nstrain transfected with the control vector [26].  \n\nDiscussion \nCurrently, chemotherapy is the only effective \n\nway to control Leishmania infection. Pentavalent \n\nantimonials are the mainstay of therapy in the \n\ntreatment of visceral leishmaniasis [27].  Increase \n\nin resistance to SAG has led to an upsurge in \n\ntherapeutic failure and in the absence of limited \n\nchemotherapeutic alternatives, it is extremely \n\nnecessary to identify biomarkers for monitoring \n\nantimony resistance.", "start_char_idx": 27255, "end_char_idx": 30239, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4ab51173-4c01-441c-9745-428bc961d73b": {"__data__": {"id_": "4ab51173-4c01-441c-9745-428bc961d73b", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "de6932c8-b273-4f8c-98c8-ed4d088c74c2", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "b526c9b6ecafb65dec7eb8093885f9d566a1a6925876d4d3847c37f4504a2efb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b7761ace-1f23-4ede-9a66-48e6f909e065", "node_type": "1", "metadata": {}, "hash": "a1bc9294078a6f82164deb4b06c35c4ec3f16685835c75eb03b1605ba525de45", "class_name": "RelatedNodeInfo"}}, "text": "Trivalent form of the antimonial drug (SbIII) is \n\nthe prodrug that is formed by conversion of \n\npentavalent antimony (SbV) by a putative \n\nmetalloid reductase present in the macrophages \n\n[28]. Antimonial resistance in both laboratory \n\nmutants and clinical isolates has been associated \n\nwith (a) decreased uptake of the drug through \n\naquaglyceroporin (AQP1) that codes for the \n\nprotein responsible for SbIII transport \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    11     Nepjol.info/index.php/njb\n\n         \n\n[11,12,20,29] (b) Over expression of ODC and \u03b3-\n\nGCS enzymes of the trypanothione biosynthetic \n\npathway [2,25] and (c) increased expression of \n\nthe ABC transporter MRPA, which sequesters \n\nSbIII-thiol conjugate [16,23]. \n\nWe had earlier reported decreased uptake of \n\nantimony in all nine SAG-R isolates used in the \n\npresent study. Down-regulation of SbIII influx \n\npump; aquaglyceroporin (AQP1) was observed \n\nin seven out of the nine resistant isolates  Strains, \n\nMC8-R and NR3A-R were an exception since \n\nthey showed up-regulation of  AQP1 gene \n\nexpression [20]. \n\nThe ABC transporter gene MRPA causes drug \n\nsequestration in Leishmania promastigotes and \n\namastigotes selected for SbIII resistance [17,30]. \n\nIncreased copy number of MRPA has been \n\nreported in SbIII resistant mutant [2]. Our earlier \n\nstudies using limited number of SAG-S and \n\nSAG-R clinical isolates showed amplification of \n\nMRPA gene as part of an extrachromosomal \n\ncircle [23]. In the present study, we found \n\ndistinct correlation between copy number of \n\nMRPA gene and antimony sensitivity in a \n\nmajority of the isolates (Figure 2A). However, \n\ntwo SAG-R strains, NR3A-R and RMP8-R were \n\nan exception. We have reported earlier that ABC \n\ntransporter gene MRPA was amplified in three \n\nout of four resistant VL isolates as part of the \n\nextrachromosmal circle and coamplifcation of \n\nPTR1 along with MRPA suggested amplification \n\nof the H locus in SAG-resistant clinical isolates \n\n[23]. In the present study, co-amplification of \n\nPTR1 gene with MRPA gene was not observed in \n\nany of the clinical isolates. CHEF gel analysis of \n\nthe SAG-S and SAG-R isolates did not show any \n\ncircular amplification in any of the SAG-R \n\nisolates. MRPA gene expression in SAG-S versus \n\nSAG-R field isolates showed correlation between \n\nMRPA gene expression and SAG susceptibility \n\nprofile of the clinical isolates. The present \n\nobservation validated our earlier results where \n\nwe have shown correlation between MRPA gene \n\nexpressions with the antimony resistant clinical \n\nprofile in the field conditions [23].  \n\nIn our previous study amplification of ODC gene \n\nwas noted in the resistant isolates but not that of \n\nthe \u03b3-GCS [23]. In another study on L. donovani \n\nisolates from Nepal, expression of \u03b3-GCS and \n\nODC was significantly decreased in the resistant \n\nisolates [31]. In the present study we observed \n\nincreased expression of ODC gene and \u03b3-GCS in \n\nall SAG-R isolates.  \n\nIt has been reported earlier that ABC transporter \n\nMRPA, confers resistance to antimonials by \n\nsequestration of metal thiol conjugates in an \n\nintracellular organelle located close to the \n\nflagellar pocket [16]. This model has been \n\ndemonstrated in promastigotes of L. tarentolae, in \n\namastigotes and also in clinical isolates from \n\nIndia [16,23,30]. These observations clearly \n\nhighlighted the importance of intracellular thiol \n\nin MRPA mediated efflux of the antimony.", "start_char_idx": 30244, "end_char_idx": 33831, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b7761ace-1f23-4ede-9a66-48e6f909e065": {"__data__": {"id_": "b7761ace-1f23-4ede-9a66-48e6f909e065", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4ab51173-4c01-441c-9745-428bc961d73b", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "66c63fb337a84c03d4fa7ec386a215e2e6a412d678fb47fa42766d904945e4a3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b1356489-9122-45a3-b319-3b6f10902653", "node_type": "1", "metadata": {}, "hash": "d2eb2c3664982206f96a3a98ca5b44f7305c0a93a01faecd6cbab0f4513c8ba0", "class_name": "RelatedNodeInfo"}}, "text": "An \n\nincrease in cysteine and glutathione levels were \n\nreported in antimony resistant L. donovani \n\nclinical isolates [23]. In the present study also, \n\nwe observed increase in cysteine and glutathione \n\nlevels in all SAG-R isolates. However, no change \n\nin trypanothione levels were observed in SAG-R \n\nisolates in comparison to SAG-S isolates. Similar \n\nobservation was made in our analysis of the \n\nmode of action of antimony in clinical isolates \n\nand earlier studies in L. infantum resistant to \n\nSb(III) [23]. It was pointed out that antimony \n\npossibly depleted trypanothione by efflux of Sb-\n\ntrypanothione conjugate [16]. It is possible that \n\nthe efflux system is increased in the resistant \n\nisolates thereby leading to increased \n\ntrypanothione efflux. This would explain the \n\nconstant levels of trypanothione in the present \n\nstudy in the resistant isolates.  \n\nRK1-S though a SAG-S isolate, was an exception \n\nto the mutlifactorial antimony resistant \n\nmechanisms reported in clinical isolates and also \n\nin the lab based resistant isolates. Clinical history \n\nof the RK1-S patient showed that the interval \n\nbetween the cure of VL and the onset of PKDL \n\nwas 2.5 years where as MS2-S and NR3A-R, \n\nPKDL isolates the interval between the cure of \n\nVL and the onset of PKDL was 7 and 11 years \n\nrespectively. It will be interesting to look at the \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    12     Nepjol.info/index.php/njb\n\n         \n\nPKDL isolates with known clinical history in \n\norder to determine if this interval has a role to \n\nplay in antimony susceptibility/resistance. \n\nThe data presented here in VL and for the first \n\ntime in PKDL isolates, establishes the relevance \n\nof overexpression of \u03b3\u2013GCS and ODC and an \n\nincreased expression of MRPA that may be \n\nresponsible for an increased efflux of thiol-Sb-III \n\nconjugate. Our data further confirms that \n\nresistance mechanisms present in the laboratory \n\nstrains can be found in the clinical isolates.  \n\nFurther work is presently going on the \n\nlaboratory to get a global overview of the \n\nresistant mechanism by proteomics approach in \n\norder to find other possible resistant \n\ndeterminants. It will further help in proper \n\nselection of therapeutic regimen and improve \n\ntreatment strategy against leishmaniasis. \n\nAcknowledgements \nThe clinical isolates used in this study were \n\nkindly provided by Dr. Mitali Chatterjee \n\n(Institute of Post Graduate Medical Education \n\nand Research, Kolkata, India) and Dr. Sarman \n\nSingh, (Division of Clinical Microbiology, All \n\nIndia Institute of Medical Sciences, New Delhi, \n\nIndia).  \n\nFunding \nThis work is supported by a grant from the \n\nDepartment of Biotechnology to Dr. Rentala \n\nMadhubala. Mahendra Maharjan and Swati \n\nMandal are supported by a fellowship from the \n\nUniversity Grants Commission, Government of \n\nIndia \n\nTransparency declarations: None to declare. \n\nReferences \n1. Guerin PJ, Olliaro P, Sundar S, Boelaert M, Croft SL, \n\nDesjeux P, Wasunna MK, Bryceson AD: Visceral \n\nleishmaniasis: current status of control, diagnosis, \n\nand treatment, and a proposed research and \n\ndevelopment agenda. Lancet Infect Dis. 2002, 2(8):494-\n\n501. \n\n2. Haimeur A, Brochu C, Genest P, Papadopoulou B, \n\nOuellette M: Amplification of the ABC transporter \n\ngene PGPA and increased trypanothione levels in \n\npotassium antimonyl tartrate (SbIII) resistant \n\nLeishmania tarentolae. Mol Biochem Parasitol. 2000, \n\n108(1):131-135. \n\n3.", "start_char_idx": 33833, "end_char_idx": 37416, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b1356489-9122-45a3-b319-3b6f10902653": {"__data__": {"id_": "b1356489-9122-45a3-b319-3b6f10902653", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b7761ace-1f23-4ede-9a66-48e6f909e065", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "1d709bb096faf4b41c7baa2667c07a71a09c629fd7b6f64e9ef53985d0c66311", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "13a48279-60f2-41a5-9b4e-dd9e8ed3e3f9", "node_type": "1", "metadata": {}, "hash": "89b2a73b843c942656b671fa88e9603285ac64a00fb95d4b97b0d67a9b696286", "class_name": "RelatedNodeInfo"}}, "text": "2000, \n\n108(1):131-135. \n\n3. Sundar S, More DK, Singh MK, Singh VP, Sharma S, \n\nMakharia A, Kumar PC, Murray HW:  Failure of \n\npentavalent antimony in visceral leishmaniasis in \n\nIndia: report from the center of the Indian \n\nepidemic. Clin. Infect. Dis. 200, 31(4): 1104-1107. \n\n4. Thakur CP, Kumar K: Post kala-azar dermal \n\nleishmaniasis: a neglected aspect of kala-azar \n\ncontrol programmes. Ann Trop Med Parasitol. 1992, \n\n86(4): 355-359. \n\n5. Zijlstra EE, Musa AM, Khalil EA, el-Hassan IM, el-\n\nHassan AM: Post-kala-azar dermal leishmaniasis. \n\nLancet Infect. Dis. 2003, 3(2): 87-98. \n\n6. Roberts WL, Berman JD, Rainey PM: In vitro \n\nantileishmanial properties of tri- and pentavalent \n\nantimonial preparations. Antimicrob Agents \n\nChemother. 1995, 39(6): 1234-1239. \n\n7. Denton H, McGregor JC, Coombs GH: Reduction of \n\nanti-leishmanial pentavalent antimonial drugs by a \n\nparasite-specific thiol-dependent reductase, TDR1. \n\nBiochem J. 2004, 381(2): 405-412. \n\n8. Ferreira CS, Martins PS, Demicheli C, Brochu C, \n\nOuellette M, Frezard F: Thiol-induced reduction of \n\nantimony(V) into antimony(III): a comparative \n\nstudy with trypanothione, cysteinyl-glycine, \n\ncysteine and glutathione. Biometals 2003, 16(3): 441-\n\n446. \n\n9. Zhou Y, Messier N, Ouellette M, Rosen BP, \n\nMukhopadhyay R: Leishmania major LmACR2 is a \n\npentavalent antimony reductase that confers \n\nsensitivity to the drug pentostam. J Biol Chem. 2004, \n\n279: 37445-37451. \n\n10. Shaked-Mishan P, Ulrich N, Ephros M, Zilberstein D: \n\nNovel Intracellular SbV reducing activity correlates \n\nwith antimony susceptibility in Leishmania \n\ndonovani. J Biol Chem. 2001, 276(6): 3971-3976. \n\n11. Gourbal B, Sonuc N, Bhattacharjee H, Legare D, \n\nSundar S, Ouellette M, Rosen BP, Mukhopadhyay R: \n\nDrug uptake and modulation of drug resistance in \n\nLeishmania by an aquaglyceroporin. J Biol Chem. \n\n2004, 279(30): 31010-31017. \n\n12. Marquis N, Gourbal B, Rosen BP, Mukhopadhyay R, \n\nOuellette M: Modulation in aquaglyceroporin AQP1 \n\ngene transcript levels in drug-resistant Leishmania. \n\nMol Microbiol. 2005, 57(6): 1690-1699. \n\n13. Wyllie S, Cunningham ML, Fairlamb AH: Dual \n\naction of antimonial drugs on thiol redox \n\nmetabolism in the human pathogen Leishmania \n\ndonovani. J Biol Chem. 2004, 279(38): 39925-39932. \n\n14. Dey S, Ouellette M, Lightbody J, Papadopoulou B, \n\nRosen BP: An ATP-dependent As (III)-glutathione \n\ntransport system in membrane vesicles of \n\nLeishmania tarentolae. Proc Natl Acad Sci U S A. \n\n1996, 93(5), 2192-2197. \n\n15. Fairlamb AH, Cerami A: Metabolism and functions \n\nof trypanothione in the Kinetoplastida.", "start_char_idx": 37388, "end_char_idx": 39989, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "13a48279-60f2-41a5-9b4e-dd9e8ed3e3f9": {"__data__": {"id_": "13a48279-60f2-41a5-9b4e-dd9e8ed3e3f9", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b1356489-9122-45a3-b319-3b6f10902653", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "cdb695d0135d6b41c3f9b13a4671dbd4b76e270eda41033371e7e9f180743c66", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9eded416-2244-46a8-aacd-55a9661eb351", "node_type": "1", "metadata": {}, "hash": "5e1a5ca7a8c7f88845b9347c727f1bac423dde8cff3e1f5fc4f4c674488c6394", "class_name": "RelatedNodeInfo"}}, "text": "Annu Rev \n\nMicrobiol. 1992, 46: 695-729. \n\n16. Legare D, Richard D, Mukhopadhyay R, Stierhof YD, \n\nRosen BP, Haimeur A, Papadopoulou B, Ouellette M: \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 6  Vol. 4, No. 1: 1-13                                    Maharjan et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal    13     Nepjol.info/index.php/njb\n\n         \n\nThe Leishmania ATP-binding cassette protein \n\nPGPA is an intracellular metal-thiol transporter \n\nATPase. J Biol Chem. 2001, 276(28): 26301-26307. \n\n17. Legare D, Papadopoulou B, Roy G, Mukhopadhyay \n\nR, Haimeur A, Dey S, Grondin K, Brochu C, Rosen \n\nBP, Ouellette M: Efflux systems and increased \n\ntrypanothione levels in arsenite-resistant \n\nLeishmania. Exp Parasitol. 1997, 87(3): 275-282. \n\n18. Mukhopadhyay R, Dey S, Xu N, Gage D, Lightbody J, \n\nOuellette M, Rosen BP: Trypanothione \n\noverproduction and resistance to antimonials and \n\narsenicals in Leishmania. Proc. Natl. Acad. Sci. U. S. A \n\n1996, 93(19): 10383-10387. \n\n19. Dey A, Singh S: Genetic heterogeneity among \n\nvisceral and post-Kala-Azar dermal leishmaniasis \n\nstrains from eastern India. Infect Genet Evol. 2007, \n\n7(2): 219-222. \n\n20. Mandal S, Maharjan M, Singh S, Chatterjee M, \n\nMadhubala R: (2010) Assessing aquaglyceroporin \n\ngene status and expression profile in antimony-\n\nsusceptible and -resistant clinical isolates of \n\nLeishmania donovani from India. J Antimicrob \n\nChemother. 2010, 65(3): 496-507. \n\n21. Sambrook J, Fritsch EF, Maniatis T: Molecular \n\ncloning: A laboratory manual. Cold Spring Harbour \n\nPress.  Int J Hyperthermia. 1989, 8: 329-340 \n\n22. Roy G, Dumas C, Sereno D, Wu Y, Singh AK, \n\nTremblay MJ, Ouellette M, Olivier M, Papadopoulou \n\nB: Episomal and stable expression of the luciferase \n\nreporter gene for quantifying Leishmania spp. \n\ninfections in macrophages and in animal models. \n\nMol Biochem Parasitol. 2000, 110(2): 195-206. \n\n23. Mukherjee A, Padmanabhan PK, Singh S, Roy G, \n\nGirard I, Chatterjee M, Ouellette, M, Madhubala R: \n\nRole of ABC transporter MRPA, gamma-\n\nglutamylcysteine synthetase and ornithine \n\ndecarboxylase in natural antimony-resistant isolates \n\nof Leishmania donovani. J Antimicrob Chemother. \n\n2007, 59(2): 204-211. \n\n24. Grondin K, Haimeur A, Mukhopadhyay R, Rosen BP, \n\nOuellette M: Co-amplification of the gamma-\n\nglutamylcysteine synthetase gene gsh1 and of the \n\nABC transporter gene pgpA in arsenite-resistant \n\nLeishmania tarentolae. EMBO J. 1997, 16(11): 3057-\n\n3065. \n\n25.", "start_char_idx": 39990, "end_char_idx": 42467, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9eded416-2244-46a8-aacd-55a9661eb351": {"__data__": {"id_": "9eded416-2244-46a8-aacd-55a9661eb351", "embedding": null, "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-29", "node_type": "4", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "c99835a1ce87d6a5446bd5bd788e81a0710446b37cc84b8c1fcd942cfd750990", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "13a48279-60f2-41a5-9b4e-dd9e8ed3e3f9", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "676304c2b24a87c30956478348e41b5f8be4fe803ee96bb7f118310af00fc6e5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "18caac93-a45b-43c7-93bc-bf5ef0600ef2", "node_type": "1", "metadata": {}, "hash": "c06e009292d38f02e4fb55f797d541f8620a123e6f824a4b473ed7006954dff4", "class_name": "RelatedNodeInfo"}}, "text": "1997, 16(11): 3057-\n\n3065. \n\n25. Haimeur A, Guimond C, Pilote S, Mukhopadhyay R, \n\nRosen BP, Poulin R, Ouellette M: Elevated levels of \n\npolyamines and trypanothione resulting from \n\noverexpression of the ornithine decarboxylase gene \n\nin arsenite-resistant Leishmania. Mol Microbiol. 1999, \n\n34(4): 726-735. \n\n26. Singh S, Mukherjee A, Khomutov AR, Persson L, \n\nHeby O, Chatterjee M, Madhubala R:  \n\nAntileishmanial effect of 3-aminooxy-1-\n\naminopropane is due to polyamine depletion. \n\nAntimicrob Agents Chemother. 2007, 51(2): 528-534. \n\n27. den Boer ML, Alvar J, Davidson RN, Ritmeijer K, \n\nBalasegaram M: Developments in the treatment of \n\nvisceral leishmaniasis. Expert Opin Emer. Drugs. \n\n2009, 14(3): 395-410. \n\n28. Sereno D, Cavaleyra M, Zemzoumi K, Maquaire S, \n\nOuaissi A, Lemesre JL: Axenically grown \n\namastigotes of Leishmania infantum used as an in \n\nvitro model to investigate the pentavalent antimony \n\nmode of action. Antimicrob Agents Chemother. 1998, \n\n42(12): 3097-3102. \n\n29. Maharjan M, Singh S, Chatterjee M, Madhubala R: \n\nRole of aquaglyceroporin (AQP1) gene and drug \n\nuptake in antimony-resistant clinical isolates of \n\nLeishmania donovani. Am J Trop Med Hyg. 2008, \n\n79(1): 69-75. \n\n30. El FK, Messier N, Leprohon P, Roy G, Guimond C, \n\nTrudel N, Saravia NG, Papadopoulou B, Legare D, \n\nOuellette M: Role of the ABC transporter MRPA \n\n(PGPA) in antimony resistance in Leishmania \n\ninfantum axenic and intracellular amastigotes. \n\nAntimicrob Agents Chemother. 2005, 49(5): 1988-1993. \n\n31. Decuypere S, Rijal S, Yardley V, De DS, Laurent T, \nKhanal B, Chappuis F, Dujardin JC: Gene expression \n\nanalysis of the mechanism of natural Sb(V) \n\nresistance in Leishmania donovani isolates from \n\nNepal. Antimicrob Agents Chemother. 2005, 49(11): \n\n4616-4621. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Persson%20L%5BAuthor%5D&cauthor=true&cauthor_uid=17101681\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Heby%20O%5BAuthor%5D&cauthor=true&cauthor_uid=17101681\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Chatterjee%20M%5BAuthor%5D&cauthor=true&cauthor_uid=17101681\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Madhubala%20R%5BAuthor%5D&cauthor=true&cauthor_uid=17101681", "start_char_idx": 42435, "end_char_idx": 44648, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "18caac93-a45b-43c7-93bc-bf5ef0600ef2": {"__data__": {"id_": "18caac93-a45b-43c7-93bc-bf5ef0600ef2", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9eded416-2244-46a8-aacd-55a9661eb351", "node_type": "1", "metadata": {"identifier": "njb-29", "author": "Maharjan, Mahendra; Mandal, Swati; Madhubala, Rentala", "title": "Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India", "date": "2016-12-31", "file": "njb-29.pdf"}, "hash": "57b6fdcbfef09b5b876a7954c2b8493dfb6f391bcf1a81bd491fab5dfe9d8859", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f86c2765-14f4-4841-b0e2-1dba3c6117a0", "node_type": "1", "metadata": {}, "hash": "1fa2af8984fe67292c11820b662d104cbb0b7a0f035360b769a89e8609300443", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 29-32  Research article DOI: https://www.doi.org/10.54796/njb.v9i2.41912 \n  \n\n\u00a9NJB, BSN  29 \n\nSero-prevalance of Cryptococcal Antigenemia in HIV Positive \nIndividual having CD4 Counts <100 Cells/mm3 \nSundar Khadka  , Samikshya Kandel , Roshan Pandit , Rosham Manjhi, Subhash Dhital, Jagat Bahadur \nBaniya, Shravan Kumar Mishra, Raj Kumar Mahato \nNational Public Health Laboratory (NPHL), Department of Health Services, Ministry of Health and Population, \nTeku, Kathmandu 44-600, Nepal \nReceived: 5th Nov 2020; Revised: 21st Sep 2021; Accepted: 17th Nov 2021; Published online: 31st Dec 2021 \n\n Abstract \nCryptococcus neoformans is one of the foremost common opportunistic infectious agents in people living with Acquired \nImmuno Deficiency Syndrome (AIDS). It has been reported to cause about 1 million cases of cryptococcal meningitis \nper year among HIV/AIDS and 600,000 deaths annually. This study was done to find the prevalence of Cryptococcal \nantigenemia among HIV positive individuals having CD4counts <100 cells/mm3.  \nA cross-sectional study was conducted in the HIV Reference unit, National public health laboratory from July to \nDecember 2015. The study comprised of 99 HIV positive individuals having CD4counts <100 cells/mm3. CD4 T cell \ncount was performed by flow cytometry (BD Biosciences, San Jose, CA, USA) and Cryptococcal antigen test by Latex \nagglutination assay. The overall prevalence of cryptococcal antigenemia was found to be 18.2%. Of the total ninety-nine \nsubjects enrolled in the study, 72 (72.8%) were males and 27 (27.2%) were females. The mean age of the patients was 38 \nyears ranging from 13 to 69 years. Higher percentage of female (22.2%) showed Cryptococcal infection in our study as \ncompared to male (16.7%).  \nThe study concludes higher prevalence of Cryptococcal antigenemia among HIV infected individuals and recommends \nCryptococcal antigen screening to be made mandatory in HIV positive patients having CD4 T cells count below 100/\u00b5l. \n\nKeywords: CD4, CrAg, Cryptococcosis, HIV, AIDS, Lateral Flow assay. \n\n Corresponding author, email: cls.sundar@gmail.com \n\nIntroduction  \nCryptococcus neoformans is one amongst the most \ncommon opportunistic pathogens infecting people with \nadvanced Human Immune Deficiency Virus (HIV). \nCryptococcal meningitis and Cryptococcal pneumonia \nare the common form of Cryptococcal infection in \npeople living with Human Immune Deficiency Virus \n(PLHIV) [1). Global data estimates in 2013 shows that \n31.8 million people live with HIV among which 1.4 \nmillion were reported for Acquired Immuno Deficiency \nSyndrome (AIDS) related death [2]. Cryptococcal \nmeningitis is supposed to cause 15% of AIDS-related \nmortality [2]. A study carried out in 2009 had estimated \napproximately 1 million cases of Cryptococcal \nmeningitis [CM) with 600,000 deaths worldwide \nannually [3].  \nCryptococcal meningitis represents a contagious form of \nthe Cryptococcal disease that necessitates \nhospitalization and administration of drugs like \namphotericin B. The infection is common mainly in \nimmunocompromised HIV infected individuals having \nCD4 T lymphocytes count less than 100/\u00b5l. The \ninfection can be treated easily if diagnosed earlier.", "start_char_idx": 98, "end_char_idx": 3349, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f86c2765-14f4-4841-b0e2-1dba3c6117a0": {"__data__": {"id_": "f86c2765-14f4-4841-b0e2-1dba3c6117a0", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "18caac93-a45b-43c7-93bc-bf5ef0600ef2", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "ab0f98a3b36b50b38d4e9c66569b91397a1ece7218578b81613d64afbc603d1b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b2794686-c361-408b-bd85-ead6cd692436", "node_type": "1", "metadata": {}, "hash": "078f6aade23b269c27436148be6f6139d4f7e3b21a258b572f02fb1bd10ca564", "class_name": "RelatedNodeInfo"}}, "text": "The \ninfection can be treated easily if diagnosed earlier. \n\nHowever, in several cases the infection is asymptomatic \nand thus go unnoticed leading to complications that \nmay further lead to death of patients [4]. \nThe World Health Organization (WHO) guideline \nrecommends two strategies for the prevention of \nCryptococcal meningitis; 1) screening of patients with \nCD4+ T cell counts below 100 cells/mm3 for \nCryptococcal antigen (CrAg) and 2) primary \nprophylaxis with fluconazole for patients with CD4+ T \ncell counts below 100 [5]. Cryptococcal antigen (CrAg) is \na biological marker of Cryptococcal infection that can be \ndetected in serum of patients in an average of 21 days \n(range 5\u2013234 days) before onset of symptoms of \nmeningitis [6]. Cryptococcal infection can be identified \nusing several laboratory tests such as Latex \nAgglutination Tests (LAT), Enzyme Immuno Assay \n(EIA), and Polymerase Chain Reaction (PCR); each \nhaving different sensitivity and specificity [6]. However, \nCryptococcal antigen detection using Latex  \nagglutination test is performed widely for screening of \nCryptocococcal infection as it is easy to perform and do \nnot require sophisticated laboratory set up. Timely \nidentification of Cryptococcosis in PLHIV in the AIDS \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n \n\nhttps://orcid.org/0000-0002-5110-4446\nmailto:cls.sundar@gmail.com\nhttps://orcid.org/0000-0001-5170-5500\nhttps://orcid.org/0000-0003-2946-1233\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 29-32 Khadka et al. \n\n\u00a9NJB, BSN 30 \n\nstage prevents them from further risk of morbidity and \nmortality. There are different prevalence rates of \nCryptococcal antigenemia from different parts of the \nworld [2, 7-9]. However, there is no such study from \nNepal till date to our knowledge. In this regard we \ncarried out this study with the objective to find out the \nprevalence of co-infection by Cryptococcus neoformans in \nimmunocompromised patients living with HIV in \nNepal. \n\nMaterials and methods \n A cross-sectional study was conducted in the HIV \nReference unit of National Public Health Laboratory \n(NPHL), Kathmandu, Nepal from July to December \n2015. Samples were collected from patients visiting \nNPHL for CD4 T cell count. All subjects enrolled in this \nstudy were HIV positive patients having CD4 T cells \ncounts less than 100 cells/mm3. A study questionnaire \nwas used to collect demographic data from each patient. \nAbout 3 ml of EDTA whole blood was collected from \neach patient by trained professionals for the purpose of \nthis study. The whole blood sample was used to \ndetermine CD4 T cell count by flow cytometry using the \nBD fluorescent-activated cell sorter system (BD \nBiosciences, San Jose, CA, USA) as per the \nmanufacturer\u2019s instructions. Briefly, 20 \u03bcl of BD Tritest \nCD3/CD4/CD45 reagent was pipetted into a BD \nTrucount tube containing bead followed by 50 \u03bcl of \nwell-mixed anticoagulated whole blood which was then \nvortexed and incubated in dark at room temperature for \n15 minutes for staining of the cells. Following staining \ncells were lysed using BD FACS lysing solution and run \nin the flow cytometer to obtain absolute count.", "start_char_idx": 3291, "end_char_idx": 6577, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b2794686-c361-408b-bd85-ead6cd692436": {"__data__": {"id_": "b2794686-c361-408b-bd85-ead6cd692436", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f86c2765-14f4-4841-b0e2-1dba3c6117a0", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "03c6b5eb2bb037e5e23b7538662d351f9818b827c56ecfbd079c25584ac89cbd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b467eb26-2321-4602-85d9-3e8f79a2eee4", "node_type": "1", "metadata": {}, "hash": "8a13f858ff6e83ec7c20921610dd70cd8f75a251eb0a13db6f5c92e07961427a", "class_name": "RelatedNodeInfo"}}, "text": "For the purpose of Cryptococcal antigen testing plasma \nsample was separated from EDTA whole blood, which \nwas then used for antigen detection using Cryptococcal \nLatex Agglutination test (Immuno Mycologics Inc, \nUSA). The test kit employed is based on antigen-\nantibody agglutination reaction that detects capsular \npolysaccharide antigen of C. neoformans. The test kit is \nbelieved to have higher specificity and greater than 95% \nsensitivity. The assay procedure involves 5-easy steps \nwith no specimen pretreatment. For test, at first a drop \nof lateral flow assay (LFA) specimen diluent was added \nto a disposable test tube. In the second step, 40 \u00b5L of the \nspecimen was added to the tube and mixed. Afterward, \na CrAg LFA test strip was inserted into the specimen \nand read at 10 min. The validity of the test was indicated \nby a single control line and the presence of line in test \nregion indicated a valid positive test [10].  \n\nInformed consent was from each patient enrolled in this \nstudy after explaining them with the objective and \noutcome of this study. Ethical approval was taken from \nNepal Health Research Council (ref no. 892) before \ncarrying out this study.  \n\nResults \nA total of Ninety-nine HIV positive patients were \nenrolled in the study during the period of six months. \nThe study comprised 72 males and 27 females which \nwere 72.8% and 27.2% of the total, respectively. The \nratio of male to female patients was 8:3. The minimum \nand maximum age of study population was 13 years \nand 69 years, respectively with the mean age of 38 years. \nThe mean CD4 T cell count was 60.4 that range from 2 to \n98 cells/mm3. Of the total patients enrolled in the study, \n18 (18.2%) were found positive for Cryptococcal antigen. \nThe positive to negative ratio for Cryptococcal antigen \nwas observed to be 2:11. The prevalence rate of \nCryptococcal antigen among male patients was found to \nbe 16.7% while that for female patients was found to be \n22.2%.  In total, the prevalence rate was 18.2%. The \nprevalence rate was observed higher among female \npatients as compared to male patients Table 1. \n\nTable 1. Prevalence of  Cryptococcal antigen among HIV \npatients (N=99) \nSex  CrAg Positive CrAg Negative Total \nMale 12 (16.7%) 60 (83.3%) 72 (100%) \nFemale 6 (22.2%) 21 (77.8%) 27 (100%) \nTotal 18 (18.2%) 81 (81.8%) 99 (100%) \n\nDiscussion \nThis study highlights the sero-prevalence of \nCryptococcal antigen in HIV positive individuals having \nCD4 counts less than 100 cells/mm3. HIV positive \npatients having CD4 count < 100 cells/mm3 is referred \nto be in the AIDS stage. These patients are in \nimmunocompromised state and are at higher risk of \nopportunistic infection. Cryptococcus neoformans is a \nfungus widely present in the environment and are able \nto infect immunocompromised host. Thus, HIV infected \nindividuals in AIDS stage are highly susceptible for \nopportunistic infection by several other agents beside C. \nneoformans. In this study, we have estimated the \nprevalence rate of opportunistic infection by C. \nneoformans in immunocompromised HIV positive \nindividuals.  \nThe prevalence of Cryptococcosis was 18.2 % among 99 \nHIV positive patients having CD4 count < 100 \ncells/mm3 in this study, which shows that a significant \nproportion of PLHIV has Cryptococcus infection in \nNepal. This finding is comparable to a study by Micol et \nal. (2007) and Oyella et al.", "start_char_idx": 6579, "end_char_idx": 9975, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b467eb26-2321-4602-85d9-3e8f79a2eee4": {"__data__": {"id_": "b467eb26-2321-4602-85d9-3e8f79a2eee4", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b2794686-c361-408b-bd85-ead6cd692436", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "273c205c90db10de133d51d50f2d3fa2b503a7ffbafb9de601150005501d7250", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "86261458-e17e-4f4c-b2f8-4d27bce425f6", "node_type": "1", "metadata": {}, "hash": "5830e48d7e5737552f67e3eb851a95a75887956240e802e1defe7ae2c39b7842", "class_name": "RelatedNodeInfo"}}, "text": "(2007) and Oyella et al. (2012)  but, much higher than \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 29-32 Khadka et al. \n\n\u00a9NJB, BSN 31 \n\nthe study by Lakoh et al. [9, 11, 12]. In one study done \nby Osazuwa et al. the prevalence rate was just 12.7% \namong 150 ART na\u00efve populations, which is less as \ncompared to our study [13]. ART na\u00efve refers to a stage \nbefore receiving antiretroviral therapy (ART). It is \nbelieved that persons in ART have lower risk of \nopportunistic infection. However, the data in our study \nis much higher as compared to study by Osazuwa et al.  \nThe difference in data might be due to the difference in \nexposure to the causative agent of Cryptococcosis. \nThe ratio of female to male HIV patients in our study \nwas 2:11.  The number of males 72 (72.8%) were higher \nthan the number of females 27 (27.2%). The data is in \ncontrary to the study of Geda et al. that reported higher \nprevalence of HIV infection in females [14]. This may be \ndue to differences of exposure as well as social stigma. \nHigher number of HIV-infected males as compared to \nfemales in Nepal might be another reason for the \ndifference in enrollment of males and females in our \nstudy [15].  \nFungal culture is the gold standard for the detection of \nCryptococcus neoformans. However, some literature \nsuggest that the latex agglutination test for Cryptococcal \nantigen has 100% sensitivity and 96% specificity \ncompared to culture [10]. Our study shows that there is \nhigh prevalence of Cryptococcal antigenemia among \nimmunocompromised HIV positive individuals in \nNepal. Though our national guidelines do not \nrecommend Cryptococcal screening and primary \nprophylaxis mandatory for every HIV infected \nindividuals having CD4 T cell count less than 100 \ncells/mm3, this study recommends testing for \nCryptococcal infection at least by latex agglutination \nassay to be made as a routine test for such cases. This \nmay be helpful in preventing morbidity and mortality \ncaused due to meningitis among HIV infected \nindividuals.  \n\nConclusion \nThe study revealed that 18.2 % of people living with \nHIV in AIDS stage in Nepal are seropositive for the \nCryptococcal antigen. Furthermore, in absence of gold \nstandard test available for detection of Cryptococcal \ninfection, serological antigen screening by latex \nagglutination assay is helpful to detect the cases of \nCryptococcosis among immunocompromised HIV \ninfected individuals.   \n\nAbbreviations \nHIV: Human immunodeficiency virus \nAIDS: Acquired Immuno Deficiency Syndrome \n\nART: Antiretroviral therapy \nCD4: Cluster of differentiation 4 \nCrAg: Cryptococcal antigen  \nLAT: Latex Agglutination Tests  \n\nConsent for publication \nNot applicable \n\nCompeting Interests \nAll the authors declare that they have no any competing \ninterests. \n\nAuthor\u2019s contributions \nSK, SD, SKM, and RKM were responsible for study \ndesign, supervision of work, and guidance. SK, SK, RP, \nRM, SD and JBB performed laboratory work and data \nanalysis. SK, SK, RP, RM and SD prepared the draft and \nwrote the manuscript. Finally, all the authors read the \nmanuscript and approved for publication. \n\nAcknowledgment \nWe would like to acknowledge the all staffs of HIV \nReference Unit, National Public Health Laboratory, \nNepal for their valuable support in completing this \nwork \n\nEthical Approval and Consent \nEthical approval was taken from Nepal Health Research \nCouncil (ref no. 892) before carrying out this study \n\nReferences \n1.", "start_char_idx": 9951, "end_char_idx": 13405, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "86261458-e17e-4f4c-b2f8-4d27bce425f6": {"__data__": {"id_": "86261458-e17e-4f4c-b2f8-4d27bce425f6", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b467eb26-2321-4602-85d9-3e8f79a2eee4", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "b45389a09865a301a8dae664b8656dc980491ebc4f6bd08f3eb560aa73ce4809", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "586bb2ab-3c08-465a-b3a5-2e4956ee8c80", "node_type": "1", "metadata": {}, "hash": "ca09f0ebe45381f9d2153994bbf583a4cdd05fb29335e46c884c8b379c617871", "class_name": "RelatedNodeInfo"}}, "text": "892) before carrying out this study \n\nReferences \n1. Osazuwa F, Dirisu JO, Okuonghae PE, Ugbebor O. Screening for\n\ncryptococcal antigenemia in anti-retroviral na\u00efve AIDS patients in\nBenin City, Nigeria. Oman Medical Journal. 2012;27(3):228. \n\n2. Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP,\nChiller TM, et al. Global burden of disease of HIV-associated\ncryptococcal meningitis: an updated analysis. The Lancet\ninfectious diseases. 2017;17(8):873-81. \n\n3. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG,\nChiller TM. Estimation of the current global burden of\ncryptococcal meningitis among persons living with HIV/AIDS.\nAids. 2009;23(4):525-30. \n\n4. Smith RM, Nguyen TA, Ha HTT, Thang PH, Thuy C, Xuan Lien T,\net al. Prevalence of cryptococcal antigenemia and cost-\neffectiveness of a cryptococcal antigen screening program\u2013\nVietnam. PloS one. 2013;8(4):e62213. \n\n5. WHO. Guideline for the diagnosis, prevention and management of\ncryptococcal disease in HIV-infected adults, adolescents and\nchildren. World Health Organization (WHO); 2018. \n\n6. Saha DC, Xess I, Biswas A, Bhowmik DM, Padma M. Detection of\nCryptococcus by conventional, serological and molecular methods.\nJournal of medical microbiology. 2009;58(8):1098-105. \n\n7. Letang E, M\u00fcller MC, Ntamatungiro AJ, Kimera N, Faini D, Furrer\nH, Battegay M, Tanner M, Hatz C, Boulware DR, Glass TR.\nCryptococcal antigenemia in immunocompromised human\nimmunodeficiency virus patients in rural Tanzania: a preventable\ncause of early mortality. InOpen forum infectious diseases 2015\nApr 1 (Vol. 2, No. 2). Oxford University Press.\n\n8. Ezenabike C, Ashaka OS, Omoare AA, Fadeyi A, Salami AK,\nAgbede OO. Cryptococcal antigen among HIV1-infected\nindividuals in north-central Nigeria. Current Medical Mycology.\n2020; 6(2):43.", "start_char_idx": 13353, "end_char_idx": 15146, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "586bb2ab-3c08-465a-b3a5-2e4956ee8c80": {"__data__": {"id_": "586bb2ab-3c08-465a-b3a5-2e4956ee8c80", "embedding": null, "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-3", "node_type": "4", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "17ed1174cff201e55f622fd876b88ced2e2c89ae95283e7269e3f089b513e306", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "86261458-e17e-4f4c-b2f8-4d27bce425f6", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "fa3ac0f018ad709859b952c07a47151a405ea8518e852da9e3df2a55c42ab303", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "352d58dd-2d7b-4187-ae0b-9bdf7b3d4953", "node_type": "1", "metadata": {}, "hash": "c57a3f59c1f9f7510e90b67ee106e6fdfdb97afb50151fcbbd7ec8ea35598e3a", "class_name": "RelatedNodeInfo"}}, "text": "Current Medical Mycology.\n2020; 6(2):43. \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 29-32 Khadka et al. \n\n\u00a9NJB, BSN 32 \n\n9. Lakoh S, Rickman H, Sesay M, Kenneh S, Burke R, Baldeh M, et al.\nPrevalence and mortality of cryptococcal disease in adults with\nadvanced HIV in an urban tertiary hospital in Sierra Leone: a\nprospective study. BMC infectious diseases. 2020;20(1):1-7. \n\n10. CrAg\u00ae LFA Cryptococcal Antigen Lateral Flow Assay For the\nDetection of Cryptococcal Antigen. IMMY.\n\n11. Micol R, Lortholary O, Sar B, Laureillard D, Ngeth C, Dousset J-P,\net al. Prevalence, determinants of positivity, and clinical utility of\ncryptococcal antigenemia in Cambodian HIV-infected patients.\nJAIDS Journal of Acquired Immune Deficiency Syndromes.\n2007;45(5):555-9. \n\n12. Oyella J, Meya D, Bajunirwe F, Kamya MR. Prevalence and factors\nassociated with cryptococcal antigenemia among severely\nimmunosuppressed HIV-infected adults in Uganda: a cross-\nsectional study. Journal of the International AIDS Society.\n2012;15(1):1-7. \n\n13. Osazuwa OF, Dirisu O, Okuonghae E. Cryptococcal antigenemia\nin anti-retroviral naive AIDS patients: prevalence and its\nassociation with CD4 cell count. Acta Medica Iranica. 2012:344-7. \n\n14. Geda N, Beyene T, Dabsu R, Mengist HM. Prevalence of\nCryptococcal Antigenemia and associated factors among\nHIV/AIDS patients on second-line antiretroviral therapy at two\nhospitals in Western Oromia, Ethiopia. PloS one.\n2019;14(12):e0225691. \n\n15. Government of Nepal MoHaP. National HIV Strategic Plan 2016-\n2021. In: control NCfAaS, editor. Nepal2017. \n\n\n\tSero-prevalance of Cryptococcal antigenemia in HIV positive individual having CD4 counts <100 cells/mm3\n\tAbstract\n\tIntroduction\n\tMaterials and methods\n\tResults\n\tDiscussion\n\tConclusion\n\tAbbreviations\n\tConsent for publication\n\tCompeting Interests\n\tAuthor\u2019s contributions\n\tAcknowledgment\n\tReferences", "start_char_idx": 15106, "end_char_idx": 16969, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "352d58dd-2d7b-4187-ae0b-9bdf7b3d4953": {"__data__": {"id_": "352d58dd-2d7b-4187-ae0b-9bdf7b3d4953", "embedding": null, "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-30", "node_type": "4", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "e9bcbf7c7a15b0551ae24ca0306a3b662703a5ecca9b422be5d9265ea36ec196", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "586bb2ab-3c08-465a-b3a5-2e4956ee8c80", "node_type": "1", "metadata": {"identifier": "njb-3", "author": "Khadka, Sundar; Kandel, Samikshya; Pandit, Roshan; Manjhi, Rosham; Dhital, Subhash; Baniya, Jagat Bahadur; Mishra, Shravan Kumar; Mahato, Raj Kumar", "title": "Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts ", "date": "2021-12-30", "file": "njb-3.pdf"}, "hash": "a5da2bc79c123de246a20a7f19fa9c945e93f4abd4549584be604924159563f7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a9c68ea8-fb6c-4dfa-8031-ff11fd34d5f3", "node_type": "1", "metadata": {}, "hash": "51f746a7d3af8db2c5a708a1ed5af16b37641540e5397e817dc65dc937f8b68a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:68-70  ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\n REVIEW ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   68     Nepjol.info/index.php/njb \n \n\nMicrobial Diversity in Freshwater and Marine Environment \n \n\nSagar Aryal1*, Gaurab Karki1, Sunil Pandey2 \n1Department of Microbiology, St. Xavier\u2019s College, Kathmandu, Nepal \n\n2 Department of Microbiology, Nobel College, Kathmandu, Nepal \n\nAbstract \nWater covers seven tenths of the Earth's surface and occupies an estimated total volume of 1,386,000,000 cubic kilometers \n\n(km3). Of all the water found on Earth, 97% is marine. Maximum of this water is at a temperature of 2 to 3\u00b0C and devoid of \n\nlight; 62% is under high pressure (>100 atm). Microscopic phytoplankton and associated bacteria generate a complex food \n\nweb that can extend over long distances and extreme depths. The marine environment looks so vast that it will not be able \n\nto be exaggerated by pollution; however, in coastal areas human activities are increasingly disrupting microbial processes \n\nand damaging water quality. \n\nKeywords: Ecology, Microorganisms, Nutrition, Water, Phototrophs. \n\n*Corresponding Author  \n\nEmail: broneps1@gmail.com \n \n\nIntroduction \nMicrobial diversity that we see today is the result of \n\nnearly 4 billion years of evolutionary change. \n\nMicrobial diversity can be seen in many forms, \n\nincluding cell size and cell morphology, physiology, \n\nmotility, pathogenicity, developmental biology, \n\nadaptation to environmental extremes, phylogeny and \n\nmechanism of cell division [1]. \n\nMicroorganisms are present everywhere on Earth that \n\nwill support life. These include habitats we are all \n\nfamiliar with- soil, water, animal, and plants-as well as \n\nvirtually any structures made by humans. Some \n\nmicrobial habitats are ones in which humans could not \n\nsurvive, being too hot or too cold, too acidic or too \n\ncaustic, or too salty. Although such environments \n\nwould pose challenges to any life forms, they are often \n\nteeming with microorganisms. Organisms inhabiting \n\nsuch extreme environments are called extremophiles, a \n\nremarkable group of microorganisms that collectively \n\ndefine the physiochemical limits to life [2]. \n\nApproximately 6000 species of prokaryotes and \n\n100,000 species of protists have been formally \n\ndescribed [3]. In the case of the diversity of \n\nmicroorganisms, even the right order of magnitude is \n\nunknown and the issue is highly controversial [4\u20136]. \n\nThe total number of prokaryotic cells in the oceans has \n\nbeen estimated to be 1029 [7]. \n\nAll prokaryotic organisms are classified as bacteria, \n\nwhereas eukaryotic organisms include fungi, \n\nprotozoa, and helminths as well as humans. \n\nProkaryotic organisms are divided into two major \n\ngroups: the eubacteria, which include all bacteria of \n\nmedical importance, and the archaebacteria, a \n\ncollection of evolutionarily distinct organisms [1]. \n\nFreshwater Microbial Diversity \nFreshwater and marine environments differ in many \n\nways including salinity, average temperature, depth, \n\nand nutrient content, but both provide many excellent \n\nhabitats for microorganisms. \n\nLarge numbers of microorganisms in a body of water \n\ngenerally indicate high nutrient levels in the water. \n\nWater contaminated by inflows from sewage systems \n\nor from biodegradable industrial organic wastes is \n\nrelatively high in bacterial numbers. Similarly, ocean \n\nestuaries (fed by rivers) have higher nutrient levels \n\nand therefore larger microbial populations than other \n\nshoreline waters [8]. \n\nIn water, particularly water with low nutrient \n\nconcentrations, microorganisms tend to grow on \n\nstationary surfaces and on particulate matter. In this \n\nway, a microorganism has contact with more nutrients \n\nthan if it were randomly suspended and floating freely \n\nwith the current. Many bacteria whose main habitat is \n\nwater often have appendages and holdfasts that attach \n\nto various surfaces. \n\nFreshwater environments are highly variable in the \n\nresources and conditions available for microbial \n\ngrowth. Both oxygen producing and oxygen-\n\nconsuming organisms are present in aquatic \n\nenvironments, and the balance between", "start_char_idx": 47, "end_char_idx": 4269, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a9c68ea8-fb6c-4dfa-8031-ff11fd34d5f3": {"__data__": {"id_": "a9c68ea8-fb6c-4dfa-8031-ff11fd34d5f3", "embedding": null, "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-30", "node_type": "4", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "e9bcbf7c7a15b0551ae24ca0306a3b662703a5ecca9b422be5d9265ea36ec196", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "352d58dd-2d7b-4187-ae0b-9bdf7b3d4953", "node_type": "1", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "dba32c8a923fa7ffe08159f0a58ae0d6f51d6c2358ba49e4576737cf679d5a0f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "94d41e40-a005-4598-992d-c2a3f33fd611", "node_type": "1", "metadata": {}, "hash": "60c6fc72a871346fa88e3332e1061eb0380346fd98726a20761a7787430de9da", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:68-70           Aryal et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   69     Nepjol.info/index.php/njb \n \n\nphotosynthesis and respiration controls the natural \n\ncycles of oxygen, carbon, and other nutrients \n\n(nitrogen, phosphorus, metals). Among \n\nmicroorganisms, oxygenic phototrophs include the \n\nalgae and cyanobacteria. These can either be \n\nplanktonic (floating) and distributed throughout the \n\nwater columns of lakes, sometimes accumulating in \n\nlarge numbers at a particular depth, or benthic, \n\nmeaning they are attached to the bottom or sides of a \n\nlake or stream. Because oxygenic phototrophs obtain \n\ntheir energy from light and use water as an electron \n\ndonor to reduce CO to organic matter, they are called \n\nprimary producers [9]. \n\nA typical lake or pond serves as an example to \n\nrepresent the various zones and the kinds of \n\nmicrobiota found in a body of fresh water. The littoral \n\nzone along the shore has considerable rooted \n\nvegetation, and light penetrates throughout it. The \n\nlimnetic zone consists of the surface of the open water \n\narea away from the shore. The profundal zone is the \n\ndeeper water under the limnetic zone. The benthic \n\nzone contains the sediment at the bottom [10]. \n\nAreas of the limnetic zone with sufficient oxygen \n\ncontain pseudomonads and species of Cytophaga, \n\nCaulobacter, and Hyphomicrobium. Photosynthetic algae \n\nare located in the limnetic zone [11]. \n\nDeeper waters of the profundal and benthic zones \n\nhave low oxygen concentrations and less light. Algal \n\ngrowth near the surface often filters the light, and it is \n\nnot unusual for photosynthetic microbes in deeper \n\nzones to use different wavelengths of light from those \n\nused by surface-layer photosynthesizers. Purple and \n\ngreen sulfur bacteria are found in the profundal zone. \n\nThese bacteria are anaerobic photosynthetic organisms \n\nthat metabolize H2S to sulfur and sulfate in the bottom \n\nsediments of the benthic zone. Clostridium species are \n\ncommon in bottom sediments and may include \n\nbotulism organisms, particularly those causing \n\noutbreaks of botulism in waterfowl [8]. \n\nMarine Microbial Diversity \nThe marine environment represents a major portion of \n\nthe biosphere and contains 97% of the Earth\u2019s water. \n\nMuch of this is in the deep sea at a depth greater than \n\n1,000 meters, representing 75% of the ocean\u2019s volume. \n\nThe ocean has been called a \u201chigh-pressure \n\nrefrigerator,\u201d with most of the volume below 100 \n\nmeters at a constant 3\u00b0C temperature. The ocean, at its \n\ngreatest depth, is slightly more than 11,000 meters \n\ndeep. The pressure in the marine environment \n\nincreases approximately 1 atm/10 meters in depth, \n\nand pressures are in the vicinity of 1,000 atm at the \n\ngreatest ocean depths [2]. \n\nMuch of the marine environment is covered by sea ice \n\nthat may comprise up to 7% of the world\u2019s surface. \n\nMicroorganisms actually grow and reproduce at the \n\ninterface between the ice and the seawater. The \n\nmicrobes that have been recovered from these ice \n\ncores have been given intriguing generic names: these \n\ninclude Polaromonas, Marinobacter, Psychroflexus, \n\nIceobacter, Polibacter, and Psychromonas antarcticus [2]. \n\nThe world\u2019s oceans are teeming with microscopic life \n\nforms. Nominal cell counts of >105 cells per ml in \n\nsurface sea water [12, 13] predict that the oceans \n\nharbor 3.6 X 1029 microbial cells with a total cellular \n\ncarbon content of 3 X 1017g [7]. Communities of \n\nbacteria, archaea, protists, and unicellular fungi \n\naccount for most of the oceanic biomass. These \n\nmicroscopic factories are responsible for 98% of \n\nprimary production [7, 14] and mediate all \n\nbiogeochemical cycles in the oceans [12]. \n\nCoastal and shelf sediments play a significant role in \n\nthe remineralization of organic matter.", "start_char_idx": 4274, "end_char_idx": 8135, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "94d41e40-a005-4598-992d-c2a3f33fd611": {"__data__": {"id_": "94d41e40-a005-4598-992d-c2a3f33fd611", "embedding": null, "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-30", "node_type": "4", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "e9bcbf7c7a15b0551ae24ca0306a3b662703a5ecca9b422be5d9265ea36ec196", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a9c68ea8-fb6c-4dfa-8031-ff11fd34d5f3", "node_type": "1", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "3ae75cb53a2e47839e8f9ead85bec86e0eb0235d2ba937bdd83234391f068264", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d497e479-2152-4afa-9b6e-65f7527974bd", "node_type": "1", "metadata": {}, "hash": "ea3fc3eab7b111cf99dc3dfa51d1d93bbd8bf35cca70d8f8c198ae234588b577", "class_name": "RelatedNodeInfo"}}, "text": "In shelf areas, \n\nan estimated 32 to 46% of the primary production \n\nsettles to the sea [15]. \n\nMuch of the primary productivity in the open oceans, \n\neven at significant depths, comes from photosynthesis \n\nby prochlorophytes, tiny prokaryotic phototrophs that \n\nare phylogenetically related to cyanobacteria. \n\nProchlorophytes contain chlorophylls a and b or \n\nchlorophylls a and d. The organism Prochlorococcus is a \n\nparticularly important primary producer in the marine \n\nenvironment. Aerobic anoxygenic phototrophs include \n\nbacteria such as Erythrobacter, Roseobacter, and \n\nCitromicrobium, all genera of Alphaproteobacteria. Deep-\n\nwater Archaea are almost exclusively species of \n\nCrenarchaeota, and many or perhaps even most are \n\nammonia-oxidizing chemolithotrophs, these \n\norganisms play an important role in coupling the \n\nmarine carbon and nitrogen cycles. Very small \n\nplanktonic heterotrophic bacteria inhabit pelagic \n\nmarine waters in numbers of 105 \u2013 106 cells/ml. The \n\nmost abundant of these is Pelagibacter, a genus of Class \n\nAlphaproteobacteria. Cells of Pelagibacter are small \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:68-70           Aryal et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   70     Nepjol.info/index.php/njb \n \n\nrods that measure only 0.2\u2013 0.5 \u00b5m, near the limits of \n\nresolution of the light microscope. Bacteroidetes and \n\nActinobacteria, and has also been found in \n\nnonhalophilic species of Archaea, such as the \n\nThermoplasma group. In the oceans, viruses are more \n\nabundant than cellular microorganisms, often \n\nnumbering over 107 virions/ml in typical seawater [1]. \n\nConclusion \nMajor bacterial groups now recognized as abundant in \n\nthe open ocean include Alpha- and \n\nGammaproteobacteria, cyanobacteria, Bacteroidetes, \n\nand to a lesser extent, Betaproteobacteria and \n\nActinobacteria; Firmicutes are only minor \n\ncomponents. With the exception of the cyanobacteria, \n\nmost marine Bacteria are thought to be heterotrophs \n\nadapted to extremely low nutrient availability, some \n\naugmenting energy conservation through \n\nproteorhodopsin or aerobic anoxygenic phototrophy. \n\nRecent anthropogenic interventions in marine \n\nenvironment have threatened all lives, including \n\nmicroorganisms. Study of marine microbial \n\nbiodiversity is of dynamic importance to the \n\nunderstanding of the different processes of the ocean, \n\nwhich may present effective novel microorganisms for \n\nscreening of bioactive compounds. \n\nReferences \n1. Madigan MT, Martinko JM, Stahl DA, Clark DP: Brock \n\nBiology of Microorganisms. 13th ed. 2012. San Francisco: \n\nPearson Benjamin Cummings. \n\n2. Prescott LM, Harley JP and Klein DA. Microbiology. 5th \n\ned. 2002. The McGraw\u2212Hill Companies. \n\n3. Corliss JO, Margulis L, Melkonian M: Handbook of \n\nProtoctista 1st ed. 1990. Jones & Bartlett Publishers. \n\n4. Finlay BJ and Esteban GF: Ubiquitous dispersal of free-\n\nliving microorganisms. Microbial Diversity and \n\nBioprospecting 2004. ASM Press. pp. 216\u2013224.  \n\n5. Hedlund BP and Staley JT: Microbial endemisms and \n\nbiogeography. Microbial Diversity and Bioprospecting 2004 \n\nASM Press. pp. 225\u2013231. \n\n6. Whitfield J: Biogeography: is everything everywhere? \n\nScience 2005. 310:960\u2013961. \n\n7. Whitman WB, Coleman DC and Wiebe WJ: Prokaryotes: \n\nthe unseen majority. Proc. Natl. Acad. Sci 1998. 95:6578\u2013\n\n6583. \n\n8. Tortoraand GJ, Funke BR: Microbiology: An \n\nIntroduction.", "start_char_idx": 8136, "end_char_idx": 11559, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d497e479-2152-4afa-9b6e-65f7527974bd": {"__data__": {"id_": "d497e479-2152-4afa-9b6e-65f7527974bd", "embedding": null, "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-30", "node_type": "4", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "e9bcbf7c7a15b0551ae24ca0306a3b662703a5ecca9b422be5d9265ea36ec196", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "94d41e40-a005-4598-992d-c2a3f33fd611", "node_type": "1", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "456daca0229001f2f1ce15f00bde30e42bffbbfc7cca5d1a767653f05c33ef96", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "82ac091d-005a-4995-9bae-b1de36b4de01", "node_type": "1", "metadata": {}, "hash": "f1069a6a7a52ed98c479e53e72e58504e570c33053cae7892ce3cb6a68af684c", "class_name": "RelatedNodeInfo"}}, "text": "Tortoraand GJ, Funke BR: Microbiology: An \n\nIntroduction. 9th ed. 2008. San Francisco: Pearson \n\nBenjamin Cummings. \n\n9. Tortora GJ, Funke BR, Case CL: Microbiology: An \n\nIntroduction. 10th ed. 2010. San Francisco: Pearson \n\nBenjamin Cummings. \n\n10. Rathi J: Microbial Physiology Genetics and Ecology. 1st \n\ned. 2009. Manglam Publishers & Distributors. \n\n11. Britannica.com: limnetic zone. ecology. Retrieved 2015-\n\n08-14. From \n\nhttp://www.britannica.com/science/limnetic-zone \n\n12. Porter KG and Feig YS: The use of DAPI for identifying \n\nand counting aquatic microflora Limnol. Oceanogr.1980, \n\n25:943\u2013948. \n\n13. Hobbie JE, Daley RJ and Jasper S: Use of nuclepore \n\nfilters for counting bacteria by fluorescence \n\nmicroscopy. Appl. Environ. Microbiol. 1977, 33:1225\u20131228. \n\n14. Atlas RM and Bartha R: Microbial Ecology: \n\nFundamentals and Applications. 1993. \n\n(Benjamin/Cummings, Redwood City, CA). \n\n15. Wollast, R: The coastal organic carbon cycle: uxes, \n\nsources, and sinks. Ocean margin processes in global change \n\n1991. pp. 365-381.", "start_char_idx": 11502, "end_char_idx": 12545, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "82ac091d-005a-4995-9bae-b1de36b4de01": {"__data__": {"id_": "82ac091d-005a-4995-9bae-b1de36b4de01", "embedding": null, "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-31", "node_type": "4", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "1fda2382f1b3f2a4b3e091a9eeb0a222866c2ce0c0aa1f5e6434ca84594330fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d497e479-2152-4afa-9b6e-65f7527974bd", "node_type": "1", "metadata": {"identifier": "njb-30", "author": "Aryal, Sagar; Karki, Gaurab; Pandey, Sunil", "title": "Microbial Diversity in Freshwater and Marine Environment", "date": "2015-12-30", "file": "njb-30.pdf"}, "hash": "c514f9488c5887035f5654264196ecaf6803bbc8b4f9fdef5382c2b62df23549", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fd7b5274-02c5-4e1d-a2ba-e1a024174b81", "node_type": "1", "metadata": {}, "hash": "3d7a9c93f3cca3336bbac9bf60cf47e7c9d8edc777b070379b40df81e011551b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 66-67   ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \nCASE STUDY \n\n\u00a9NJB, Biotechnology Society of Nepal   66    Nepjol.info/index.php/njb \n\nFasciolopsis buski Vomited Out by a Child; The First Case \nReported from Nepal \n\nNarayan Dutt Pant1*, Manisha Sharma2, Saroj Khatiwada3 \n\n1Department of Microbiology Grande International Hospital, Dhapasi, Kathmandu, Nepal \n2Department of Microbiology, Kathmandu medical college, Kathmandu, Nepal \n\n3CIST College, Kathmandu, Nepal \n\nAbstract \nLive adult worms of Fasciolopsis buski are rarely seen in humans except in autopsy. Only a few such cases have been \n\nreported in the world literature. We reported a case of fasciolopsiasis in a child of age 14 months who coughed out the live \n\nadult Fasciolopsis buski after administration of antihelminthic drug. The patient was a resident of Terai (Far Western) \n\nregion of Nepal and had history of travelling to India. This is the first case of fasciolopsis reported from Nepal. \n\nKey words: Fasciolopsiasis, Nepal, antihelminthic drug, diarrhea. \n\n*Corresponding author \n\nEmail: ndpant1987@gmail.com \n\nIntroduction \nFasciolopsiasis is a gastrointestinal infestation by a \n\ntrematode; Fasciolopsis buski mainly involving \n\nduodenum and jejunum. The fluke is the largest \n\nintestinal fluke parasitizing humans and was first noted \n\nby Busk in 1943 from the duodenum of a deceased \n\nIndian sailor. Fasciolopsiasis is prevalent in various \n\nparts of South East Asia including the neighboring \n\ncountries China and India. The infections by Fasciolopsis \n\nbuski are common in impoverished countries where \n\nproper sanitation systems are lacking [1]. The disease \n\noccurs due to ingestion of encysted metacercariae on \n\naquatic vegetation or direct water [2]. Mostly the \n\ninfection is asymptomatic but in severe infection the \n\ncommon symptoms are abdominal pain, diarrhea, low-\n\ngrade fever, toxemia, allergy, anemia, ascites, \n\ngeneralized edema, obstruction of intestine sometimes \n\nleading to death [1, 3]. Diagnosis is made by detection \n\nof eggs in stool but the differentiation between \n\nFasciolopsis buski and Fasciola hepatica is very difficult in \n\nroutine examination of stool [1]. Here we report a case \n\nof fasciolopsiasis in a child of age 14 months who \n\ncoughed out the live adult Fasciolopsis buski after \n\nadministration of antihelminthic drug. This is the first \n\ncase of fasciolopsis reported from Nepal.  \n\nCase report: \n\nA 14 months male child attended outpatient \n\ndepartment with the chief complain of diarrhea,  \n\nvomiting, refusal to eat, fever, irritability, and \n\nweakness. The child was a resident of Terai (Far \n\nWestern) region of Nepal and had travel history to \n\nLucknow, India for treatment of urinary tract infection \n\nand follow up for the treatment of epilepsy. The patient \n\nwas under medication (ofloxacin and carbamazepine). \n\nHe had significant leucocytosis (14000 cells/mm3) with \n\neosinophillia (12%). Stool routine examination and \n\nculture didn\u2019t reveal any significant findings. Since the \n\npatient was already in broad spectrum antibiotic for \n\ntreatment of urinary tract infection the possibility of \n\nbacteria being the cause of the illness was quite low. So \n\nthe patient was given metronidazole and mebendazole \n\nto cover all other possible causes of the diarrhea. After \n\naround 12 hours of administration of the medication \n\nthe patient vomited out a moving worm of size 30\u00d719 \n\nmm2, leaf shaped with anterior end narrower and the \n\nposterior end broadly rounded, dorsoventrally \n\nflattened, unsegmented and flesh colored (Figure 1).", "start_char_idx": 47, "end_char_idx": 3687, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fd7b5274-02c5-4e1d-a2ba-e1a024174b81": {"__data__": {"id_": "fd7b5274-02c5-4e1d-a2ba-e1a024174b81", "embedding": null, "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-31", "node_type": "4", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "1fda2382f1b3f2a4b3e091a9eeb0a222866c2ce0c0aa1f5e6434ca84594330fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "82ac091d-005a-4995-9bae-b1de36b4de01", "node_type": "1", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "b9f90104ed80a99176702c91395e6d65f779e917506e3041f8dafb092619ae35", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91842a86-7400-4bb5-9049-aa0e4511fdb3", "node_type": "1", "metadata": {}, "hash": "504e91d1473b35b1818fb73ecee4eec04e1c62dfddf447fd12499b76ed385e9e", "class_name": "RelatedNodeInfo"}}, "text": "The worm was identified as Fasciolopsis buski on the \n\nbasis of the morphological characteristics such as lack \n\nof cephalic cone, poorly-developed suckers (oral and \n\nventral) and the unbranched ceca. All symptoms \n\nsubsided after the full course of treatment and total \n\nleucocyte counts and eosinophil counts became normal. \n\n \n\nFigure 1. Fasciolopsis buski vomited out by the child. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:66-67         Pant et al. \n\n  \n\n \n\n \n\n \n\u00a9NJB, Biotechnology Society of Nepal   67    Nepjol.info/index.php/njb \n\n \n\nDiscussion \nFasciolopsiasis is endemic in India, cases being \n\nreported mainly from areas including Bihar and Uttar \n\nPradesh but no cases have been yet reported from \n\nNepal which is surprising as those areas are connected \n\nwith the Terai region of Nepal with open borders and \n\nshares cultural and geographical similarities [4]. The \n\ncases may have been underdiagnosed due to poor \n\nhealth facility and hence unreported. In the country like \n\nNepal where the open defecation around the water \n\nbodies is common and the pigs are kept in close contact \n\nwith humans the prevalence of the disease may be \n\nalarming as the habit of eating aquatic vegetation and \n\ndrinking untreated water is common in Nepal. So a \n\nstudy is necessary to determine the prevalence of the \n\nFasciolopsis buski infection at least in the areas of Nepal \n\nwhich are connected with the high prevalence area of \n\nIndia.  \n\nThe child had history of drinking water from a pond \n\nduring his stay in India. So it is high chance that the \n\nchild may have got infection from the water he drank \n\nfrom the pond as no other history which might involve \n\nthe risk of getting infection by Fasciolopsis buski could be \n\nelucidated. Live adult worms of this parasite are very \n\nrarely seen in humans except in autopsy [5]. There are \n\ntwo more reports of live adult worms being vomited \n\nout [1, 5]. Other cases of live adult worms causing \n\ndifferent clinical conditions are reported by Cao et al. \n\n[2], Mahajan et al. [4] and Lee et al. [6]. The present case \n\nraises the possibility of unidentified cases of \n\nfasciolopsiasis in Nepal.  \n\nConclusion \nWith reporting of this case it can be concluded that the \n\ncases of fasciolopsiasis are possible in Nepal and it \n\nshould also be considered as differential diagnosis in \n\ncase of the suspected patients with gastrointestinal \n\nsymptoms. \n\nCompeting interests \n The authors declare that they have no competing \n\ninterests. \n\nStatement Regarding the Patient\u2019s \nConsent \n Patient\u2019s guardian\u2019s consent was taken for the \n\npublication of this case report. \n\nReferences \n1. Mohanty I, Narasimham MV, Sahu S, Panda P, Parida B: \n\nLive Fasciolopsis buski vomited out by a boy. Ann Trop \n\nMed Public Health 2012,5:403-405. \n\n2. Cao YH, Ma YM, Qiu F, Zhang XQ: Rare cause of \n\nappendicitis: Mechanical obstruction due to Fasciolopsis \n\nbuski infestation. World J Gastroenterol 2015,21(10):3146-\n\n3149. \n\n3. Sen\u2013Hai Y, Mott KE: Epidemiology and morbidity of \n\nfood borne intestinal trematodes infections. Trop Dis Bull \n\n1994, 91:r126-r150. \n\n4. Mahajan RK, Duggal S, Biswas NK, Duggal N, Hans C:  A \n\nfinding of live Fasciolopsis buski in an ileostomy \n\nopening. J Infect Dev Ctries 2010,4(6):401-403. \n\n5. Le TH, Nguyen VD, Phan BU, Blair D, McManus DP: Case \n\nreport: unusual presentation of Fasciolopsis buski in a \n\nVietnamese child. Trans R Soc Trop Med Hyg 2004, \n\n98(3):193-194. \n\n6.", "start_char_idx": 3690, "end_char_idx": 7160, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91842a86-7400-4bb5-9049-aa0e4511fdb3": {"__data__": {"id_": "91842a86-7400-4bb5-9049-aa0e4511fdb3", "embedding": null, "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-31", "node_type": "4", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "1fda2382f1b3f2a4b3e091a9eeb0a222866c2ce0c0aa1f5e6434ca84594330fa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fd7b5274-02c5-4e1d-a2ba-e1a024174b81", "node_type": "1", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "47fd327890f096f37f1af85939370186998f813977e840f8c155ab7a20bda203", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "855e662b-a74a-40d0-b0a7-0fc6a0b8d017", "node_type": "1", "metadata": {}, "hash": "869b38ce1b409ee431bd93c935287323d53345fe0619197b0cf48620f46bba20", "class_name": "RelatedNodeInfo"}}, "text": "6. Lee TH, Huang CT, Chung CS: \n\nGastrointestinal: Fasciolopsis buski infestation \n\ndiagnosed by upper gastrointestinal endoscopy. Journal \n\nof Gastroenterology and Hepatology 2011, 26(9):1464. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Le%20TH%5BAuthor%5D&cauthor=true&cauthor_uid=15024930\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Nguyen%20VD%5BAuthor%5D&cauthor=true&cauthor_uid=15024930\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Phan%20BU%5BAuthor%5D&cauthor=true&cauthor_uid=15024930\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Blair%20D%5BAuthor%5D&cauthor=true&cauthor_uid=15024930\nhttp://www.ncbi.nlm.nih.gov/pubmed/?term=McManus%20DP%5BAuthor%5D&cauthor=true&cauthor_uid=15024930\nhttp://www.ncbi.nlm.nih.gov/pubmed/15024930", "start_char_idx": 7158, "end_char_idx": 7933, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "855e662b-a74a-40d0-b0a7-0fc6a0b8d017": {"__data__": {"id_": "855e662b-a74a-40d0-b0a7-0fc6a0b8d017", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91842a86-7400-4bb5-9049-aa0e4511fdb3", "node_type": "1", "metadata": {"identifier": "njb-31", "author": "Pant, Narayan Dutt; Sharma, Manisha; Khatiwada, Saroj", "title": "Fasciolopsis buski Vomited Out by a Child; The First Case Reported from Nepal", "date": "2015-12-30", "file": "njb-31.pdf"}, "hash": "8e67dd7ef72789bc9c3fd1a8a9d119105dbe6c8eda8db825ef3626b8e8f205da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6277a22a-9651-4706-a951-69885ec8f7f5", "node_type": "1", "metadata": {}, "hash": "f068a9b498b304f37b7526306f61567b1a4d09e693c8c2db59d277aa8ce94658", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65   ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   60     Nepjol.info/index.php/njb \n\nStudy on Phytochemical, Antibacterial, Antioxidant and \nToxicity Profile of Viscum album Linn Associated with  \n\nAcacia catechu \nMeena Kusi1,3*, Kanti Shrestha2, Rajani Malla1  \n\n1Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal;  \n2Nepal Academy of Science and Technology, Faculty of Science, Khumaltar, Nepal;  \n\n3Deerwalk Services Pvt. Ltd., Kathmandu, Nepal \n\nAbstract \nThis study focuses on antibacterial, antioxidant and toxic potentials of Viscum album Linn, commonly known as \nEuropean mistletoe associated with Acacia catechu (Khayer in Nepali). Methanol extract of the aerial parts of the \nMistletoe was prepared by cold percolation method. The resulting extract was simultaneously subjected to \nphytochemical screening; anti-microbial activity; anti-oxidant potential and Brine shrimp toxicity test. The major \nbiologically active phyto-constituents observed were alkaloids, glycosides, saponins, polyphenols, flavonoids, tannins, \nterpenoids and cardiac glycosides. Upon antibacterial activity screening, the plant extract was found to be highly \neffective against Pseudomonas aeruginosa with the zone of inhibition 16\u00b11mm compared to 17\u00b11mm of chloramphenicol \n(50 mcg). The antioxidant activity as EC50 value by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging \nactivity was found to be 1.58 mg/ml while the ferric reducing capacity was measured to be 282.83\u00b119.55 mg \nFeSO4.7H2O eqvt/g dry wt. of the extract during Ferric Ion Reducing Antioxidant Power (FRAP) Assay. The LC50 value \nfor Brine Shrimp Toxicity Assay was found to be 31.62 ppm. This study shows the medicinal value of the mistletoe \nassociated with Acacia catechu.  Further meticulous analysis of this plant might lead to identification of active \nbiomolecules effective as drugs for various ailments. \n\nKey words: alkaloids, glycosides, polyphenols, flavonoids, tannins, terpenoids, toxicity. \n\n*Corresponding author \n\nEmail: meena.kusi.917@gmail.com \n\nIntroduction \nViscum album Linn, commonly known as European \n\nmistletoe is a semi-parasitic shrub, growing on large \n\nvariety of woody trees especially on their stems and \n\nbranches [1].  The plant is distributed along sub-\n\ntropical and temperate Himalayas at an altitude of \n\n900-2100m [2]. The most common host plants include \n\nSalix, Populus, Acer, Malus, Crataegus, Prunus, Sorbus, \n\nAbies, and Pinus [3].  \n\nViscum album in Nepal has been used as a medicinal \n\nherb since very long. Traditionally the paste of \n\nmistletoe bark is applied to relieve muscular \n\nswelling, boils and wounds and even in the \n\nmanagement of joint dislocation; the berries act as \n\nlaxative, tonic, diuretic and aphrodisiac while the \n\nplant has been used in complications like \n\nenlargement of spleen and tumors [2, 4]. The plant \n\nhas also been used in case of epilepsy, vertigo, \n\nanxiety, exhaustion and hypertension [5].", "start_char_idx": 47, "end_char_idx": 3156, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6277a22a-9651-4706-a951-69885ec8f7f5": {"__data__": {"id_": "6277a22a-9651-4706-a951-69885ec8f7f5", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "855e662b-a74a-40d0-b0a7-0fc6a0b8d017", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "b526e2fc4ebb7d15e4a84ca17db0b0b359f45938e9810f6c922ea2acec86383b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f57fda2b-48d0-4d39-80be-def094c976d3", "node_type": "1", "metadata": {}, "hash": "b188dd6159aada60b4ef6222f91e0a5a985656edb74739b7c12cea3fe2c84af1", "class_name": "RelatedNodeInfo"}}, "text": "Bussing \n\n(2004) has compiled wide use of this plant in various \n\ncountries as: In ancient Greek in spleen diseases and \n\ncomplications associated with menstruation while in \n\nArgentina; mistletoe has been used as sedative and as \n\nstabilizer in bone fractures; in India the mistletoe tea \n\nin diabetes; in Africa in treatment of various stomach \n\ntroubles of children including diarrhea. The German \n\nCommission E Monographs, a guide to herbal \n\nmedicine approved the use of the plant in \n\nrheumatism and tremor diseases. The evergreen \n\nmistletoe was regarded as a symbol of fertility and \n\ngood luck and thus was believed to avoid any fertility \n\nproblems [7]. \n\nDespite the long traditional use of this herb in \n\nmedicine, the chemical and biological study of this \n\nplant has only been initiated lately. The active \n\nchemical constituents reported from the mistletoe are \n\nGlycoproteins- mistletoe lectin I, II, and III; protein- \n\nviscotoxin; polysaccharides- galacturonan, \n\narabinogalactan and alkaloids [5]. Recently two novel \n\namino-alkaloids namely 4,5,4\u2019-trihydroxy-3,3\u2019-\n\niminodibenzoic acid and 4,5,4\u2019,5\u2019-tetrahydroxy-3,3\u2019-\n\niminodibenzoic acid have been isolated and \n\ncharacterized [8]. Other classes of secondary \n\nmetabolites from Viscum album are flavonoids, \n\nphenylpropanoids, triterpenes, phytosterols, cyclic \n\npeptides and cyclitols [6]. Being a parasitic plant, the \n\nphenolic content and the antioxidant capacity of \n\nmistletoe have been found to vary depending upon \n\nthe host trees and the time of harvest [9,10]. New \n\ncompounds have been also reported in mistletoe \n\ngrowing on different hosts [11].   \n\nThe organic solvent fraction of mistletoe extract from \n\nits twigs and leaves showed good antimicrobial \n\nactivity against both  gram positive and gram \n\nnegative bacteria [12, 13] .The hexane extract of the \n\nplant has shown anti-fungal activity against C. \n\nalbicans as well  [14]. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65                    Khusi et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   61     Nepjol.info/index.php/njb \n\nThough the plant contains various cytotoxic toxins \n\nthere are no reported toxicity to humans. Some \n\nstudies have shown that direct ingestion of the plant \n\nparts especially berries might result in mild effects \n\nlike nausea, vomiting, bloody diarrhea and shock \n\ninduced hypertension [6] Application of recombinant \n\nmistletoe lectins are reported to cause some sort of \n\nreversible hepatotoxicity [15]. \n\nThis plant is in high demand by the traditional \n\nhealers and herbal companies for manufacturing \n\nvarious medicine based on this herb. In Nepal herbal \n\ndrug manufacturers use this plant as an important \n\nconstituent in various formulations. However the \n\nexact chemical compounds or the proven therapeutic \n\napplications of this plant have not been stated \n\nanywhere. Since this plant is parasitic in nature it has \n\nbeen stated that the host plant also plays important \n\nrole in the active biomedical content of this plant. So, \n\nthis study of Viscum album associated with Acacia \n\ncatechu is unique in itself. The aim of this study is to \n\nidentify active biomedical components of this plant \n\nwhich is associated with Acacia catechu and to assert \n\nany associated toxicity. We carried out phytochemical \n\nanalysis of this plant which was followed by \n\nantibacterial activity screening, estimation of anti-\n\noxidant capacity and toxicity analysis. \n\nMaterials and Methods \nAll the procedures were carried out in Natural \n\nProducts Chemistry Laboratory, Faculty of Science, \n\nNepal Academy of Science and Technology, Lalitpur, \n\nNepal. \n\nPlant Materials:  \nThe aerial parts of Viscum album Linn. associated with \n\nAcacia catechu were collected from Sarlahi District, \n\nNepal (1,000 - 3,300ft).", "start_char_idx": 3158, "end_char_idx": 6969, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f57fda2b-48d0-4d39-80be-def094c976d3": {"__data__": {"id_": "f57fda2b-48d0-4d39-80be-def094c976d3", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6277a22a-9651-4706-a951-69885ec8f7f5", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "28f8b01278d09d7c19afd548c35b3f32d350e5447f4dddc7a1e5b1308fd27382", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "41095605-90f5-4ea2-b731-c857eab73924", "node_type": "1", "metadata": {}, "hash": "8c5b9b1bc6d30257744a7376c3facdaae58694242193a7482c35d55e61f14c06", "class_name": "RelatedNodeInfo"}}, "text": "It was shade dried and \n\ncrushed into powder using a crusher. The resulting \n\npowder was stored in plastic bag for further work at \n\nroom temperature.  \n\nExtraction of phytochemicals: \nFor extraction of phytochemical compounds, cold \n\npercolation method followed by intermittent \n\nsonication was followed. First the solvent methanol \n\nwas added in the ratio of 8:1 (plant powder weight in \n\ngram) and shaken well then kept on stand for 3 days \n\nat room temperature. From day four onwards, the \n\nresulting solution was subjected for intermittent \n\nsonication (Rocker Scientific Co. Taiwan) - a \n\ncontinuous cycle of sonication at 20 kHz at 60\u02daC for \n\n15 minutes followed by 2 hour standing at room \n\ntemperature, till day six. On day six, the supernatant \n\nwas slowly poured into the round bottom flask \n\nthrough cotton plug filtration which was then \n\nevaporated at reduced pressure in rotatory vacuum \n\nevaporator (Hanshin Scientific Co., Korea). Resulting \n\ndry extracts were then sealed in small glass \n\ncontainers and stored at 4\u02daC until use. \n\nPhytochemical Screening:  \nThe procedures for phytochemical screening has been \n\nfollowed in reference to the book \u201cPhytochemical \n\nTechniques\u201d and the research paper [16, 17]. Mayer\u2019s \n\nTest and Dragendorff\u2019s Reagent Test were employed \n\nfor detection of alkaloids while carbohydrates and \n\ncardiac glycosides were detected with Molisch\u2019s Test \n\nand Fehling\u2019s Test. Proteins and amino acids were \n\ntested by applying Biuret Test and Ninhydrin Test. \n\nLiebermann-Burchard\u2019s Test gave results for presence \n\nof Phytosterols. Ferric Chloride Test, Alkaline reagent \n\nTest and Magnesium-Hydrochoric acid Reduction \n\nTest detected phenols and tannins. Bramer\u2019s Test was \n\nused for specific detection of tannins. Liebermann\u2019s \n\nBurchard test and Salkowski\u2019s Test detected \n\nterpenoids. Shinoda Test for flavonoids and Kellar-\n\nKilliani Test for cardiac glycosides were used. \n\nSaponins, Gums and Mucilages were also \n\nsubsequently detected. \n\nAntibacterial Screening:  \nThe antimicrobial activity of the crude extract was \n\nstudied by Agar well diffusion Technique [18]. \n\nFive standard strains of Gram negative bacteria \n\n(Escherichia coli ATCC 25922, Klebsiella pneumoniae \n\nATCC 700603, Salmonella typhimurium ATCC 14028, \n\nSerratia marcescens ATCC 13880 and Pseudomonas \n\naeruginosa ATCC 27853) and a single standard strain \n\nof Gram positive bacterium (Staphylococcus aureus \n\nATCC 25923) were used for antibacterial assay. The \n\nabove mentioned strains of bacteria were obtained \n\nfrom National Public Health Laboratory, Teku, Nepal \n\nand Institute of Medicine, Maharajgunj, Nepal. \n\nA standard antibiotic disc (Chloramphenicol, 50mcg) \n\nas positive control and DMSO as solvent control was \n\nused. \n\nDetermination of Anti-oxidant Activity:  \nDPPH (1,1-diphenyl-2-picrylhydrazyl) Free Radical \nScavenging Activity: \nA range of concentration varying from 0-10mg/ml of \n\nthe sample was taken for the assay. A 50\u00b5l of the \n\ncrude extract was added to 450\u00b5l of Tris-HCl buffer \n\n(0.05M, pH7.4) and 1ml of 0.1mM DPPH was added to \n\nthe resulting mixture and incubated in dark at \n\nambient temperature for 30min. Absorbance value of \n\nresulting solution at 517nm was recorded [19, 20] \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65                    Khusi et al.", "start_char_idx": 6970, "end_char_idx": 10304, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "41095605-90f5-4ea2-b731-c857eab73924": {"__data__": {"id_": "41095605-90f5-4ea2-b731-c857eab73924", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f57fda2b-48d0-4d39-80be-def094c976d3", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "9e277d3a014d2277d89d362c233dac22350469f664031ad3e5fcfae6043b8807", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "20682b39-5d69-41ec-8f33-aae7837dbc53", "node_type": "1", "metadata": {}, "hash": "4945829716925073430a93d107131bfa6fbd27927edb31b688aaf9031a22b7e7", "class_name": "RelatedNodeInfo"}}, "text": "1: 60-65                    Khusi et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   62     Nepjol.info/index.php/njb \n\nThe free radical scavenging activity (RSA) of the \n\nsamples was calculated in percentage by using the \n\nformula: \n\nRadical Scavenging Activity (RSA) = \n\n \n\nThe EC50 values of each extract were calculated by \n\nusing the formula given below: \n\nEC 50 = \n\n \n\nFerric Ion Reducing Antioxidant Power \n(FRAP) Assay:  \n10\u00b5l of the extract was added to 30\u00b5l of distilled \n\nwater and 300\u00b5l of FRAP reagent; the resulting \n\nsolution was incubated at room temperature for \n\n5min. Then after the intensity of the blue colored \n\ncomplex formed was measured \n\nspectrophotometrically using Jenway UV-Vis \n\nSpectrophotometer at 593nm. The FRAP Reagent was \n\nprepared by mixing 25 ml of Acetate buffer (300mM \n\nSodium acetate at pH3.6) and  2.5ml of TPTZ (10mM \n\n2,4,6-Tripyridyl-s-triazine in 40mM HCl) with 2.5ml \n\nof Ferric chloride solution (20mM FeCl3.6H2O in \n\ndistilled water). All the chemicals were prepared \n\nfresh and the FRAP reagent was warmed at 37\u02daC \n\nbefore use. Then the reducing power of each extract \n\nwas expressed as equivalent to that of 1mM of Fe (II) \n\ni.e. FRAP unit. All the tests were carried out in \n\ntriplicates (n=3). [21- 23]  \n\nBrine Shrimp Lethality Assay:  \nCrude plant extract was prepared in three different \n\nconcentrations of 1000ppm, 100ppm and 10ppm. \n\n100ul of each of the extracts of each concentration \n\nwere taken in wells of micro-titer plate. Freshly \n\nhatched brine shrimp (Artemia salina) nauplii were \n\nplaced in each well at the rate of 5 in each and the \n\nplate was incubated at 22-28\u02daC for 24hrs. Potassium \n\ndichromate (K2Cr2O7 at 1mg/ml) was used as \n\npositive control [24]. \n\nThe percentage viability of the larvae was observed \n\nand the percentage mortality was calculated as:  \n\nPercentage of Mortality (PM) =   \n\n \n\nThen the LC50 values for each of the extracts were also \n\ncalculated according to the formula given below: \n\nLC 50 =  \n\nStatistical Analysis \nThe final data were presented as mean \u00b1 standard \n\ndeviation from the three independent assays. The \n\nDPPH radical scavenging activity and brine shrimp \n\ntoxicity values were calculated using Microsoft Excel \n\n2007 software while these data were exported to \n\nGraphPad Prism V.5.0 for generation of graphs and \n\nfurther analysis.  \n\nResults \nThe dried aerial parts upon extraction with methanol \n\nresulted into light brown dry powder. The yield \n\npercentage was 26.67% as shown in Table 1. \n\n \n\nTable 1: Percentage Yield and Physical characteristics of the \n\ncrude methanolic extract \n\nPlant \nparts \nused \n\nDry \nwt. \ntaken \n(gm) \n\nWt.  of \nextract \n(gm) \n\nPercentage \nyield (%) \n\nCharacteristics of \nextract \n\n \nColor Consistency \n\nStem \nand \nbranches \n\n8.51 2.27 26.67  \nLight \nbrown \n\nPowdery \n\nThe qualitative testing of bioactive phytochemicals in \n\nthe methanolic extract of the aerial parts of Viscum \n\nalbum Linn. showed rich presence of alkaloids, \n\nsaponins, phenolics and tannins. However, the \n\nflavonoid content in this extract was found to be very \n\nlow. These phytochemical compounds are known to \n\nexert bioactive properties for  medicinal plants. Other \n\nphytochemicals detected were steroids, terpenoids \n\nand cardiac glycosides as shown in Table 2.", "start_char_idx": 10264, "end_char_idx": 13549, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "20682b39-5d69-41ec-8f33-aae7837dbc53": {"__data__": {"id_": "20682b39-5d69-41ec-8f33-aae7837dbc53", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "41095605-90f5-4ea2-b731-c857eab73924", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "42d65279977ceced9be47adbe7c6ac3bac15feb084505cac7dd4bf025b8c8239", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "24541a87-4971-426a-a5d6-f5f40edac8ec", "node_type": "1", "metadata": {}, "hash": "d492a22d3d6bcbde674b7c34103333de01f0b74cc5ee9ebc5511301f2807f97b", "class_name": "RelatedNodeInfo"}}, "text": "Table 2: Preliminary Phytochemical Screening of \n\nmethanolic extract  \n\nActive Compounds Tests Results \n\nAlkaloids \nMayer\u2019s Test ++ \n\nDragendorff\u2019s Test ++ \nCarbohydrates and \n\nGlycosides \nMolisch\u2019s Test - \nFehling Test - \n\nSaponin  +++ \n\nProtein and Amino Acids \nBiuret Test - \n\nNinhydrin Test - \n\nPhenolics and Tannins \nFerric Chloride Test ++ \n\nAlkaline Reagent Test +++ \nMg Conc. HCl Test - \n\nGums and Mucilages  - \nTannins Braemer\u2019s Test +++ \n\nSteroids \nLiebermann-buchard\u2019s \n\nTest \n\n+ \n \n \n\nTerpenoids \nLiebermann-buchard\u2019s \n\nTest \n+ \n\ns ++ \nFlavonoids Shinoda Test - \n\nCardiac Glycosides Kellar-Killiani Test ++ \n\n \n\nThe anti-bacterial activities of methanolic extract of \n\nViscum album were evaluated against 6 bacteria (5 \n\ngram negative and one gram positive). The results are \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65                    Khusi et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   63     Nepjol.info/index.php/njb \n\npresented as zone of inhibition by the extract (mm in \n\ndiameter) against the growth of the test \n\nmicroorganisms. The bacterium Pseudomonas \n\naerouginosa was the most susceptible bacterium to the \n\nplant extract for which the zone of inhibition was \n\nmarked to be 16mm while the rest of the \n\nmicroorganisms were not inhibited to significant \n\nextent, shown in Table 3. Chloramphenicol (50mcg) \n\nwas employed as the positive control which showed \n\nsignificant inhibition to all the test organisms. \n\nTable 3: Anti-bacterial sactivity of the crude methanolic extract    \n\n \nThe crude methanolic extract was then subjected to in \n\nvitro test to evaluate antioxidant activity using two \n\ndifferent tests: DPPH Free radical Scavenging Assay \n\nand FRAP assay. During DPPH Assay the EC50 value \n\nof the methanolic extract of Viscum album was found \n\nto be 1.58 mg/ml which was only 50 times lower than \n\nthat of the standard - Gallic acid as shown in Table 4. \n \n\nTable 4: EC50 values of Crude methanolic extracts in DPPH Free \n\nRadical Scavenging Assay \n\nS.N Sample methanolic extract EC50 values (mg/ml) \n\n1. Gallic acid 0.039 \n\n2. V. album 1.58 \n\n \n\nThe value of FRAP assay was expressed as mg \n\nFeSO4.7H2O per gm dry wt. of the sample. In table 5, \n\nthe FRAP activity value of the plant found to be \n\n282.83\u00b119.55 mg FeSO4.7H2O/gm dry wt. has been \n\nshown. \nTable 5: Estimation of Antioxidant Activity of Crude Methanolic \n\nExtracts by FRAP Assay \n\nSample Methanolic \n\nExtract \n\nFRAP activity (mg \n\nFeSO4.7H2Oeqvt/gm) \n\nV. album 282.83\u00b119.55 \n\n In the brine shrimp toxicity assay, the degree of \n\nlethality was found to be directly proportional to the \n\nconcentration of the extract.  \n\nAll the brine shrimp larvae were found to be alive \n\nafter 24 hours in the extract concentration of 10ug/ml \n\nwhile higher concentration was toxic enough to kill \n\nall the larvae before 24hrs.(Table 6) The plant extract \n\nshowed significant toxicity which was measured to \n\nbe an LC50 of 31.62 ppm (Table 7). \nTable 6: Brine shrimp lethality assay \n\n \n\nTable 7: LC50 values in Brine shrimp cytotoxicity assay \n\n \n\nDiscussion \nPreliminary Phytochemical screening of the aerial \n\nparts of V. album Linn.", "start_char_idx": 13552, "end_char_idx": 16714, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "24541a87-4971-426a-a5d6-f5f40edac8ec": {"__data__": {"id_": "24541a87-4971-426a-a5d6-f5f40edac8ec", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "20682b39-5d69-41ec-8f33-aae7837dbc53", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "8a202c082729ba9e7b7dd3a046e3c1491a0840d6c3d0fd8522f43feac4626da4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "642db308-9582-4b42-94d6-4b1873758c0f", "node_type": "1", "metadata": {}, "hash": "524465413a9e1549968280498b647bea183cf8a3263aaf49db9c8f84101906d1", "class_name": "RelatedNodeInfo"}}, "text": "associated with Acacia catechu, \n\nespecially the stem parts showed presence of rich \n\nbioactive secondary metabolites like Alkaloids, \n\nCardiac Glycosides, Saponins, Phenolics, Tannins and \n\nSteroids. This result was in positive correlation with \n\nfindings from Bussing (2004) and Kunwar (2010). \n\nThese rich bioactive molecules have potential to be \n\nused in various ailments as tannins are capable of \n\nbinding protein molecules which might have been \n\nexploited in treatment of diarrhea and skin bleedings \n\nby traditional healers. Anabolic steroids are known to \n\nretain nitrogen in osteoporosis and various wasting \n\nillnesses [25]. Alkaloids are known for their inherent \n\ntoxicity to cells [26]. Flavonoids and phenolics are \n\nuniversally known as potent anti-oxidants and anti-\n\ninflammatory agents and therefore have been found \n\nuseful against tumors and cancers [27]. Regulations of \n\nNa+/K+-ATPase-pumps are assisted by cardiac \n\nglycosides while saponins are well known for their \n\nimmune-modulatory and anti-neoplastic effects [28, \n\n29]. The rich presence of the phytochemicals of \n\nmedical value thus proves the effective use of this \n\nplant in various formulations in ethno medicine.  \n\nThe exciting results from the  qualitative test of \n\nbioactive phytochemicals continued to show their \n\nstrong presence in antibacterial activity assay as well. \n\nThe crude extract was able to form a clear and \n\ndistinct halo zone of inhibition measuring a diameter \n\nof 16mm (Table 3). This was quite comparable with \n\nthat of the positive control used. Ps. aeruginosa is \n\nconsidered one of the most rapidly growing bacteria \n\nin its resistance to existing antibiotics [30]. This \n\nbacterium has been one of the major causes of \n\nhospital acquired infections all around the globe [31]. \n\nSo, the excellent inhibition of this bacterium by V. \n\nConcentration of \nViscum album \nextract (\u03bcg/ml) \n\nTotal Number of \nBrine shrimp \nlarvae used \n\nNo. of Brine Shrimp \nlarvae alive after \n\n24hrs 48hrs \n\n1000 5 0 0 \n500 5 0 0 \n100 5 0 0 \n10 5 5 0 \n\nS.N. Crude Extracts LC50  ppm \u00b1 SD \n\n1. V. album 31.62  \u00b1 0.00 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65                    Khusi et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   64     Nepjol.info/index.php/njb \n\nalbum Linn extract could lead to discovery of potent \n\nnovel antibiotics. Although the results against other \n\nbacteria were not much promising, an increment of \n\nthe concentration of the extract could have improved \n\nthe inhibition ability. Since the extract used in this \n\nstudy is crude one, use of refined extract would have \n\ngiven sharper and better results.  \n\nDPPH-free RSA and FRAP assay exhibit anti-oxidant \n\npower based on electron transfer reactions. The \n\nprogressive fading of the violet color of DPPH \n\nreagent to yellow indicates the scavenging of free \n\nradicals. Lower the EC50 value, greater is the anti-\n\noxidant activity. The EC50 value of Viscum album in \n\nthis study is 1.58 which describes better free radical \n\nscavenging ability of this plant. The co-efficient of \n\ndetermination (R2 value) value of 77.30% as shown in \n\nFigure 1 explains that the radical scavenging activity \n\nis in positive correlation with the concentration of the \n\nextract used. So, the RSA value can be enhanced \n\nfurther by increasing the purity of the extract. \n\nSimilarly the FRAP activity has been calculated as \n\n282.83\u00b119.55 which is also very high value which \n\nindicates significant reduction potential of the extract. \n\nThe polyphenols and flavonoids might be responsible \n\nfor this active anti oxidant activities of this plant. This \n\ntherefore provides evidence for the use of this plant \n\nin treatment of free radicals associated diseases like \n\ndiabetes.", "start_char_idx": 16715, "end_char_idx": 20478, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "642db308-9582-4b42-94d6-4b1873758c0f": {"__data__": {"id_": "642db308-9582-4b42-94d6-4b1873758c0f", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "24541a87-4971-426a-a5d6-f5f40edac8ec", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "8b5a80fd64bc68ab77f370badd7537f0a2984990a1e0b123620151b60a6457f8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "008b197d-e88b-441c-ac22-c759c9ed7c08", "node_type": "1", "metadata": {}, "hash": "da7bdf2a45d7467a600088f4cfde5c760e151ef5db2cbcc1ec4913962dd1e5a3", "class_name": "RelatedNodeInfo"}}, "text": "Toxicity is yet another important parameter to qualify \n\nany drug or drug formulation/preparation regarding \n\nsafety. Preliminary monitoring of the toxicity of \n\nnatural products has been carried out via Brine \n\nShrimp Toxicity assay. The freshly hatched nauplii \n\nbetween 24-48 hours of hatching are considered \n\nhighly sensitive to toxins and therefore this stage of \n\nbrine shrimp larvae are used for toxicity assay. V. \n\nalbum extract was found to have LC50 31.62 \u00b1 0.00 \n\nppm which is below 1000ppm and the standard chart \n\nexplains that only those plant extracts having LC50 \n\nlesser than 1000ppm are practically cytotoxic. This \n\nobserved toxicity might be attributed to high alkaloid \n\ncontent of the plant. Duke, 2010 has mentioned \n\ntoxicity of the lectin fraction, viscotoxin and the juice \n\nof the plant with the LC50 value of 80\u00b5g/kg, \n\no.7mg/kg, 32mg/kg of mouse while the capability of \n\nmistletoe lectins in inactivating ribosomes thereby \n\nhalting protein synthesis has been discussed by \n\nBussing, 2004. Therefore care must be taken while \n\nformulating this herb for drug preparation. Instead \n\nthe cytotoxicity of this plant might be exploited for \n\ntheir use as anticancer drugs. However, Viscum album \n\nLinn should be subjected to further rigorous \n\nbioassays for confirmation of specific toxicity. \n\nConclusion \nThe Viscum album aerial parts associated with Acacia \n\ncatechu showed rich presence of bioactive \n\nphytochemical constituents like alkaloids, glycosides, \n\nsaponins, flavonoids and phenolics. The significant \n\ninhibition of Ps. aeruginosa presents this plant a \n\npromise for screening active constituent against its \n\nresistant forms. The significant toxicity of the extract \n\ndemands careful analysis of the plant before use in \n\nmedical formulations in one hand while in other it \n\nalso directs its potential use for targeting cancer cells. \n\nAcknowledgement \nThe authors would like to express sincere \n\ngratefulness to University Grants Commission, \n\nSanothimi, Bhaktapur, Nepal for financial assistance \n\nand Nepal Academy of Science and Technology, \n\nNAST, Lalitpur, Nepal for providing laboratory \n\nfacilities. \n\nReferences \n1. Zuber DK: Biology and evolution for the \n\nEuropean misletoe (Viscum album). PhD Thesis \n\nUniversity of Zurich, Switzerland (ETH Zurich); \n\n2008 \n\n2. Nepal DoMPo: Medicinal Plants of Nepal, His \n\nMajesty's Government of Nepal,    Ministry of \n\nForest, Department of Plant Resources, Thapathali, \n\nKathmandu, Nepal; 1970, pp. 86 \n\n3. Barney CW, Hawksworth FG, Geils BW: Hosts \n\nof Viscum album. Forest Pathology, 2007, 28(3): \n\n187-208 \n\n4. Rajbhandari KR: Ethnobotany of Nepal, \n\nEthnobotany Society of Nepal 2001, pp. 146-147 \n\n5. Loeper ME: Mistletoe (Viscum album L.). \n\nLongwood Herbal Task Force, 1999  \n\n6. Bussing A:  Mistletoe: The Genus Viscum, \n\nHarwood Academic Publishers, 2004  \n\n7. Klein S, Rister R, Riggins: The Complete \n\nGerman Commission E Monographs: \n\nTherapeutic Guide to Herbal Medicines, \n\nAmerican Botatnical Council, First Editon,1998 \n\n8. Amer B, Juvik OJ, Dupont F, Francis GW, \n\nFossen T: Novel aminoalkaloids from \n\nEuropean mistletoe (Viscum album L.). \n\nPhytochemistry Letters. 2012,  5(3): 677-681 \n\n9. \u00d6nay-U\u00e7ar E, Karag\u00f6z A, Arda N: Antioxidant \n\nactivity of Viscum album ssp. album. \n\nFitoterapia. 2006, 77(7\u20138): 556-560 \n\n10.", "start_char_idx": 20481, "end_char_idx": 23810, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "008b197d-e88b-441c-ac22-c759c9ed7c08": {"__data__": {"id_": "008b197d-e88b-441c-ac22-c759c9ed7c08", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "642db308-9582-4b42-94d6-4b1873758c0f", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "629021b336fbd549c24672d5c7ef01ebb62e59395507a2c01a689eac91aff07c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9b799fe0-2e11-45b8-9482-1afc27e7f33d", "node_type": "1", "metadata": {}, "hash": "2f1a5cd8120cafb6ec9fdb178b4d8133e8b8b0c33915e5704c1c8a175c3bdeb7", "class_name": "RelatedNodeInfo"}}, "text": "2006, 77(7\u20138): 556-560 \n\n10. Vicas S, Prokisch J, Rugina D, Socaciu C: \n\nHydrophilic and Lipophilic Antioxidant \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 60-65                    Khusi et al. \n\n \n \n\n\u00a9NJB, Biotechnology Society of Nepal   65     Nepjol.info/index.php/njb \n\nActivities of Mistletoe (Viscum album) as \n\nDetermined by FRAP Method,. Not. Bot. Hort. \n\nAgrobot. Cluj 2009, 32(2): 112-116 \n\n11. Luczkiewicz M, Cisowski W, Kaiser P, Ochocka \n\nR, Piotrowski A: Comparative analysis of \n\nphenolic acids in mistletoe plants from \n\nvarious hosts. Acta Pol Pharm 2001, 58(5): 373-9 \n\n12. Hussain MA, Khan MK, Hussain N, Habib T: \n\nAntibacterial and antifungal potential of \n\nleaves and twigs of Viscum album L. Journal of \n\nMedicinal Plants Research 2011, 5(23): 5545-5549 \n\n13. Oguntoye SO, Olatunji GA, Kolawole OM, \n\nEnonbun KN: Phytochemical Screening and \n\nAntibacterial Activity of Viscum album \n\n(Mistletoe) Extracts. Plant Science Research. 2008, \n\n1(3): 44-46 \n\n14. Erturk O,  Kati H, Yayli N, Demibrag Z: \n\nAntimicrobial Activity of Viscum album L. \n\nsubsp. abietis (Wiesb). Turkish Journal of Biology \n\n2003, 27(4): 255-258 \n\n15. Kienle GS, Grugel R, Kiene H: Safety of higher \n\ndosages of Viscum album L. in animals and \n\nhumans--systematic review of immune changes \n\nand safety parameters. BMC Complement Altern \n\nMed 2011, 11: 72 \n\n16. Raaman N: Phytochemical Techniques, New \n\nIndia Publishing Agency 2006 pp. 19-24 \n\n17.  Sasidharan S, Chen Y, Saravanan D, Sundram \n\nKM, Yoga LL: Extraction, isolation and \n\ncharacterization of bioactive compounds from \n\nplants' extracts. Afr J Tradit Complement Altern \n\nMed. 2011, 8(1): 1-10 \n\n18. Perez C PM, Bazevque P: An antibiotic assay by \n\nthe agar well diffusion method. Acta Biologiae et \n\nMedicine Experimentalis 1990,15:113-115 \n\n19. Gyamfi MA, Yonamine M, Aniya Y: Free-radical \n\nscavenging action of medicinal herbs from \n\nGhana: Thonningia sanguinea on \n\nexperimentally-induced liver injuries. Gen \n\nPharmacol 1999, 32(6): 661-7 \n\n20. Hinneburg I, Damien Dorman HJ, Hiltunen R: \n\nAntioxidant activities of extracts from Selected \n\nculinary herbs and spices. Food Chemistry. 2006, \n\n97(1): 122-129 \n\n21.  Benzie IF, Strain JJ: The ferric reducing ability of \n\nplasma (FRAP) as a measure of \"antioxidant \n\npower\": the FRAP assay. Anal Biochem. 1996,  \n\n239(1): 70-6 \n\n22. Huang D, Ou B, Prior RL: The chemistry behind \n\nantioxidant capacity assays. J Agric Food Chem. \n\n2005, 53(6): 1841-56 \n\n23.", "start_char_idx": 23782, "end_char_idx": 26272, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9b799fe0-2e11-45b8-9482-1afc27e7f33d": {"__data__": {"id_": "9b799fe0-2e11-45b8-9482-1afc27e7f33d", "embedding": null, "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-32", "node_type": "4", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "4611d595d5b9eb92549c79884465bb0ff80ae59d72cee470b4d0f0500e7c90bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "008b197d-e88b-441c-ac22-c759c9ed7c08", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "7c75cd9d61b71d49198ca852d931c60b405fa81dcecddf576daf10dc20ddf99c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8ca3a449-e147-47e7-b585-b532dc0f71f3", "node_type": "1", "metadata": {}, "hash": "dd37879a68a7a2c78e099d620d20f273c1e99dc3021bc3636e76d564d159931c", "class_name": "RelatedNodeInfo"}}, "text": "2005, 53(6): 1841-56 \n\n23. Pfundstein B, El Desouky SK, Hull WE, Haubner \n\nR, Erben G, Owen RW: Polyphenolic compounds \n\nin the fruits of Egyptian medicinal plants \n\n(Terminalia bellerica, Terminalia hebula and \n\nTerminalia horrida): Characterization, \n\nquantitation and determination of antioxidant \n\ncapacities. Phytochemistry. 2010, 71(10): 1132-1148 \n\n24. Rahman AU, Choudhary MI, Thomsen WJ: \n\nBioassay techniques for drug development, \n\nHarwood Academic Publishers; 2001, pp. 9-10 \n\n25. Adachi M, Takayanagi R: Effects of anabolic \n\nsteroids on osteoporosis. Clinical Calcium 2008, \n\n10: 1451-9 \n\n26. Aniszewski T: Biological Significance of \n\nAlkaloids.Alkaloids-Secrets of Life: Alkaloid \n\nChemistry, Biological Significance, Applications \n\nand Ecological Role. Elsevier; 2007:141-180 \n\n27. Dubios M: Bioactive saponins with cancer \n\nrelated and immunomodulatory activity: Recent \n\nDevelopments: Studies in Natural Products \n\nChemistry: Bioactive Natural Products (Part L), \n\nVolume 32; Edited by Atta-ur-Rahaman: Elsevier; \n\n2011: 209-230 \n\n28. Chahar MK, Sharma N, Dobhal MP, Joshi YC: \n\nFlavonoids: A versatile source of anticancer \n\ndrugs; Pharmacognosy Review 2011, 9: 1-12 \n\n29. Katz AM: Effects of digitalis on cell \n\nbiochemistry: sodium pump inhibition. Journal \n\nof the American College of Cardiology 1985, 5: 16A-\n\n21A \n\n30. CDC: Antibiotic Resistance Threats in the \n\nUnited States 2013. Centers for Disease Control and \n\nPrevention  Report 2013, 69  \n\n31. Kollef  MH, Napolitano LM, Solomkin JS, \n\nWunderink RG, Bae IG, Folwer VG, Balk RA, \n\nStevens DL, Rahal JJ, Shorr AF, Linden PK, \n\nMickek ST; Health Care-Associated Infection \n\n(HAT):A Critical Appraisal of the Emerging \n\nThreat. Proceedings of the HAI Summit; Clinical \n\nInfectious Diseases 2008, 47 (Suppl 2):  S55-S99", "start_char_idx": 26246, "end_char_idx": 28043, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8ca3a449-e147-47e7-b585-b532dc0f71f3": {"__data__": {"id_": "8ca3a449-e147-47e7-b585-b532dc0f71f3", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9b799fe0-2e11-45b8-9482-1afc27e7f33d", "node_type": "1", "metadata": {"identifier": "njb-32", "author": "Kusi, Meena; Shrestha, Kanti; Malla, Rajani", "title": "Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu", "date": "2015-12-30", "file": "njb-32.pdf"}, "hash": "c0564f05e032c22368bd4c4447e2da029e97a55b8d9f753696206d31d3439d0c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b6343bd5-82b4-429d-8434-626e6e6a0fdf", "node_type": "1", "metadata": {}, "hash": "103fac6bf627c80cb71c32d8d857a9d47a5ac5d55761ac9ab172ef5a691d8ed3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:55-59   ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n \n\u00a9NJB, Biotechnology Society of Nepal   55    Nepjol.info/index.php/njb \n\nPrevalence of Multidrug Resistant Bacteria in Causing Community \nAcquired Urinary Tract Infection Among the Patients Attending \nOutpatient Department of Seti Zonal Hospital, Dhangadi, Nepal \n\nTek Raj Awasthi1, Narayan Dutt Pant2*, Puspa Raj Dahal3 \n1Department of Microbiology, Siddhanath Science Campus, Kanchanpur , Nepal \n\n2Department of Microbiology, Grande International Hospital, Dhapasi, Kathmandu, Nepal \n3Department of Microbiology, Trichandra Multiple College, Kathmandu, Nepal \n\nAbstract \n\nInvolvement of multidrug resistant bacteria in causing community acquired infection is very serious public health issue. The \n\nmain objective of our study was to determine the prevalence of multidrug resistant bacteria in causing community acquired \n\nurinary tract infection. In this study we cultured the 384 mid stream urine samples collected aseptically from the patients \n\nattending outpatient department of Seti zonal hospital and having no past history of hospitalization. The organisms isolated \n\nwere identified by using conventional biochemical tests and antimicrobial susceptibility testing was performed by Kirby Bauer \n\ndisc diffusion technique. Out of total 384 samples 98 (25.52%) samples showed significant bacterial growth. The most prevalent \n\nbacterium isolated was Escherichia coli. 42.86% of the bacteria isolated were found to be multidrug resistant (MDR). The \n\ninvolvement of such large numbers of multidrug resistant bacteria in causing community acquired urinary tract infection is \n\nvery serious issue and cannot be neglected. And some abrupt initiatives should be taken by the responsible authorities to \n\nimprove or at least avoid the further worsening of the situation. \n\nKey Words: Pyelonephritis, bacteriuria Cystine Lactose Electrolyte Deficient (CLED) agar, CLSI \n\n*Corresponding author \n\nEmail: ndpant1987@gmail.com \n\nIntroduction \n\nUrinary tract infection (UTI) is the presence of \n\nmultiplying bacteria within the urinary tract. The \n\npresence of significant numbers of bacteria in \n\naseptically collected urine is the indication of urinary \n\ntract infection. Despite the presence of different host \n\ndefense mechanisms against microbial infection in \n\nurinary tract, UTI is present as one of the commonest \n\nbacterial infections [1]. It is an important global health \n\nproblem affecting millions of peoples annually, from \n\nall age groups. \n\nWomen are more vulnerable to UTI due to presence of \n\nshorter urethra and its proximity to perianal region \n\n[2]. UTI in men is less common in comparision to that \n\nin women but may be very serious when occurred [1]. \n\nPeople with defects which cause the retention of the \n\nurine are at high risk of getting UTI. Further increased \n\nrate of UTI is seen in patients with catheters or tubes \n\nplaced in urinary tract and patients with problems \n\nwith the body\u2019s natural defense mechanisms. The \n\nmost common cause of UTI is Escherichia coli. Other \n\nbacteria include the other members of the  \n\nEnterobacteriaceae, Pseudomonas aeruginosa, \n\nStaphylococcus saprophyticus, Enterococcus spp. etc [2].  \n\nMultidrug resistant bacteria are defined as the bacteria \n\nwhich are resistant to three or more than three \n\ndifferent structural classes of the antibiotics [3]. The \n\ninvolvement of multi drug resistant bacteria in \n\ncausing UTI has created a serious problem for its early \n\nproper management. For the proper management of \n\nthe infections hence to prevent the possible \n\ncomplications like chronic pyelonephritis, chronic \n\nrenal failure; timely appropriate treatment as \n\nsuggested by urine culture and sensitivity report is \n\nessential. But in most of the part of Nepal including \n\nthe Farwestern region the urine culture and sensitivity \n\nis not performed due to lack of resources and \n\ncompetent manpower.", "start_char_idx": 47, "end_char_idx": 4066, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b6343bd5-82b4-429d-8434-626e6e6a0fdf": {"__data__": {"id_": "b6343bd5-82b4-429d-8434-626e6e6a0fdf", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8ca3a449-e147-47e7-b585-b532dc0f71f3", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "ba800ac598222b150839415aae9e26f536fca347bdf5328eedd5cd0af5aaa87a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7a6daba0-2f3f-4014-9381-93ef2f76c75b", "node_type": "1", "metadata": {}, "hash": "3ac6e1d87b8cea03706a46b09eb6c53f7b4e6ca06cb1c93244c314c706dd929f", "class_name": "RelatedNodeInfo"}}, "text": "Studies on the prevalence of UTI due to multidrug \n\nresistant bacteria have been carried out frequently in \n\ndifferent parts of Nepal but no such studies have been \n\ncarried out in Farwestern region. Hence the \n\nprevalence of UTI caused due to multidrug resistant \n\nbacteria is unknown in this region of Nepal. Further \n\nthe antimicrobial susceptibility patterns of different \n\nbacteria for commonly used antibiotics are not also \n\nclear in this region. Therefore the suspected patients of \n\nUTI are treated just on the basis of guess most of times \n\nmailto:ndpant1987@gmail.com\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:55-59      Awasthi et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal   56    Nepjol.info/index.php/njb \n\nresulting in treatment failure and development of \n\nmore drug resistance among the bacteria. Hence this \n\nstudy will present the clear picture of antimicrobial \n\nsusceptibility patterns of different bacteria for \n\ncommonly used antibiotics and the involvement of the \n\nmultidrug resistant bacteria in causing UTI in far \n\nwestern region of Nepal. Seti zonal hospital is the \n\nreferral center for whole Farwestern region.  \n\nAntimicrobial susceptibility pattern of the bacteria \n\nisolated from the patients (having no past history of \n\nhospitalization) with UTI attending the outpatient \n\ndepartment of Seti zonal hospital was determined and \n\nprevalence of UTI due to multidrug resistant bacteria \n\nwas calculated. \n\nMaterials and methods \n\nA cross sectional study was conducted among the \n\npatients suspected of urinary tract infection visiting \n\noutpatient department of Seti zonal hospital from June \n\n2013 to December 2013. The samples were collected \n\nfrom only those patients who do not have past history \n\nof hospitalization. Total 384 midstream urine \n\nspecimens collected aseptically were cultured on \n\nCystine Lactose Electrolyte Deficient (CLED) agar by \n\nusing semi-quantitative culture technique. The \n\ncolonies of the bacteria from the samples with \n\nsignificant growth (\u2265105cfu/ml) after 48 hrs of aerobic \n\nincubation at 37o C were isolated and were identified \n\nup to species level with the help of colony \n\nmorphology, staining reactions and conventional \n\nbiochemical tests. The common biochemical tests used \n\nwere oxidase test, catalase test, urease test, sulphide \n\nindole motility test, citrate utilization test, triple sugar \n\niron test, lysine decarboxylase test, methyl-red voges \n\nproskauer test, coagulase test etc. During isolation and \n\nidentification purity plate culture was used for quality \n\ncontrol. Antimicrobial susceptibility testing was \n\nperformed according to the Clinical Laboratory \n\nStandard Institute (CLSI 2013) guidelines by modified \n\nKirby-Bauer disc diffusion method using Mueller \n\nHinton agar (MHA). The diameter of each zone of \n\ninhibition (in mm) was measured and results were \n\ninterpreted with the help of zone size interpretive \n\nchart. Control strains E. coli (ATCC 25922) and \n\nStaphylococcus aureus (ATCC 25923) were used for the \n\nstandardization of the antibiotic susceptibility testing. \n\nAnalysis for multidrug resistant bacteria: \nBased on susceptibility patterns of isolates, bacteria \n\nresistant to \u22653 classes of antibiotic were considered as \n\nMulti Drug Resistant (CLSI 2013). \n\nData Analysis \nThe data obtained was entered into MS excel and \n\nanalyzed using SPSS version 11.0. P-values <0.05 were \n\nconsidered statistically significant.  \n\nResults \n\nOut of total 384 midstream urine samples, 98 (25.52%) \n\nsamples showed significant bacterial growth. 29/154 \n\n(18.83%) males and 69/230 (30.00%) females had \n\nsignificant bacteriuria. The association of significant \n\nbacteriuria in male and female patients was found to \n\nbe statistically significant (p<0.05). \n\nAmong 98 significant bacteriuria cases, 6 different \n\nmicroorganisms were isolated.", "start_char_idx": 4070, "end_char_idx": 7940, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7a6daba0-2f3f-4014-9381-93ef2f76c75b": {"__data__": {"id_": "7a6daba0-2f3f-4014-9381-93ef2f76c75b", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b6343bd5-82b4-429d-8434-626e6e6a0fdf", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "b1ffc55020423aa15433bf2cd7b8bab7c4c5dac81c4522552d6e696de0ac77e4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "01be77f1-ca75-47fd-be9c-f654c40799f9", "node_type": "1", "metadata": {}, "hash": "7274a328493a9db920eabfb8617333d4b91584af71c0a99239596f3ee5b2f074", "class_name": "RelatedNodeInfo"}}, "text": "Among 98 significant bacteriuria cases, 6 different \n\nmicroorganisms were isolated. Among these isolates, \n\nE. coli (53.06%) was found to be the most predominant \n\norganism followed by Klebsiella pneumoniae (21.43%), \n\nPseudomonas aeruginosa (12.24%), Proteus vulgaris \n\n(7.14%), Staphylococcus aureus (4.08%) and Proteus \n\nmirabilis (2.04%) (Table no.1).  \n\nTable no.1: Different bacteria isolated from urine of the \n\npatients. \n\n \n\nSusceptibility of gram negative bacteria \n\ntowards different antibiotics \nAmong the common antibiotics used against all Gram \n\nnegative bacteria, the most effective antibiotic was \n\nfound to be gentamicin (57.45%) followed by \n\nceftriaxone (51.06%), nitrofurantoin (45.75%), Co-\n\ntrimoxazole (32.98%), ofloxacin (29.79%), nalidixic \n\nacid (15.96%), and  ampicillin (5.32%) (Table no. 2).   \n\nName of the bacteria Numbers isolated (%) \n\nEscherichia coli 52 (53.06%) \n\nKlebsiella pneumoniae 21 (21.43%) \n\nPseudomonas aeruginosa 12 (12.24%) \n\nProteus vulgaris 7 (7.14%) \n\nStaphylococcus aureus 4(4.08%) \n\nProteus mirabilis 2 (2.04%) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:55-59      Awasthi et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal   57    Nepjol.info/index.php/njb \n\nTable no.2: Susceptibility of gram negative bacteria towards \n\ndifferent commonly used antibiotics. \n\nAntibiotics \nNumbers of sensitive gram negative \n\nbacteria (%) \n\nGentamicin 57.45 \nCeftriaxone 51.06 \n\nNitrofurantoin 45.75 \nCo-trimoxazole 32.98 \n\nOfloxacin 29.79 \nNalidixic acid 15.96 \n\nAmpicillin 5.32 \n\n \n\nAmong the 4 gram positive bacteria all isolates were \n\nStaphylococcus aureus. All of them were susceptible to \n\ngentamicin and 3(75.00%) were susceptible to \n\nceftriaxone. Half of the S. aureus isolates were \n\nsusceptible to ampicillin, co-trimoxazole and ofloxacin \n\neach. 1(25.00%) isolate was found to be susceptible to \n\nnalidixic acid and nitrofurantoin each. \n\nMultidrug resistance among various \n\nBacteria  \nOut of 98 isolates, 42 (42.86%) were found to be MDR. \n\n48.08% of the E. coli, 19.05% of the K. pneumonia, 50% \n\nof the P. aeruginosa, 85.71% of the P. vulgaris and 25% \n\nof the Staphylococcus aureus were found to be MDR. No \n\nisolates of P. mirabilis were MDR (Table no. 3). \n\nTable no.3: Multidrug resistance among different bacteria. \n\nName of bacteria Number of multi drug \nresistant bacteria (%) \n\nEscherichia coli 48.08 \nKlebsiella pneumoniae 19.05 \nPseudomonas aeruginosa 50 \nProteus vulgaris 85.71 \nStaphylococcus aureus 25 \nProteus mirabilis 0 \n\n \n\nDiscussion \n\nOut of total 384 urine samples 98 (25.52%) samples \n\nshowed significant growth. Similar type of result was \n\nobtained by Sharma et al (27.3%) [4]. Females are more \n\nprone to UTI than males. In the present study also, \n\nsame fact was observed where the rate of growth \n\npositivity was found to be 30.00% (69/230) in females \n\nand 18.83% (29/154) in males.", "start_char_idx": 7857, "end_char_idx": 10749, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "01be77f1-ca75-47fd-be9c-f654c40799f9": {"__data__": {"id_": "01be77f1-ca75-47fd-be9c-f654c40799f9", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7a6daba0-2f3f-4014-9381-93ef2f76c75b", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "903f77b363ec6264180aa7f7de9cbb069d66938433f48a668c1ce248ce759bd8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ce53b06f-8f07-420f-a6cb-2e9e78bfd7fe", "node_type": "1", "metadata": {}, "hash": "eee02c40cca0bd596b6929a52c6560e0bb65246b31a5400e3084312b18ebfc7f", "class_name": "RelatedNodeInfo"}}, "text": "Our findings were \n\nconsistent with the findings by Shrestha et al [5] who \n\nreported the culture positivity of 29.8% in females and \n\n15.2% in males. In a similar study by Baral et al. [6] the \n\ngrowth positivity was 33.5% among female patients \n\nand 23.7%, in male patients. This higher growth \n\npositivity seen in females was found to be statistically \n\nsignificant (p<0.05) and is due to their anatomical \n\nstructure (short urethra and proximity to anal orifice) \n\nleading to easy access for enteric bacteria. Among the \n\ntotal 98 bacterial isolates, 94(95.92%) were Gram \n\nnegative bacilli and only 4 (4.08%) were found to be \n\nGram positive cocci. The results were in the favor of \n\nthe findings of Shrestha et al [5] and Karki et al [7].  In \n\nthe study by Shrestha et al [5] among the total 80 \n\nbacterial isolates, 75 (93.8%) were Gram negative \n\nbacilli and only 5 (6.3%) were Gram positive cocci. \n\nSimilarly Karki et al [7] found 91.1% of the isolates \n\nfrom urine to be Gram negative bacilli and 8.8% of \n\nthem to be Gram positive cocci. \n\nAmong 98 significant bacteriuria cases, 6 different \n\nspecies of the bacteria were isolated. Among these \n\nisolates, E. coli 52 (53.06%) was found to be the most \n\npredominant organism followed by Klebsiella \n\npneumoniae 21(21.43%), Pseudomonas aeruginosa \n\n12(12.24%), Proteus vulgaris 7(7.14%), Staphylococcus \n\naureus 4(4.08%) and Proteus mirabilis 2(2.04%). In a \n\nsimilar study done by Khanal et al, out of 41 isolates \n\nisolated from mid stream urine samples 8 different \n\nspecies were isolated among which E. coli (65.85%) \n\nwas found to be most predominant organism followed \n\nby Klebsiella pneumoniae (9.75%) [8]. The high \n\nprevalence of E. coli in causing UTI also resembled \n\nwith the studies by Raza et al in Kathmandu, Nepal \n\n[9] and Patel et al in India [10].  E. coli can bind to the \n\nglycoconjugate receptor (Gal \u03b1 1-4 Gal) of the \n\nuroepithelial cells of human urinary tract with its \n\nunique virulence determinant, the p pilus (Gal-Gal \n\nreceptor) so as to initiate the infection [11].  \n\nAs in the study by Gautam et al [12] all the Gram \n\npositive isolates were Staphylococcus aureus and were \n\nobtained from female patients only. \n\nAntibiotic resistance is a serious public health concern \n\nand is associated with prolonged hospitalization, high \n\nhealth-care cost, increased morbidity and mortality. In \n\nour study, gentamicin (57.45%) was found to be the \n\nmost effective antibiotic against Gram negative \n\nbacteria followed by ceftriaxone (51.06%) and \n\nnitrofurantoin (45.75%).  In a similar study carried out \n\nby Jha and Bapat at Sukhraraj Tropical Hospital, \n\nKathmandu, Nepal 92.5% of urinary isolates were \n\nfound to be susceptible to gentamicin [13]. In a study \n\nby Khanal 50% of gram negative organisms were \n\nsensitive to ceftriaxone and 58.34% of the gram \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:55-59      Awasthi et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal   58    Nepjol.info/index.php/njb \n\nnegative isolates were sensitive towards \n\nNitrofurantoin [8]. On the other hand, in our study \n\nampicillin was found to be the least effective drug \n\nagainst Gram negative bacteria (5.32% sensitive).", "start_char_idx": 10750, "end_char_idx": 13973, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ce53b06f-8f07-420f-a6cb-2e9e78bfd7fe": {"__data__": {"id_": "ce53b06f-8f07-420f-a6cb-2e9e78bfd7fe", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "01be77f1-ca75-47fd-be9c-f654c40799f9", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "c5731036affb9cbd3b6f3de4f7a8759d659766069865d74575859ee176b337df", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "738f89f0-e067-4ff6-ae20-4526b18fff60", "node_type": "1", "metadata": {}, "hash": "631dc918a4487a7cb9ded613192853ad96e3aabe5999107bc8a1925501cad319", "class_name": "RelatedNodeInfo"}}, "text": "Resistance to penicillins may be determined by the \n\norganisms due to the production of penicillin \n\ndestroying enzymes (\u03b2- lactamase). 15.96%, 29.79% \n\nand 32.98% of the gram negative bacilli were \n\nsusceptible to nalidixic acid, ofloxacin and co-\n\nTrimoxazole respectively. These results are in \n\naccordance with the results of Shrestha [5]. Similar \n\nfindings were also given by Gautam et al [12] and \n\nSharma et al [4]. Among gram positive cocci \n\nGentamicin was found to be 100% effective where as \n\nNalidixic acid and nitrofurantion were found least \n\neffective with 25% of the isolates being sensitive to \n\neach antibiotic.  \n\nThe exposure of the bacteria to antibiotic causes \n\nselective pressure causing the killing of susceptible \n\nbacteria allowing the resistant ones to survive. The \n\nrapid development of the antibiotic resistance among \n\nthe bacteria is attributed to the haphazard use of \n\nantibiotics [14]. The problem of the drug resistance \n\namong bacteria is more prevalent in developing \n\ncountries due to lack of awareness and lack of \n\neffective implementation of the policy that regulates \n\nthe use of antibiotics.  \n\nOut of 98 isolates, 42.86% were found to be multi drug \n\nresistant. The finding of the present study was \n\nsupported by the results of the study done by Khanal \n\n[8] and Upadhaya et al [15] noted the MDR causing \n\nUTI to be 56.09% and 48% respectively. In our study \n\n48.08% of the E. coli, 19.05% of the K. pneumonia, 50% \n\nof the P. aeruginosa, 85.71% of the P. vulgaris and 25% \n\nof the Staphylococcus aureus were found to be MDR. \n\nSimilar findings were obtained in the study done by \n\nTuladhar et al [16] in a hospital in Kathmandu, where \n\nMDR bacterial strains were detected in 35.21% with \n\nthe most predominant MDR bacterium being E. coli \n\nfollowed by Klebsiella spp. Increasing haphazard use of \n\nantibiotics and sales of substandard drugs are \n\nresponsible for development of multi drug resistance \n\namong the bacteria [17]. Due to development of drug \n\nresistance against commonly used antibiotics among \n\nthe bacteria the therapeutic options have become \n\nlimited.  \n\nThe isolation of the multi drug resistance bacteria \n\nfrom the patients with no past history of \n\nhospitalization indicates that the infection was \n\ncommunity acquired. The involvement of such large \n\nnumber of multidrug resistant bacteria in causing \n\ncommunity acquired urinary tract infections is very \n\nserious issue and cannot be neglected. Some abrupt \n\ninitiatives should be taken by the responsible \n\nauthorities to improve or at least avoid the further \n\nworsening of the situation. \n\nConclusion \n\n Involvement of multidrug resistant bacteria in \n\ncausing large numbers of community acquired \n\ninfections is a very serious public health concern. \n\nSome necessary initiatives should be taken \n\nimmediately to control the situation. \n\nCompeting interests \n\n The authors declare that they have no competing \n\ninterests. \n\nReferences \n\n1. Leigh DA, Smith GR, Easman CSF: Topley and \n\nWilson's principles of bacteriology, urology and \n\nimmunology, Bacterial diseases, 8th edition, frome \n\nand London; Butter and Tanner Ltd 1990,3:197-214. \n\n2. Forbes BA, Sahm DF, Weissfeld AS: Baily and \n\nScott's Diagnostic Microbiology, 11th edition. \n\nMosby Inc, USA; 2002. \n\n3. Tuladhar NR: A Report: Surveillance of Multiple \n\nDrug Resistant (MDR) bacterial infection among \n\nthe patients attending to different Out Patient \n\nDepartments (OPD) and Hospitalized Patients in \n\nTribhuvan University Teaching Hospital; 2001. \n\n4. Sharma AR, Bhatta DR, Shrestha J, Banjara MR: \n\nAntimicrobial Susceptibility Pattern of \n\nEscherichia coli Isolated from Urinary Tract \n\nInfected Patients Attending Bir Hospital.", "start_char_idx": 13976, "end_char_idx": 17696, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "738f89f0-e067-4ff6-ae20-4526b18fff60": {"__data__": {"id_": "738f89f0-e067-4ff6-ae20-4526b18fff60", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ce53b06f-8f07-420f-a6cb-2e9e78bfd7fe", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "00cc8fd053a71b13c334fbe23d0c61087ba529606881e6b652c9c3c78e61522c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "59f371c0-aec0-4d39-9a51-e48138199bc3", "node_type": "1", "metadata": {}, "hash": "d0ced95df071a31916cf19a5bfdb19eed0343609074d9bd2cb8270067004b515", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J \n\nScience and Technology 2013, 14(1): 177-184. \n\n5. Shrestha P: Study of bacteria causing urinary tract \n\ninfection and their antimicrobial resistance trend \n\nat National Public Health Laboratory. MSc thesis. \n\nTribhuvan University, Central Department of \n\nMicrobiology; 2007. \n\n6. Baral P, Neupane S, Marasini BP, Ghimire KR, \n\nLekhak B, Shrestha B: High prevalence of \n\nmultidrug resistance in bacterial uropathogens \n\nfrom Kathmandu, Nepal. BMC Research Notes \n\n2012,5:38.", "start_char_idx": 17697, "end_char_idx": 18180, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "59f371c0-aec0-4d39-9a51-e48138199bc3": {"__data__": {"id_": "59f371c0-aec0-4d39-9a51-e48138199bc3", "embedding": null, "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-33", "node_type": "4", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "5cf01350703b2438a3ba40a3f54a99f68517312feea57f970cba8cc18cfae0f8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "738f89f0-e067-4ff6-ae20-4526b18fff60", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "7c3e8fd8bbc7689f408a23298e71082014d8d4ef8179a85f2e712c25928ee211", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5d0c85c4-5dc7-4cef-ae97-7e6d2ed8326c", "node_type": "1", "metadata": {}, "hash": "23c91196a625b63d57a4aeba139d97dd0207d07bb9515bba351bae98e9f42aa2", "class_name": "RelatedNodeInfo"}}, "text": "BMC Research Notes \n\n2012,5:38. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:55-59      Awasthi et al. \n\n \n \n\n \n\u00a9NJB, Biotechnology Society of Nepal   59    Nepjol.info/index.php/njb \n\n7. Karki A, Tiwari BR, Pradhan SB: Study of Bacteria \n\nisolated from Urinary tract infection and their \n\nsensitivity Pattern. J Nepal Med Assoc 2004,43:200-\n\n203. \n\n8. Khanal S: A study on Microbiology of urinary \n\ninfection at Tribhuvan University Teaching \n\nHospital, Kathmandu, Nepal. MSc thesis. \n\nTribhuvan university, central department of \n\nMicrobiology; 2006. \n\n9. Raza S, Pandey S, Bhatt CP:  Microbiological \n\nAnalysis of the Urine Isolates in Kathmandu \n\nMedical College Teaching Hospital, Kathmandu, \n\nNepal. Kathmandu Univ Med J 2011,36(4):295-7. \n\n10. Patel S, Taviad PP, SinhaM, Javadekar TB, \n\nChaudhari VP: Urinary Tract Infections (UTI) \n\nAmong Patients at G.G. Hospital & Medical \n\nCollege, Jamnagar. National J Community Medicine \n\n2012, 3(1): 138-41. \n\n11. Johnson JR: Virulence factors in Escherichia coli \n\nurinary tract infection. Clin Microbiol Rev 1991,4:80-\n\n128. \n\n12. Gautam K, Pokhrel BM: Prevalence of Urinary \n\nTract Infection at Kanti Children\u2019s Hospital. J \n\nChitwan Medical College 2012, 1(2):22-5. \n\n13. Jha N, Bapat SK: A study of sensitivity and \n\nresistance of pathogenic microorganisms causing \n\nUTI in Kathmandu valley. KUMJ 2005,3:123-9. \n\n14. Dalhoff A: Global fluoroquinolone resistance \n\nepidemiology and implictions for clinical use. \n\nInterdisciplinary Perspectives on Infectious Diseases \n\n2012, 12:37. \n\n15. Upadhyay G, Shakya G, Upadhyaya BP, Shrestha S, \n\nAnsari S, Ghimire P et al: Comparative evaluation \n\nof urine isolates among kidney transplanted and \n\nother UTI suspected patients visiting National \n\nPublic Health Laboratory, (NPHL) Teku, Nepal. \n\nInt J Biomedical And Advance Research 2013,4(6):369-\n\n75. \n\n16. Tuladhar NR, Banjade N, Pokhrel BM, Rizal B, \n\nManandhar R, Shrestha S, Shah A and Chaurasia S. \n\nAntimicrobial resistant bacterial strains from \n\ninpatients of Tribhuvan University teaching \n\nhospital Kathmandu. J Inst Med 2003, 25:19-26. \n\n17. Gautam R, Chapagain ML, Acharya A, Rayamajhi \n\nN, Shrestha S, Ansari S, Upadhaya G, Nepal HP. \n\nAntimicrobial susceptibility patterns of \n\nEscherichia coli from various clinical sources. \n\nJCMC 2013,3:14-7. \n\n18. Baral R, Timilsina S, Jha p, Bhattarai NR, Poudyal \n\nN, Gurung R, Khanal B, Bhattachary Sk. Study of \n\nAntimicrobial Susceptibility pattern of Gram \n\nPositive Organism causing UTI in a tertiary care \n\nhospital on Eastern region of Nepal. Health \n\nRenaissance 2013, 11(1): 119-24.", "start_char_idx": 18149, "end_char_idx": 20765, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5d0c85c4-5dc7-4cef-ae97-7e6d2ed8326c": {"__data__": {"id_": "5d0c85c4-5dc7-4cef-ae97-7e6d2ed8326c", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "59f371c0-aec0-4d39-9a51-e48138199bc3", "node_type": "1", "metadata": {"identifier": "njb-33", "author": "Awasthi, Tek Raj; Pant, Narayan Dutt; Dahal, Puspa Raj", "title": "Prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of Seti Zonal Hospital, Dhangadi, Nepal", "date": "2015-12-30", "file": "njb-33.pdf"}, "hash": "e12e9a5eb111cb32ad1ed64417f6fa5de3d6f0d410ab6783fe1ace8409999bef", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6c41e571-e914-4683-b568-831d287e5eb3", "node_type": "1", "metadata": {}, "hash": "8e9d9ff572c929673f353b028fe4752822f58113f29056299959a1111124e7bb", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:48-54    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\n     ORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   48     Nepjol.info/index.php/njb \n\n \n\nPhytochemical Screening, Antimicrobial Activity and Cytotoxicity of \nNepalese Medicinal Plants Swertia chirayita and \n\n Dendrobium amoenum \nPritish Shrestha, Manisha Bista, Prativa Sharma, Shristi Shrestha, Basanta Lamichhane, Sandeep Adhikari, Binayak \n\nRaj Pandey, Bhupal Govinda Shrestha* \nDepartment of Biotechnology, School of Science Kathmandu University, Dhulikhel, Nepal \n\nAbstract \nResearch on medicinal plants are important to Nepal because most of its rural population relies on it as mode of medicine. \n\nMedicinal plants namely Swertia chirayita and Dendrobium amoenum were collected from mid hills of Nepal. The present \n\nstudy was undertaken to find the antimicrobial activity, phytochemical presence and their cytotoxicity in different \n\nextraction medium. The percentage yield from the plants were highest in warm methanol extraction with 12.6%, followed \n\nby ethyl acetate and lowest was for cold methanol. Plant extract showed the presence of antioxidants like alkaloid, \n\nterpenoids, flavonoids, tannin, glycosides. The Brine Shrimp Bioassay of methanol and ethyl acetate extract showed \n\ncytotoxicity. Chiraito extract showed LC50 of 199 ppm for Dhunche sample, 128.82 ppm for Daman sample and 131.82 ppm \n\nof Illam sample. The antibacterial activity of methanol extract of Chiraito and Dendrobium amoenum showed significant \n\nbioactivity by inhibiting growth of microbial species selected for the test. The zone of inhibition shown by the extracts was \n\ncomparable to the standard antibiotics. Similarly, methanol extract of Chiraito also showed significant antifungal activity \n\nwith the zone of inhibition comparable to amphotericin. \n\nKeywords:  Antioxidant, LC50 (Lethal concentration-50), nauplii, ZOI (Zone of Inhibition) \n\n*Corresponding author \n\nEmail: bgs@ku.edu.np \n\nIntroduction \nFor centuries, medicinal plants are being used as \n\nAyurveda or traditional medicine in Nepal by local \n\ntribes and also in most of the rural areas of the Indian \n\nsubcontinent. Medicinal plants and Ayurveda \n\npractice in the sub continent are intertwined. \n\nDifferent parts of plant are used in different herbal \n\nmedicine as one of the constituents of final \n\ncomposition. Some phytochemicals present in these \n\nmedicinal plants, have medicinal values and are \n\nexpected to yield positive biological activities. Nepal, \n\nin the middle of the Himalayan belt which extends \n\nfrom Myanmar in the East to Karakorum in the West, \n\npossess vast diversity of plants because of its \n\ngeographical distribution [1,2,3,4]. Some of these \n\nMedicinal plants are used in indigenous rural \n\nremedies, homoeopathic medicines, and allopathic \n\npharmacopeia [5,6,7,8]. The exploitation of locally \n\navailable medicinal plants in health care and \n\neconomic advancement is a necessity of Nepal. \n\nAmong all those plants Swertia chirayita and \n\nDendrobium amoenum is the focus of this research. \n\nAmong 100 species of Swertia genus, 27 species are \n\nfound in Nepal. Among them around nine species are \n\nreported to be traded for medicinal purpose. Swertia \n\nchirayita is biennial erect herb which is approximately \n\n50 to 125 cm tall.", "start_char_idx": 47, "end_char_idx": 3412, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6c41e571-e914-4683-b568-831d287e5eb3": {"__data__": {"id_": "6c41e571-e914-4683-b568-831d287e5eb3", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5d0c85c4-5dc7-4cef-ae97-7e6d2ed8326c", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "731d3f7a9eff2fd0d2a6e8c8869388b927f1f310db86070e9499b3996488bdec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ef533fd3-902e-40f6-9f4b-cc2cd1a0a771", "node_type": "1", "metadata": {}, "hash": "5ae5423035c546c2e8f6abe76817bad2500b5ee178717f233b8fee5ae3765f50", "class_name": "RelatedNodeInfo"}}, "text": "The plant is a native of temperate \n\nHimalayas, found at an altitude of 1200\u20133000 m. Its \n\nstem is robust, branched and cylindrical below, four \n\nangled upward and containing large pith, broadly \n\nlanceolate leaves with 5-nerve and sub-sessile. It has \n\nlurid greenish yellow flowers tinged with purple in \n\nlarge panicles, with egg-shaped capsules and minute \n\nseed which are smooth and many angled [13]. \n\nChiraito grows mainly in temperate Himalayas and is \n\nreported from 40 districts of Nepal. Chiraito grows \n\nbetween open forest and margin of cultivated land, it \n\nis predominant in Dolakha and spreads mainly in \n\naltitude of 1500 m to 3000 m of Eastern and Central \n\nregion of Nepal [2,9].  \n\nDendrobium contains about 1,200 species and is also \n\nknown as orchid [10]. This genus also occurs in \n\ndiverse habitats throughout much of South and \n\nEast Asia, stretching to Oceania and some of Pacific \n\nislands. This plant is commonly distributed between \n\n660 m to 2000 m temperate forest of Himalayan \n\nregion and grow in little light exposure [11,12]. The \n\norchids are mostly found in terrestrial, epiphytic and \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   49    Nepjol.info/index.php/njb \n\nsaprophytic habitat. Dendrobium amoenum grows in \n\nclustered pendulous five to six slender stems. \n\nMorphologically, this species is highly evolved, tall, \n\nstraight with elongated pseudobulbs covered by \n\nmodest sized leaves. It has unique floral pattern \n\nwhich is fairly constant varying in size from very \n\nsmall to large, the shape and form of the stems and \n\nleaves are divergent. This traditional medicinal \n\nspecies is also ornamental plant and have been \n\ncultivated for decorative purpose.  The flowers \n\nflourish on an older stem in cluster of two to three \n\nper node and the flower is amusingly perfumed \n\n[13,14,15]. \n\nMaterials and Methods \nSample preparation \nSwertia chirayita plants were collected from or near \n\nfrom Dhunche, Daman and Illam and Dendrobium \n\namoenum from Pokhara, during their flowering \n\nseason. The plants were air dried under room \n\ntemperature. The dried plant samples were cut and \n\ngrinded to make it in powder form and kept for \n\nstorage at room temperature. \n\nExtraction \nThree types of extraction method were carried out, \n\nviz. warm methanol extraction, cold methanol \n\nextraction and ethyl acetate extraction.  \n\nFor warm extraction, Soxhlet apparatus was used. 10 \n\ng of the crushed sample along with 200 ml of \n\nmethanol was put into the soxhlet apparatus. The \n\nsoxhlet was run for 28 hours at 65\u2070C. The methanol \n\nextract was taken out from the soxhlet apparatus. The \n\npigment was removed using Hexane in the \n\nseparating funnel. The methanol fraction was then \n\ndried using water bath. \n\nFor cold extraction methanol at room temperature \n\nwas used. 100 g of powdered form of sample was \n\ntaken and 450 ml methanol, that was just enough to \n\ncover the upper layer of sample, was poured on it \n\nand was shaken regularly.  After 48 hrs in room \n\ntemperature, filtration was done and the filtrate was \n\nstored at room temperature. \n\nFor ethyl acetate extraction, 10 g of powdered \n\nmaterial was dissolved in 25 ml ammonium \n\nhydroxide.  300 ml of ethyl acetate was then added \n\nand left for 72 hrs at room temperature. Extract was \n\nthen filtered. Ethyl acetate was then left to dry in \n\nwater bath [16,17,18].", "start_char_idx": 3413, "end_char_idx": 6865, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ef533fd3-902e-40f6-9f4b-cc2cd1a0a771": {"__data__": {"id_": "ef533fd3-902e-40f6-9f4b-cc2cd1a0a771", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6c41e571-e914-4683-b568-831d287e5eb3", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "1bb9e1a70e15346639c1158e3196918732bdc5d52309173b0a1bc12fdb9399db", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3ffc9d42-7d5d-4550-90b5-623712969c7e", "node_type": "1", "metadata": {}, "hash": "a45c7658e4243fb1cd2b6d60e10253abb2e2c3d80bfd96bea14f22fbb362f715", "class_name": "RelatedNodeInfo"}}, "text": "Phytochemical Screening was done to check the \n\npresence of alkaloid, sterols, triterpenes, tannins and \n\npolyphenols, reducing sugar, saponins, flavonoids, \n\nglycosides and coumarin according to protocol \n\ndescribed in  [19,20,21]. \n\nBrine Shrimp Bioassay \nPreparation of Test sample \n\nStock solution was prepared by dissolving 100 mg of \n\nthe extract in little amount of dimethyl sulfoxide \n\n(DMSO) for initial solublization and then addition of \n\nwater to final volume of 25 ml to make a stock of \n\nconcentration of 4000 ppm. The stock solution was \n\nfurther diluted to 1000, 100, 10 ppm concentration. \n\nFor Hatching of Brine shrimp, 50 mg of Brine shrimp \n\neggs were sprinkled in a beaker with 300 ml of sea \n\nwater. The transferred sample as then allowed \n\nincubating at 32 \u2013 35oC for 24 hrs. \n\nBioassay \nCleaned test tubes were divided into four groups \n\neach group consisting of five test tubes.  After 24 hrs \n\nof incubation, the nauplii were recovered with a \n\npipette and 10 nauplii were transferred in each test \n\ntube. The groups were then treated with different \n\ndilutions of sample. The test tubes were then \n\nincubated at 32\u2013 35oC overnight. The incubated tubes \n\nwere observed for the number of survived nauplii \n\nand graph was plotted for death percentage versus \n\nLog of concentration of the extract. This gives linear \n\nequation in the form of y = mx + c. \n\nCalculation of LC50 \nThe death of nauplii was calculated as death \n\npercentage as,  \n\n% death = deaths/initial x 100.  The % death was \n\ncorrected for any control deaths by subtracting the -\n\n%death control from % death test.  The Lethal \n\nconcentration 50 (LD50) was derived from the graph \n\nfrom the equation of the straight line [19]. \n\nAntimicrobial Screening \nMicro-organisms used Six clinical samples of bacteria \n\nwere collected from Kathmandu University Teaching \n\nHospital (KUTH), Dhulikhel namely Staphylococcus \n\naureus, Escherichia coli, Kleibsella pneumonia, Salmonella \n\nparatyphi, Salmonella typhi, Pseudomonas aerugenosa. \n\nThe Yeast Saccharomyces cerevisiae (YCS2) were taken \n\nfrom microbiology lab of Department of \n\nBiotechnology, Kathmandu University.", "start_char_idx": 6868, "end_char_idx": 9019, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3ffc9d42-7d5d-4550-90b5-623712969c7e": {"__data__": {"id_": "3ffc9d42-7d5d-4550-90b5-623712969c7e", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ef533fd3-902e-40f6-9f4b-cc2cd1a0a771", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "a44cd03482e53763c5a135cdcbb0100cd843ae9e7eb6731dd3af14e47384dcda", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4bf5ec91-397b-4e92-8e11-fb88bc9b2a01", "node_type": "1", "metadata": {}, "hash": "d981d0e72946cd2f72f0413a32e6150d5361c605c909460d4c376403a05cf15b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   50    Nepjol.info/index.php/njb \n\nPreparation of Sample \nThe different extraction of plant was dissolved in \n\nDMSO and water, which was made into different \n\nconcentrations. \n\nInoculation \n\nSterilized petri dishes were filled to a uniform depth, \n\nwith respective sterilized medium (Nutrient agar for \n\nbacteria and Muller Hinton agar for fungus \n\nrespectively). The solidified media was then \n\ninoculated with respective organisms. Holes were cut \n\ninto the medium using a sterile borer. Solution of \n\nvarious concentrations of the extracts under test was \n\npoured into the bored holes with the help of a \n\nmicropipette and the standard antibiotic discs or \n\nsolutions were gently placed at the different sites of \n\nthe petri-dish containing the inoculated medium in \n\nthe case of cup plate method. In case of filter disc \n\nmethod, filter paper discs prepared from Watman\u2019s \n\nfilter paper were soaked with the test solution and \n\nthe filter discs are then gently placed into the \n\ninoculated medium with the help of a sterile forceps.  \n\nThe plates were then maintained at room \n\ntemperature for few minutes to allow the antibiotic \n\nand the extract solution to diffuse into the medium. \n\nIn the uniform media, diffusion of the extract \n\nsolution and the antibiotic discs will occur uniformly \n\naround the cup and the concentration gradient will \n\nbe established around it and at a certain distance \n\nfrom the cup. The plates were then incubated at a \n\nsuitable temperature (usually 37-39\u2070C) for 24-48 \n\nhours. After 48 hours, the zones of inhibition were \n\nmeasured with the help of a measuring ruler or \n\nantibiotic zone reader [22]. \n\nResult \nThe percentage yield for a given plant is calculated \n\nas: \n\n \n \n\n \n \n\n \n \n\n \nFigure 1: Yield percentage of different plant from various solvents. \n\n[A] representsyield percentage from cold extract (Methanol), [B] \n\nrepresents yield percentage from cold extract (Ethyl Acetate) and \n\n[C] represents yield percentage from warm extract (Methanol).  \n\n \n\nThe various phytochemicals tested for Chiraito and \n\nDendrobium amoenum extracts with their \n\ncorresponding results can be tabulated in Table 1 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   51    Nepjol.info/index.php/njb \n\n \n\n \n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \nFigure 2: Zone of Inhibition (ZOI) of different plant samples from \n\ncold methanol extract of 2.5 % and 7.5 % against different bacteria. \n\nA, B, C, D, E and F represents ZOI of different plant extract against \n\nS. paratyphi, E. coli, K. pneumoniae, S. typhi, P. aerugenosa and S. \n\naureus respectively.", "start_char_idx": 9024, "end_char_idx": 11804, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4bf5ec91-397b-4e92-8e11-fb88bc9b2a01": {"__data__": {"id_": "4bf5ec91-397b-4e92-8e11-fb88bc9b2a01", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3ffc9d42-7d5d-4550-90b5-623712969c7e", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "1ee141bf68534e919c6bd8bc80568007b70e7450469721cf6720b0aacf517f41", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7798ae4d-36f5-4a65-906f-2b85a760812a", "node_type": "1", "metadata": {}, "hash": "51c77d056ebcc7609a10f8336a90add77f4b16cf7f31e9a8f59d71c63bc82a84", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   52    Nepjol.info/index.php/njb \n\n \n\n \nFigure 3: Zone of Inhibition (ZOI) of different Chiraito samples. A \n\nrepresents ZOI of Chiraito (Dhunche) prepared from ethyl acetate \n\nagainst 5 microbes and B represents ZOI of Chiraito (Daman) \n\nagainst Saccharomyces cerevisiae. \n\nZOI (in mm) of plant extract against following fungus \n\nand comparison with fungicide solution Figure 3 \n\n Brine Shrimp Bioassay \n\n \n\n \nFigure 4: Brine Shrimp assay graph for Chirato sample of different \n\nplaces Graph A= Dunche, Graph B= Illam and Graph C=Daman \n\n \n\n \nFigure 5: Brine Shrimp Assay for different Chirato samples in \n\ndifferent concentration  \n\nCalculation: \n\nGraph was plotted as death percentage versus Log of \n\nextract concentration in ppm. This shows the linear \n\nequation in the form of y=mx+c. By substituting the \n\nvalue of y=50, the corresponding value of x gives the \n\nLog value of LC50 value and antilog of that value \n\ngives the LC50 value in ppm. \n\n \n\nFrom Fig 4, Graph 1, equation: y=22x -1.33 \n\nFor LC50, Y=50, then X=2.3, Antilog of X is \n\nAntilog(2.3) = 199ppm \n\nTherefore, LC50 of Chirato (Dhunche) methanol \n\nextract for brine shrimp is 199ppm. \n\nFrom Fig 4, Graph 2, From equation: Y= 26.5x-6 \n\nFor LC50, Y=50, then X=2.11, Antilog of X is \n\nAntilog(2.11) = 128.82ppm \n\nTherefore, LC500 of Chirato (Daman) methanol \n\nextract for brine shrimp is 128.82 ppm. \n\nFrom Fig 4, Graph 3, From equation: Y= 20x + 7.6 \n\nFor LC50, Y=50, then X=2.12, Antilog of X is Antilog \n\n(2.12) = 131.82ppm \n\nTherefore, LC50 of Chiraito (Illam) methanol extract \n\nfor brine shrimp is 131.82ppm. \n\n 5 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   53    Nepjol.info/index.php/njb \n\nDiscussion \nThe % yield from the Chiraito extract was highest in \n\nwarm methanol extraction with 12.6% as compared \n\nto cold methanol extraction and that for ethyl acetate \n\nwas 9% as shown in Figure 1. However in the case of \n\nDendrobium amoneum the % yield was higher for cold \n\nextraction at 4.4 % as compared to warm extraction at \n\n3.4 %.This indicates that there are many compounds \n\npresent in the plants soluble in methanol and ethyl \n\nacetate and that there is higher amount of bioactive \n\ncomponent in Chiraito compared to Dendrobium \n\namoneum. Generally all alkaloids are highly soluble in \n\nmethanol. High extract yield for methanol also \n\nsuggests that there are high amount of alkaloids in \n\nthe plant. \n\nThe Phytochemical screening of the plant extract \n\nshowed the presence of alkaloid, terpenoids, tannins, \n\ncoumarins, flavanoids and sterols, as shown in Table \n\n1, indicating that there are high value natural \n\ncompounds in the plants.", "start_char_idx": 11812, "end_char_idx": 14658, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7798ae4d-36f5-4a65-906f-2b85a760812a": {"__data__": {"id_": "7798ae4d-36f5-4a65-906f-2b85a760812a", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4bf5ec91-397b-4e92-8e11-fb88bc9b2a01", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "1bab87d00dbd9e4821ba25ac5859b448a0fd9533ddfd22204767c913ee2e18b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3937508f-1629-49db-82cd-8a15a6e62987", "node_type": "1", "metadata": {}, "hash": "1eb4ddcc152adbd28f8d1525933308532bf252783914fee3c2b8613732f8b3d1", "class_name": "RelatedNodeInfo"}}, "text": "Since there are presence of \n\nalkaloids, terpenoids, coumarins, flavanoids and \n\nsterols these plants could have anti cancer activity. \n\nThe antibacterial activity of methanol and ethyl \n\nacetate extract of Chiraito and Dendrobium amoenum \n\nshowed bioactivity by inhibiting growth of microbial \n\nspecies selected for the test as shown in Figure 2 and \n\n3. The zone of inhibition shown by the extracts was \n\ncomparable to the standard antibiotics. Cold \n\nmethanol extract of Chiraito didn\u2019t show any activity \n\nagainst E Coli. Cold methanol extract of Chiraito from \n\nDhunche had activity higher than that of the control \n\nantibiotic tetracycline. Bioactivity of ethyl acetate \n\nextract was not significant as that of methanol extract \n\nand showed activity against only Pseudomonas \n\nauregenosa. This may be due to absence of alkaloids. \n\nCold methanol extract of Chiraito (Daman) showed \n\nsignificant activity against Saccharomyces cerevisiae. \n\nThe zone of inhibition was significantly higher than \n\nthat of control, amphotericin at 25\u00b5g/ml. \n\n \n\nThe Brine Shrimp Bioassay of methanol extract \n\nshowed cytotoxic nature of plant extracts. If LC50 \n\nvalue of test sample is less than 1000 ppm the extract \n\nis considered to be biologically active. Chirato extract \n\nshowed LC50 of 199 ppm for Dhunche sample, 128.82 \n\nppm  for  Daman sample and 131.82 ppm for Illam \n\nsample. Since cytotoxicity value given by Brine \n\nShrimp assay directly correlates with its cytotoxicity \n\nability, toxicity at low concentration of Chiraito \n\nextracts can have toxicity against cancer cell lines and \n\nhas the potential to be developed as drugs for cancer. \n\nConclusion: \nResearchers have identified compounds used in \n\nmainstream medicine derived from plant resources. \n\nSimilarly, this research has assessed the \n\nphytochemical, biological screening of Chiraito and \n\nDendrobium amoenum. This natural product can bring \n\nnew and effective antimicrobial agents and serve as \n\nalternate source of combating infections in human \n\nbeings. Hence this research can have a promising \n\npotential in various traditional, complementary and \n\nalternate systems of treatment of human diseases. \n\nFurther work on isolation and characterization of \n\nthese plants and their pharmacodynamics study can \n\nbe a great contribution. The research also proved to \n\nbe beneficial in exploiting medicinal plants having \n\nbiological activities namely cancer. \n\nReferences \n1. Radcliffe-Smith A, Hara H, Steam WT, Williams LHJ: An \n\nEnumeration Of The Flowering Plants Of Nepal. Kew \n\nBulletin 1979, 34(1):198.  \n\n2. Joshi K: Swertia L. (Gentianaceae) In Nepal: \n\nEthnobotany and Agenda For Sustainable \n\nManagement. Ethnobotanical Leaflets 2008, 12:1-6.  \n\n3. Cseke JL, Kirakosyan A, Kaufman PB, Warber SL, Duke \n\nJA, Brielmann HL: Natural Products From Plants. In 2nd \n\nedition. CRC Press Taylor and Francis Group, 2006.  \n\n4. Siddiqui S, Verma A, Rather AA, Jabeen F, Meghvansi \n\nMK: Preliminary Phytochemicals Analysis of Some \n\nImportant Medicinal And Aromatic Plants. Advances in \n\nBiological Research 2009, 3(5-6):188-195.  \n\n5. Joshi B, Lekhak S, Sharma A: Antibacterial Property of \n\nDifferent Medicinal Plants: Ocimum Sanctum, \n\nCinnamomum Zelanicum, Xanthoxylum Armatum and \n\nOriganum Majorana. Kathmandu University Journal of \n\nScience, Engineering and Technology 2009, 5:1-6.  \n\n6.", "start_char_idx": 14659, "end_char_idx": 18013, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3937508f-1629-49db-82cd-8a15a6e62987": {"__data__": {"id_": "3937508f-1629-49db-82cd-8a15a6e62987", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7798ae4d-36f5-4a65-906f-2b85a760812a", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "79d2db27938244d98753be63e2d7c41845c642d95d82a53f9c026de830292a91", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "acbcc076-f4f7-4688-b458-37e5773f9413", "node_type": "1", "metadata": {}, "hash": "69fec51783751876be5611dc8bdfd1fdaa8ec644906f13c32d70b72392a72822", "class_name": "RelatedNodeInfo"}}, "text": "6. Bhattarai KR, Ghimire M: Commercially Important \n\nMedicinal and Aromatic Plants of Nepal and Their \n\nDistribution Pattern and Conservation Measure Along \n\nthe Elevation Gradient of the Himalayas. Banko Jankari \n\n2007, 16(1):1-6. \n\n7. Okamura T, Ayajiki K, Fujioka H, Toda M, Fujimiya M, \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 48-54      Shrestha et al.  \n\n\u00a9NJB, Biotechnology Society of Nepal   54    Nepjol.info/index.php/njb \n\nToda N: Effects of Endothelial Impairment by Saponin \n\nOn the Responses to Vasodilators and Nitrergic Nerve \n\nStimulation in Isolated Canine Corpus Cavernosum. \n\nBritish Journal of Pharmacology 1999, 127(3):802-808.  \n\n8. Sharma M, Chapagain N, Sah S: Phytochemicals as \n\nEmerging Antimicrobials. Plant Resources (A Scientific \n\nPublication) 2010, 32:96-101.  \n\n9. Joshi P, Dhawan V: Swertia chirayita \u2013 an overview, \n\nCurrent Science, 2005, 89(04):635-640 \n\n10. Venkateswarlu S, Mudunuri SSR, Gottumukkala VS: \n\nSynthesis and Biological Activity of Isoamoenylin, A \n\nMetabolite of Dendrobium Amoenum. Bioscience, \n\nBiotechnology and Biochemistry 2002, 66(10):2236-2238.  \n\n11. Rajkarnikar K; In Vitro Propagation of Dendrobium \n\nAmoenum Wall. Ex Lindi. From Seed Culture. Plant \n\nResources (A Scientific Publication) 2010, 32:90-92. \n\n12. Shrestha P: Antibacterial Activity of Medicinal Plant \n\nExtracts. Plant Resources (A Scientific Publication) 2010, \n\n32:51-54.  \n\n13. Martha R, Gutierrez P: Orchids: A Review of Uses in \n\nTraditional Medicine, Its Phytochemistry and \n\nPharmacology. Journal of Medicinal Plants Research 2015, \n\n4(8):592-638.  \n\n14. Zhang CF, Wang M, Wang L, Iinuma M, Zhang M, Xu \n\nLS, Wang ZT: Chemical Constituents of Dendrobium \n\nGratiosissimum and Their Cytotoxic Activities. Indian \n\nJournal of Chemistry 2008, 47B:952-956.  \n\n15. Buckingham J: Dictionary of Natural Products.  CD-\n\nROM 13:1. Boca Raton, Florida: Chapman & Hall/CRC \n\nPress, 2005.  \n\n16. Bhandari J, Naqvi A, Niraula P, Thapa P, Thapa N, \n\nShrestha N and Shrestha BG: Phytochemical Screening, \n\nAntioxidant Assay of Junipers recurva and study of its \n\nInvitro cytotoxicity against Breast Cancer Cell Lines. \n\nInternational Journal of Pharma and Bio Sciences. 2015, \n\n6(3B):1134-1145. \n\n17. Lamichhane B, Adhikari S, Shrestha P, Shrestha BG: \n\nStudy of Phytochemical, Antioxidant, Antimicrobial \n\nand Anticancer Activity of Berberis Aristata. Journal of \n\nTropical Life Science. 2014, 4(1): 1-7. \n\n18. Shrestha P, Adhikari S, Lamichhane B, Shrestha BG: \n\nPhytochemical Screening of the Medicinal Plants of \n\nNepal. IOSR Journal of Environmental Science, Toxicology \n\nand Food Technology. Special Issue 2015, 1(6):11-17. \n\n19.", "start_char_idx": 18011, "end_char_idx": 20692, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "acbcc076-f4f7-4688-b458-37e5773f9413": {"__data__": {"id_": "acbcc076-f4f7-4688-b458-37e5773f9413", "embedding": null, "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-34", "node_type": "4", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "02866eda2f5d03a3c34635e26374f89b37e1bdedcf32fe38d42247a380ea9284", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3937508f-1629-49db-82cd-8a15a6e62987", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "afb557d994f2b15bd8ba17242976eee1a6d96a0a1102e7790d113e58c7b2829e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "850475e4-6a07-4925-927f-f7e07ac29815", "node_type": "1", "metadata": {}, "hash": "06017274a907ddb05d39a4da2df63b051056590a76e5430ff8d4a2eb4cf682c7", "class_name": "RelatedNodeInfo"}}, "text": "Special Issue 2015, 1(6):11-17. \n\n19. Chhetri HP, Yogol NS, Sherchan J, Anupa KC, Mansoor \n\nS, Thapa P: Phytochemical and Antimicrobial \n\nEvaluations of Some Medicinal Plants of Nepal. \n\nKathmandu University Journal of Science engineering and \n\ntechnology. 2008, 1:49-54.  \n\n20. Setzer WN, Flair MN, Byler KG, Huang J, Thompson \n\nMA, Setzer AF, Moriarity DM, Lawton RO and \n\nWindham-Carswell.: Antimicrobial and cytotoxic \n\nactivity of crude extracts of Araliaceae from \n\nMonteverde. Costa Rica. Brenesia 1992, 38:123-130.  \n\n21. Zabri H, Kodjo C, Anoubil\u00e9 B, B\u00e9kro M, Bekro Y A: \n\nPhytochemical Screening and Determination of \n\nFlavonoids in Secamone Afzelii (Asclepiadaceae) \n\nExtracts. African Journal of Pure and Applied Chemistry \n\n2008, 2:80-82. \n\n22. Reuben KD, Abdulrahman FI, Akan JC and Egwu \n\nGO: Phytochemical Screening and in Vitro \n\nAntimicrobial Investigation of the Methanolic Extract \n\nof Croton Zambesicus Muell ARG. Stem Bark. European \n\nJournal of Scientific Research 2008, 23:134-140.", "start_char_idx": 20655, "end_char_idx": 21660, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "850475e4-6a07-4925-927f-f7e07ac29815": {"__data__": {"id_": "850475e4-6a07-4925-927f-f7e07ac29815", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "acbcc076-f4f7-4688-b458-37e5773f9413", "node_type": "1", "metadata": {"identifier": "njb-34", "author": "Shrestha, Pritish; Bista, Manisha; Sharma, Prativa; Shrestha, Shristi; Lamichhane, Basanta; Adhikari, Sandeep; Pandey, Binayak Raj; Shrestha, Bhupal Govinda", "title": "Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum", "date": "2015-12-30", "file": "njb-34.pdf"}, "hash": "859dd9568908371e4b42b865191281eacc5be41bdf113aa97c7e403467c5747d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "acbfa93f-d44e-4952-a9d8-6f20f7c2a318", "node_type": "1", "metadata": {}, "hash": "63480f0516dcbcf8147ba2441edcc6172101c2fefa76688a2d8c218bd5e85ee1", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   40              Nepjol.info/index.php/njb \n\nMolecular Markers for Septoria Leaf Spot (Septoria lycopersici Speg.) \nResistance in Tomato (Solanum lycopersicum L.) \n\nBal K. Joshi1*, Frank J. Louws2, G. Craig Yencho3, Bryon R. Sosinski3, Consuelo Arellano4, Dilip R. Panthee3 \n1Genebank-NARC, Khumaltar, PO Box 3055, Kathmandu, Nepal \n\n2Department of Plant Pathology, North Carolina State University, North Carolina \n3Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina. \n\n4Department of Statistics, North Carolina State University, Raleigh, North Carolina. \n\nAbstract \nMarker assisted selection (MAS) has not been initiated in tomato (Solanum lycopersicum L.) for \n\nseptoria leaf spot (SLS) resistance caused by Septoria lycopersici Speg due to lack of molecular \n\nmarkers. We studied the inheritance of SLS resistance and identified molecular markers linked to \n\nSLS resistance using bulked segregant analysis (BSA) in a segregating F2 population. Tomato \n\ninbred lines, NC 85L-1W (2007), susceptible to SLS and NC 839-2(2007)-1, resistant to SLS were \n\nused to develop the segregating population. A total of 250 F2 plants, and 10 plants each of P1, P2 \n\nand F1 were grown at the Mountain Horticultural Crops Research and Extension Center \n\n(MHCREC), Mills River NC in the summer of 2009. Disease severity was scored using a scale of 0 to \n\n5, where 0 = no disease and 5 = complete development of disease. DNA was extracted from 2-3 \n\nweek old plants and parental lines were screened with a total of 197 random amplified \n\npolymorphic DNA (RAPD) primers, of which 34 were polymorphic. Two DNA bulks, called \n\nresistant bulk (RB) and susceptible bulk (SB) were prepared from the F2 individuals. The RB and SB \n\nconsisted of 8 individuals each with disease scores of 0, and 4.0 or 4.5, respectively. The segregation \n\nratio of resistant and susceptible plants in F2 generation fit the expected Mendelian ratio of 3:1 for a \n\nsingle dominant gene. Five RAPD markers were linked to the SLS disease reaction, of which two \n\nwere linked to susceptibility and three to the resistance. Subject to verification in independent \n\npopulations, these markers may be useful for MAS of SLS resistance in tomato. \n\nKey words: Bulked segregant analysis, resistant bulk, septoria leaf spot, Solanum lycopersicum, susceptible bulk \n\n*Corresponding author \nE-mail: joshibalak@yahoo.com \n\nIntroduction \nTomato (Solanum lycopersicum L., 2n = 2x = 24) is one \n\nof the most important vegetable crops worldwide. \n\nAmong the foliar diseases of tomato, septoria leaf spot \n\n(SLS) caused by Septoria lycopersici Speg is one of the \n\nmost devastating diseases. It occurs worldwide \n\nincluding Canada and Northeast America.  It can \n\ncause complete defoliation leading to a significant \n\ncrop loss under favorable environmental conditions, \n\nparticularly in humid regions during periods of heavy \n\nrainfall, frequent dew or over-head irrigation [1,2]. \n\nAlthough fungicides are effective to control this \n\ndisease, breeding for resistance is preferred by tomato \n\ngrowers due to the costs involved in the management \n\nof the disease and their associated environmental \n\nhazards.", "start_char_idx": 47, "end_char_idx": 3430, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "acbfa93f-d44e-4952-a9d8-6f20f7c2a318": {"__data__": {"id_": "acbfa93f-d44e-4952-a9d8-6f20f7c2a318", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "850475e4-6a07-4925-927f-f7e07ac29815", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "f492f7cc194e0b9aee0e108ec7388126e16ab598b6b396289c90abb7e42ca8aa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ebe30047-7607-47a3-bf2c-31e957449e4d", "node_type": "1", "metadata": {}, "hash": "58c2f4d09f81563add097251136451d10c47f5858d1f14700d69aa7279b98ba9", "class_name": "RelatedNodeInfo"}}, "text": "However, because SLS is relatively easy to \n\ncontrol with fungicides this disease has not been an \n\nimportant breeding priority in the past [3,4].  \n\nIt has been reported that resistance to SLS is controlled \n\nby a single dominant gene [3]. While the majority of \n\nthe source of resistance lines belongs to wild species \n\nincluding S. peruvianum, S. glandulosum and S. \n\npimpinellifolium, the highest degree of resistance was \n\nfound in S. habrochaites [1, 4]. In this study, 22 out of \n\n700 accessions, mostly from S. habrochaites and S. \n\nperuvianum, had a score of 2.0 and 3.9 when scored on \n\na scale of 0 to 9, where 0 = no disease and 9 = severe \n\ndisease. The resistance was found to be associated \n\nwith small fruit size and late maturity[5].  Useful \n\nlevels of resistance have also been found in S. pennelli, \n\nS. pimpinellifolium, S. chilense, and S. lycopersicum var. \n\ncerasiforme. Breeding lines of interspecific crossing \n\nwith S. habrochaites accessions have shown high level \n\nof resistance. However, these interspecific lines had \n\none or more undesirable horticultural traits such as \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   41              Nepjol.info/index.php/njb \n\nindeterminate growth habit, late maturity, small fruits \n\nor low yield. \n\nBreeding for SLS resistance was not a priority for \n\ntomato breeders for a long time. However, SLS has \n\nbecome a major problem in Canada and Northeastern \n\nAmerica [6,7,8] and North Carolina(NC) (Randy \n\nGardner, personal communication). The level of intensity \n\nof the disease has become so high that it may be even \n\nmore severe than early blight (Randy Gardner, personal \n\ncommunication). Because of the magnitude of the \n\nproblem, breeders at Cornell University have begun to \n\nintrogress SLS resistance into tomato breeding lines \n\nand NC State is following suit.  As discussed above, \n\nsources of SLS resistance are available but resistance is \n\nlinked with horticulturally unacceptable traits. One of \n\nthe ways to mitigate this problem is to use molecular \n\nmarkers. Molecular markers linked to the gene(s) of \n\ninterest can be used to select the plants that are \n\ngenetically similar to the recurrent parent possessing \n\nthe desired horticultural traits. However, due to lack \n\nof molecular markers linked to the SLS resistance in \n\ntomato, marker assisted selection (MAS) has not been \n\ninitiated for SLS resistance.  \n\nMichelmore et al. (1991) developed a rapid and simple \n\nPCR based method, which was called bulked \n\nsegregant analysis (BSA), to identify single genes \n\nlinked  to a trait [9]. Using this approach, they \n\nidentified random amplified polymorphic DNA \n\n(RAPD) markers linked to the downy mildew \n\nresistance gene in lettuce. For BSA, any kind of \n\nmapping population (e.g. recombinant inbred lines \n\n(RIL), backcross (BC), F2 or double haploid (DH) that \n\nare segregating for a trait of interest can be used.  \n\nMany disease resistance genes have been identified in \n\ntomato using RAPD following the BSA approach. For \n\nexample, De Giovanni et al. (2004) identified RAPD \n\nmarker linked to the ol-2 gene conferring resistance to \n\npowdery mildew using BSA in F2 population [10]. \n\nStevens et al. (1995) and Chague et al. (1996) identified \n\nRAPD markers linked to the Sw-5 gene, resistance to \n\ntomato spotted wilt virus (TSWV) [11,12]. Smiech et al.", "start_char_idx": 3431, "end_char_idx": 6873, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ebe30047-7607-47a3-bf2c-31e957449e4d": {"__data__": {"id_": "ebe30047-7607-47a3-bf2c-31e957449e4d", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "acbfa93f-d44e-4952-a9d8-6f20f7c2a318", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "128c29c9874a97107c3b0c58debcdc21694510103b70664210eef38178042a7e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a68ba52b-87eb-4601-8579-b16a89758a67", "node_type": "1", "metadata": {}, "hash": "769cf88cf1d794673834f5381ba46a4c593994c95d2a215702eefc3b40df13fc", "class_name": "RelatedNodeInfo"}}, "text": "Smiech et al. \n\n(2000) used BSA in an F2 segregating population and \n\nfound five primers that distinguished resistant and \n\nsusceptible bulks. In this study, we used BSA \n\ntechnique to identify RAPD markers linked to SLS \n\nresistance in tomato using an F2 population [13]. As \n\nexplained by Michelmore et al. (1991), this is an \n\nappropriate starting point for molecular studies of \n\ndisease like SLS in tomato [9]. \n\nMaterial and Methods  \nPlant materials  \nTwo tomato inbred lines, NC 85L-1W (2007) (referred \n\nonward as NC 85L) and NC 839-2(2007)-1 (referred \n\nonward as NC 839) were used to produce an F2 \n\npopulation in the greenhouse. NC 85L, was used as a \n\nfemale and is susceptible to SLS (susceptible parent, \n\nSP) and NC 839, was the male and is resistant to SLS \n\n(resistant parent, RP). The source of resistance in NC \n\n839 traces back to LA3707, a S. pimpinellifolium line \n\n(Randy Gardner, personal communication). A total of \n\n250 F2 plants, and 10 plants each of SP, RP and F1 were \n\ngrown at the Mountain Horticultural Crops Research \n\nand Extension Center (MHCREC), Mills River, NC. \n\nAmong F2 plants, data could not be recorded from 16 \n\nplants, which were used as missing points. Therefore, \n\nwe used observations from 234 F2 plants for data \n\nanalysis. The fruits of NC 85L were mini-roma type \n\nwith dark red color whereas NC 839 was a grape \n\ntomato with light red fruit color (Table 1).  \n\nThe NC 85L selection was made for late blight and \n\nearly blight resistance in the disease nursery at \n\nWaynesville, NC and the NC 839 selection was made \n\nat Mills River for outstanding fruit and plant type \n\nalong with SLS  resistance. \n\nField evaluation  \nSeeding was done on June 1, 2009 in 30.5 x 45.5 cm \n\ntrays containing peat moss and vermiculite. Trays \n\nwere kept in the greenhouse at an average \n\ntemperature of 21.1oC. Twelve-day old seedlings were \n\ntransplanted in a 12.7 x 24.4 cm 50-cell tray. Six-week \n\nold seedlings were transplanted in the field with silty-\n\nloam soil with a row-to-row and plant-to-plant \n\nspacing of 150 cm and 45 cm, respectively. The beds \n\nwere raised and covered with black plastic. Other \n\nrecommended cultural practices were followed as \n\ndescribed in the Southern US 2009 Vegetable Crop \n\nHandbook [14]. A total of 280 plants consisting of 10 \n\nplants each of SP, RP and F1, and 250 F2 plants were \n\nplanted in a hotspot for SLS at the MHCREC, Mills \n\nRiver, NC in summer of 2009.  \n\nData scoring and analysis \nDisease severity was scored at 60 days after \n\ntransplanting (August 17, 2009). Individual disease \n\nrating  scores  were based  on visual  assessment  of \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   42              Nepjol.info/index.php/njb \n\nTable 1. Parental description along with their partial pedigree and coefficient of parentage in the population used for tagging septoria \nleaf spot resistance gene in tomato. \n\n \n\nseverity.", "start_char_idx": 6860, "end_char_idx": 9862, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a68ba52b-87eb-4601-8579-b16a89758a67": {"__data__": {"id_": "a68ba52b-87eb-4601-8579-b16a89758a67", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ebe30047-7607-47a3-bf2c-31e957449e4d", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "40d384acf65e3462393700a82ed0c2a16fe665a2a4e1abdb69e78cde64ca9291", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "97398878-f391-4388-baae-c5f0993c53ee", "node_type": "1", "metadata": {}, "hash": "a7c0b53c0a026c49ccceb20eb19197c8093403848f70ba193ab4919309859627", "class_name": "RelatedNodeInfo"}}, "text": "severity. The following scoring criteria were \n\ndeveloped based on [15,16] and used in this study: \n\n0  = no disease symptoms  \n\n0.5 = Less than 10% leaf area with symptoms \n\n1  = 10-20% leaf area with symptoms \n\n1.5 = 20-30% leaf area with symptoms \n\n2  = 30-40% leaf area with symptoms \n\n2.5 = 40-50% leaf area with symptoms \n\n3 = 50-60% leaf area with symptoms \n\n3.5 = 60-70% leaf area with symptoms \n\n4 = 70-80% leaf area with symptoms \n\n4.5 = 80-90% leaf area with symptoms \n\n5 = 90-100% leaf area with symptoms \n\nFor the inheritance study, we grouped the \n\nsegregating plants into resistance groups with scores \n\nfrom 0 to 2, and susceptible groups with a score \n\nfrom 2 to 5. Scores of parental lines and F1 were an \n\naverage of individual plants. Frequency of different \n\nscore categories was estimated for F2 populations \n\nusing SAS v.9.1 for segregation analysis and \n\nfrequency distribution. Skewness was estimated \n\nusing SAS v.9.1. Frequency data were analyzed by \uf0632 \n\nto test the goodness of fit for a single dominant gene \n\nusing SAS v.9.1 [17].  \n\nDNA extraction, quantification and \ndilution  \nDNA was extracted from 2-3 weeks old plants \n\nfollowing the method of Fulton et al. (1995). \n\nApproximately 100 mg of young leaves from 2-3 \n\nweek old tomato seedlings were collected from the \n\ngreenhouse in 1.5 ml Eppendorf tubes.[18] The tubes \n\nwere dipped into liquid nitrogen and the samples \n\nwere ground by glass rod. After adding 200 \u00b5L \n\nmicroprep buffers, samples were incubated in a 65oC \n\nwater bath for about 60 min and filled with \n\nchloroform/isoamyl (24:1) solution. Samples were \n\nthen centrifuged at 10,000 rpm for 5 minutes. The \n\naqueous phase was pipetted out into a new micro-\n\ncentrifuge tube and 2/3 to 1 times the volume of \n\ncold isopropanol was added to precipitate the DNA. \n\nAfter centrifuging this sample at 10,000 rpm for 5 \n\nminutes, the DNA pellet remaining was separated \n\nand washed with 70% ethanol. The dry DNA pellet \n\nwas re-suspended in 100 \u00b5L of TE buffer and stored \n\nat -20oC. The concentration of DNA was determined \n\nby Nanodrop (NanoDrop 1000, Thermo Scientific, \n\nDE, USA). Working solutions of DNA samples with \n\na concentration of 20 ng/ \u00b5L were prepared from \n\noriginal DNA samples in TE buffer.  \n\nRAPD screening and Bulked \nSegregant Analysis \nA total of 197 10-mer random amplified \n\npolymorphic DNA (RAPD) primers were used to \n\nscreen parental lines using 20 ng DNA template. \n\nPrimers polymorphic to parental lines were then \n\nused to screen resistant and susceptible bulks. \n\nAmplification reactions were performed in 10 \uf06dL \n\nreaction volume containing 1x buffer (10 mM Tris-\n\nHCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 200 \uf06dM of \n\neach dNTP, 0.2 \uf06dM primer and 1 U Taq polymerase. \n\nAbout 15 \uf06dL mineral oil was overlaid on the reaction \n\nmixture. DNA amplifications were performed in \n\nthermal cycler (Eppendorf, NY) using the following \n\ncycling condition: one cycle of 92oC for 3 min; 45 \n\ncycles of 92oC for 30 seconds, 42oC for 1 min and \n\n72oC for 30 seconds; one cycle of 72oC for 8 min \n\nfollowed by holding at 4oC. \n\nBulked segregant analysis (BSA) was performed \n\nfollowing the method of Michelmore et al. (1991) [9].", "start_char_idx": 9853, "end_char_idx": 13042, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "97398878-f391-4388-baae-c5f0993c53ee": {"__data__": {"id_": "97398878-f391-4388-baae-c5f0993c53ee", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a68ba52b-87eb-4601-8579-b16a89758a67", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "72e732ca052dc95b5ecf4a06282871f0d74aaf136a82f9578ef9868cec289369", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4c55dbfb-5bac-49bc-8c78-47b58ad33723", "node_type": "1", "metadata": {}, "hash": "4b1ea30aa965db3ffac5624fd38ea0d1bb7696c8f3524202ad3f5e8cba151b1e", "class_name": "RelatedNodeInfo"}}, "text": "(1991) [9]. \n\nTwo DNA bulks, called resistant bulk (RB) and \n\nsusceptible bulk (SB) were prepared from F2 \n\nindividuals. The RB consisted of 8 individuals with \n\ndisease score of 0 and the SB contained 8 individuals \n\nwith the score of 4 or 4.5 (Figure 1). DNA bulks were \n\nParent Maturity Fruit \ncharacters \n\nSeptoria leaf \nspot reaction \n\nPedigree Common \npedigree \n\nCOP \n\nNC 85L-1W \n(2007) \n\nEarly Mini roma \ntype, dark red \n\nSusceptible NC051(x)-\n18//0463/9722(x)-18 \n\nNC0179(x)-1-\n18-4, NC215E-\n1(93), \nNC9722(x)-18, \nNC051, \nNC03220, \nLA3707 \n\n0.23 \n\nNC  839-2 \n(2007)-1 \n\nAverage Grape type, \nlight red \n\nResistant NC051(x)-18//CB25(x)-\n18-3/9722(x)-18/0464 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   43              Nepjol.info/index.php/njb \n\nprepared by pooling equal amounts of DNA of eight \n\nresistant and eight susceptible F2 individual plants \n\nfor RAPD analysis. PCR was run with polymorphic \n\nprimers on the bulked samples using the same \n\nreaction conditions as described above. PCR was \n\nrepeated for at least two times for those primers that \n\nwere polymorphic between bulks. \n\nGel electrophoresis \nAll RAPD PCR products were analyzed in 2% \n\nagarose gels containing ethidium bromide in TBE \n\nbuffer (40 mM Tris-borate, pH 8.0, 1 mM EDTA) \n\nwith a 100 bp ladder. Electrophoresis was run at 135 \n\nV for 2 hr. Gels were rinsed with water to enhance \n\ncontrast and photographed under UV light. RAPD \n\nfragments were scored as 1 for presence and 0 for \n\nabsence. Bands size was estimated based on the 100 \n\nbp DNA ladder. Simple statistics based on the DNA \n\nbands were calculated using MS Excel 2007. \n\n \n\n \n\n \nFigure 1. Frequency distribution of 234 F2 individuals derived \nfrom NC 085L-1W(2007) x NC839-2(2007)-1 based on the score of \ninfestation of septoria leaf spot in tomato at Mills River, NC in \n2009. Figure shows the bulked segregant analysis method \nadopted in this study and schematic representation of RAPD \nband linked to resistance gene. The average phenotypic values of \nthe parents and F\n\n1\n are shown by arrow. SP = Septoria leaf spot \n\nsusceptible parent, NC 85L-1W(2007). RP = Septoria leaf spot \nresistant parent, NC 839-2(2007)-1. R = Resistant. S = Susceptible. \nP = Parent. B = Bulk. \n\nResults  \nSegregation of resistance \nSeverity of SLS infestation was assessed in 234 \n\nindividual plants at 60 days after transplanting, \n\nbased on the percentage of total leaf area infected.   \n\nOnly 234 plants out of 250 were scored as some of \n\nthe plants were dead or malformed. No symptoms \n\nwere observed in the resistant parent, but the \n\nsusceptible parent had intermediate levels of SLS \n\ninfection (Figure 1). Comparing the SLS scores of F2 \n\nindividuals with their parents, it was clear that \n\ntransgressive segregation was found towards \n\nsusceptibility. The distribution of disease reaction \n\nwas highly left-skewed (Figure 1). This suggests that \n\nthe susceptible parent may also have contribution to \n\nresistance. Based on the distribution of F2 \n\nindividuals, we found two distinct groups of \n\nresistant and susceptible plants.", "start_char_idx": 13031, "end_char_idx": 16200, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4c55dbfb-5bac-49bc-8c78-47b58ad33723": {"__data__": {"id_": "4c55dbfb-5bac-49bc-8c78-47b58ad33723", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "97398878-f391-4388-baae-c5f0993c53ee", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "e0827e2d4ffcc1cf5cc294e260a11d6cb8a34c0900420cd1a1a334ed260dd8ae", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "89826c0e-302c-4ebf-a11d-79543d2cc6f2", "node_type": "1", "metadata": {}, "hash": "378747479ff4ec5635ad9d89318c93e192c5abb93ae642672ca9516a2d7065ef", "class_name": "RelatedNodeInfo"}}, "text": "This allowed us to \n\nreadily perform BSA to identify the linked markers. \n\nAbout fifty percent of the F1 plants had a disease \n\nscore of 0 indicating that resistance to SLS was \n\nincomplete dominant. Among 234 F2 individuals, \n\n164 were resistant (0-2 score) and 70 were \n\nsusceptible (2-5 score). The segregation ratio of \n\nresistant and susceptible plants fit the expected ratio \n\nof 3:1 for a single dominant gene (\uf0632 = 3.014, p > \n\n0.05) which indicated that the inheritance of \n\nresistance to SLS was based on a single dominant \n\ngene in the present study.  \n\nRAPD markers and Bulked \nSegregant Analysis \nOut of the 197 RAPD primers used to screen parent \n\nlines, 34 (17.26%) were polymorphic (Data not \n\nshown). A total of 176 bands with a maximum \n\nfragment size of 1500 bp and minimum fragment \n\nsize of 100 bp were amplified using 34 primers. \n\nAmong these fragments, 84 were polymorphic \n\nbetween parents. The 34 polymorphic RAPD \n\nprimers were used to screen the resistant and \n\nsusceptible bulks and 11 exhibited polymorphisms \n\nbetween resistant and susceptible bulks (Data not \n\nshown). A total of 87 bands were amplified by 34 \n\nRAPD primers. Among these bands, 34 and 20 \n\nbands were polymorphic between the parents and \n\nbulks, respectively. The size of bands ranged from \n\n150 to 2000 bp. Five primers were linked to SLS \n\nreaction (Table 2).  \n\nF2\n\nRB (8 individuals \nwith 0 score)\n\nSB (8 individuals \nwith 4-4.5 score)\n\nSP RP RB SB\n\nX\n\nNC 85L NC 839\n\nF1\n\nRAPD analysis\n\nBand linked to \nresistance gene\n\n93\n\n24\n19\n\n28\n20 20 19\n\n10\n1\n\n0 1 1.5 2 2.5 3 3.5 4 4.5\n\nF\nre\n\nq\nu\n\ne\nn\n\nc\ny\n\nScore of septoria leaf spot in F2 population\n\nF1 (1.1)RP SP Skewness = 2.46\nN = 234\n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   44              Nepjol.info/index.php/njb \n\nOne band of each of two primers, namely \n\nMRTOMR-121 and MRTOMR-031 (Figure 2) was \n\nfound only in the susceptible parent NC 085L and \n\nthe susceptible bulk. Similarly, one band of each of \n\nthree RAPD primers (MRTOMR-022, MRTOMR-117 \n\nand MRTOMR-121) was amplified only in the \n\nresistant parent NC 839 and the resistant bulk. \n\nAmplified band sizes linked to susceptibility were \n\n800 and 600 bp whereas those linked to resistance \n\nranged from 600 to 1000 bp (Figure 3).    \n\nSix primers were not linked to any of the loci (Figure \n\n4). These primers distinguished only the parents and \n\nnot the bulks. Some of the amplified bands were \n\nonly found in bulks but not in either parent (Figure \n\n4). This may be due to recombination in F2 \n\npopulation. \n\nTable 2. Polymorphic bands of RAPD markers linked to \n\neither resistance or susceptible genes of tomato to septoria \n\nleaf spot.", "start_char_idx": 16201, "end_char_idx": 18954, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "89826c0e-302c-4ebf-a11d-79543d2cc6f2": {"__data__": {"id_": "89826c0e-302c-4ebf-a11d-79543d2cc6f2", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4c55dbfb-5bac-49bc-8c78-47b58ad33723", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "0590ec79ae3b0f6e833cc164ef4d8f62da2304f414acba2b1b9c0bc6e75a0624", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2ad7d33d-455a-4103-98ec-42aef097acd2", "node_type": "1", "metadata": {}, "hash": "ce447ec6b4fe359967f5d3d6d458ea807e8b471ee6942ffb299c64f1a7422e11", "class_name": "RelatedNodeInfo"}}, "text": "Marker Sequence PBN Size, \nbp \n\nSP RP RB SB \n\nMRTOMR-\n022 \n  \n  \n   \n\nAGGGC\nCAGC      \n\n1 1000 0 1 1 0 \n\n2 800 0 1 0 0 \n3 600 1 0 1 1 \n7 250 1 0 1 1 \n8 150 1 0 1 1 \n\nMRTOM\nR-031  \n\nGGGAC\nGTCGC      \n\n1 1100 0 1 1 1 \n\n3 600 1 0 0 1 \n\nMRTOM\nR-117 \n\nCCGAA\nCAATC \n\n2 850 0 1 1 0 \n\nMRTOM\nR-118 \n  \n   \n\nTGCTTG\nGGGG \n\n3 800 1 0 0 0 \n\n4 750 0 1 0 0 \n5 650 1 1 1 1 \n\n6 600 0 1 1 0 \n\nMRTOM\nR-121 \n  \n  \n  \n   \n  \n\nGGCGTC\nGTAA \n\n1 1100 0 0 0 1 \n3 900 1 1 0 0 \n4 850 1 1 0 1 \n5 800 1 0 0 1 \n6 650 1 1 1 0 \n7 420 1 0 0 0 \n8 380 1 0 0 1 \n\nDiscussion  \nResistance to SLS in tomato was found to be \n\ncontrolled by a single incomplete dominant gene in \n\nthis study. Andrus and Reynard (1945) also reported \n\nthat SLS resistance was dominant and named it the \n\nSe gene.[1]  However, Wright and Lincoln. (1940)  \n\nhave reported recessive gene conferring resistance to \n\nthe SLS in the field observation in the past \n\nstudies.[19] The differences observed in the \n\ninheritance of resistance in the present study from \n\nthe  past  studies  might  be  due to  use  of  different  \n\n \nFigure 2. Electrophoresis pattern of DNA fragments generated by \n\nRAPD markers (A. MRTOMR-031, B. MRTOMR-121). \n\n Polymorphic band (i.e. linked to susceptible) between parents, \n\nand between resistant and susceptible bulks are indicated by \n\narrow. SP = Susceptible parent, NC 085L. RP = Resistant parent, \n\nNC 839. RB = Resistant bulk. SB = Susceptible bulk. M = Marker. \n\n \n\n \n \n\nFigure 3. Electrophoresis pattern of DNA fragments generated by \n\nRAPD marker (A. MRTOMR-022, B. MRTOMR-117 and C. \n\nMRTOMR-118). Polymorphic band (i.e. linked to resistance) \n\nbetween parents and between resistant and susceptible bulks are \n\nindicated by arrow. SP = Susceptible parent, NC 085L. RP = \n\nResistant parent, NC 839. RB = Resistant bulk. SB = Susceptible \n\nbulk. M = Marker. \n\nsources of resistance. The susceptible parent used in \n\nthis study did not appear completely susceptible \n\nsuggesting that there may be its allelic difference in \n\nthe expression of resistance. In fact both parents, NC \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   45              Nepjol.info/index.php/njb \n\n \nFigure 4. RAPD marker (A. MRTOMR-130) showing polymorphic \n\nband (indicated by arrow) only to parents, i.e. band with \n\nunlinked loci and RAPD marker (MRTOMR-146) showing band \n\n(indicated by arrow) only in two bulks. SP = Susceptible parent, \n\nNC 085L. RP = Resistant parent, NC 839. RB = Resistant bulk.", "start_char_idx": 18957, "end_char_idx": 21504, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2ad7d33d-455a-4103-98ec-42aef097acd2": {"__data__": {"id_": "2ad7d33d-455a-4103-98ec-42aef097acd2", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "89826c0e-302c-4ebf-a11d-79543d2cc6f2", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "92ffab90a2b6eb92be3416513295b8e565d1e2a27ad55a25e9ba00abe4bdd91a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ce1d2cf9-05ed-40d0-99c8-2b11265f9ee7", "node_type": "1", "metadata": {}, "hash": "f330bc4d581a792632ebb0d46994430b1e83827d727c7ead468e989105f19b5f", "class_name": "RelatedNodeInfo"}}, "text": "RP = Resistant parent, NC 839. RB = Resistant bulk. SB \n\n= Susceptible bulk. M = Marker \n\n85L and NC 839 have a coefficient of parentage \n\n(COP) of 0.23 (Table 1) indicating that they have \n\ncommon parentage. This fact has been confirmed \n\nbased on their common pedigree (Randy Gardner, \n\npersonal communication).  \n\nBased on the field screening of the F2 population \n\nwith 197 RAPD primers, we identified three RAPD \n\nmarkers linked to resistance alleles and two RAPD \n\nmarkers linked to susceptible alleles. Through the \n\nbulking of the extreme individuals segregating in \n\nthe F2 population we were able to rapidly tag the \n\nmarkers associated with chromosomal segment that \n\nhas a role in reaction to SLS in tomato. For BSA \n\nconsisting of eight individuals in each bulk, five \n\nprimers yielded different banding patterns, which \n\nwere useful markers in SLS screening in tomato. \n\nBands of two of these markers were only present in \n\nsusceptible parent and bulk, and bands of three \n\nmarkers were present only in resistant parent and \n\nbulk. Therefore, these bands were considered \n\nassociated either susceptible allele or resistant allele. \n\nTagging of resistance genes using BSA is very fast, \n\nwhich facilitates the screening of new alleles of \n\nresistance for a particular disease, especially for one \n\nthat does not have background information \n\navailable such as SLS in tomato. The two parental \n\nlines used in this study are closely related to each \n\nother (COP=0.23). However, we found RAPD to \n\ndistinguish these parents at the molecular level. \n\nRAPDs are multi locus-based markers. Therefore, \n\nthe primers identified might be from the same \n\nregions of the chromosome. For example, \n\nMRTOMR-022 produced a 1000 bp band and \n\nMRTOMR-118 produced a 600 bp band. The band \n\nproduced by MRTOMR-118 might be the part of the \n\nband produced by MRTOMR-022. The \n\ndisadvantages associated with RAPDs include the \n\nfact that they anneal in multiple sites, and they are \n\ndominant in nature, and sensitive to reaction \n\nconditions, which may limit their use directly in \n\nMAS. Therefore, these RAPD markers need to be \n\nconverted to sequence characterized amplified \n\nregion (SCAR) or cleaved amplified polymorphic \n\nsequence (CAPS), which are much more useful for \n\nMAS.   \n\nThrough BSA, marker development and MAS has \n\nbeen used for the selection of resistance to a number \n\nof diseases in tomato. For example, De Giovanni et \n\nal. (2004) identified RAPD marker linked to the ol-2 \n\ngene conferring resistance to powdery mildew.[10] \n\nA single RAPD marker, OPU31500 with 1500 bp in \n\nsize was detected in the susceptible bulk, which was \n\nconverted into a CAPS marker. Stevens et al. (1995) \n\nand Chague et al. (1996) identified RAPD markers \n\nlinked to the Sw-5 gene conferring resistance to \n\ntomato spotted wilt virus (TSWV).[11][12] Among \n\nthe four RAPD markers, two were tightly linked to \n\nSw-5 gene. Linkage analysis mapped these markers \n\nwithin a distance of 10.5 cM from Sw-5. Czech et al. \n\n(2003) have used MAS using a co-dominant marker \n\nthrough BSA for developing TSWV resistant \n\ntomato.[20] Smiech et al. (2000) used BSA in F2 \n\nsegregating population and found 5 primers that \n\ndistinguished resistant and susceptible for TSWV. \n\n[13] A PCR-based co-dominant marker, tightly \n\nlinked to Mi was developed using the information \n\nfrom BSA [21](Williamson et al. 1994). In light of \n\nthese past reports, the five RAPD primers identified \n\nin the present study may be informative to develop \n\nco-dominant markers for SLS resistance breeding.", "start_char_idx": 21453, "end_char_idx": 25018, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ce1d2cf9-05ed-40d0-99c8-2b11265f9ee7": {"__data__": {"id_": "ce1d2cf9-05ed-40d0-99c8-2b11265f9ee7", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2ad7d33d-455a-4103-98ec-42aef097acd2", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "20d507fac5dbfd6932c1abfe1c0c78e31367ac135e51fece965834a315d0225f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "438bd900-40fc-4f5a-992a-3d246a8f0079", "node_type": "1", "metadata": {}, "hash": "5ae30de3d83096b25cfeeb92589525fe58fa2d7354d54a82cd43fc60857f2871", "class_name": "RelatedNodeInfo"}}, "text": "RAPD markers identified here needs to convert into \n\nSCAR or CAPS marker for MAS of resistance to SLS \n\nin tomato. The MAS is cost effective and more \n\nreliable for screening, because it does not need to \n\nhave a pathological evaluation and can genotype at \n\nany growth stage. Molecular markers linked to the \n\nSLS resistance in tomato may also have a potential \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   46              Nepjol.info/index.php/njb \n\nrole on gene pyramiding. To our knowledge, there \n\nare no any molecular markers reported associated \n\nwith SLS resistance in tomato. Molecular markers \n\nidentified in this study are novel, and provide \n\nenough background to develop different group of \n\nmarkers (SCAR or CAPS) which may be useful for \n\nspeeding up the tomato breeding program aiming to \n\nimprove SLS resistance.   \n\nREFERENCES \n\n1. Andrus CF , Reynard GB: Resistance to septoria \n\nleaf spot and its inheritance in tomatoes. \n\nPhytopathology 1945,35:16-24. \n\n2. Delahaut K , Stevenson W: Tomato disorders: \n\nEarly blight and septoria leaf spot. The \n\nUniversity of Wisconsin  2004, Madison, WI \n\n3. Barksdale TH , Stoner AK: Resistance in tomato \n\nto Septoria lycopersici. Plant Disease Reporter \n\n1978,62:844-847. \n\n4. Locke SB: Resistance to early blight and \n\nseptoria leaf spot in the genus Lycopersicon. \n\nPhytopathology 1949,39:829-836. \n\n5. Poysa V and Tu JC: Response of cultivars and \n\nbreeding lines of Lycopersicon spp. to Septoria \n\nlycopersici. Canadian Plant Disease Survey 1993, \n\n73:9-13. \n\n6. Mutschler MA, SM Zitter, D Dejong, TA Zitter \n\nand K Ivors: Performance of hybrids \n\ncombining genetic control to early blight and \n\nlate blight with and without resistance to \n\nSeptoria leaf spot.  Tomato Disease Workshop, \n\n2011, Ithaca, NY. \n\n7. Zitter TA, SM Zitter and MA Mutschler: \n\nComparing the performance of early blight \n\nand Septoria leaf spot resistant materials in the \n\npresence and absence of fungicides.  Tomato \n\nDisease Workshop, 2011a, Ithaca, NY. \n\n8. Zitter TA, SM Zitter, MA Mutschler and SP \n\nMckay: Using host resistance and reduced-risk \n\nfungicides to control early blight and Septoria \n\nleaf spot on tomato, 2010.  Plant Disease \n\nManagement Report, 2011b, Cornell University. \n\n9. Michelmore RW, Paran I and  Kesseli RV: \n\nIdentification of markers linked to disease-\n\nresistance genes by bulked segregant analysis: \n\nA rapid method to detect markers in specific \n\ngenomic regions by using segregating \n\npopulations. Proceedings of National Academic of \n\nSciences, USA 1991,88:9828-9832. \n\n10. De Giovanni C, Orco P Dell', Bruno A, Ciccarese \n\nF, Lotti C and Ricciardi L. Identification of \n\nPCR-based markers (RAPD, AFLP) linked to a \n\nnovel powdery mildew resistance gene (ol-2) \n\nin tomato. Plant Science 2004, 166:41-48. \n\n11. Stevens MR, Lamb EM and Rhoads DD:Mapping \n\nthe Sw-5 locus for tomato spotted wilt virus \n\nresistance in tomatoes using RAPD and RFLP \n\nanalysis. Theoretical and Applied Genetics \n\n1995,90:451-456. \n\n12.", "start_char_idx": 25021, "end_char_idx": 28108, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "438bd900-40fc-4f5a-992a-3d246a8f0079": {"__data__": {"id_": "438bd900-40fc-4f5a-992a-3d246a8f0079", "embedding": null, "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-35", "node_type": "4", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "4d766cef4729ba6a979b79f4512cf4867dc9e5153b909a357b25335150ffd6dc", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ce1d2cf9-05ed-40d0-99c8-2b11265f9ee7", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "7cc088c5358d0ca3d94b31582c360f65a75925e0f21f34b953d090103ba5bce8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "71130145-75f6-4f3c-ad2c-eef6772671eb", "node_type": "1", "metadata": {}, "hash": "30ffe719c8f9ece59da0ae776e9ef5b9165f4a4b927ddcefcd2df43845ffbafa", "class_name": "RelatedNodeInfo"}}, "text": "Theoretical and Applied Genetics \n\n1995,90:451-456. \n\n12. Chague V, Mercier JC, Guenard M, Decourcel A \n\nand Vedel F:Identification and mapping on \n\nchromosome 9 of RAPD markers linked to Sw-\n\n5 in tomato by bulked segregant analysis. \n\nTheoretical and Applied Genetics  1996: 92:1045-\n\n1051. \n\n13. Smiech M, Rusinowski Z, Malepszy S and \n\nNiemirowicz-Szczytt K: New RAPD markers of \n\ntomato spotted wilt virus (TSWV) resistance in \n\nLycopersicon esculentum Mill. Acta \n\nPhysiologiae Plantarum 2000, 22:299-303. \n\n14. Kemble JM (ed):Southeastern Vegetable Crops \n\nHandbook. Auburn University, Auburn, AL \n\n2009. \n\n15.  Tu JC and Poysa V: A brushing method of \n\ninoculation for screening tomato seedlings for \n\nresistance to Septoria lycopersici. Plant Disease \n\n1990, 74:294-297. \n\n16. Winstead NN , Kelman A: Inoculation technique \n\nfor evaluating resistance to Pseudomonas \n\nsolanacearum. Phytopathology 1952, 42:628-634. \n\n17. Sas Institute Inc:The SAS System, version 9.1.3 for \n\nwindows, 9th ed. SAS Institute, Cary, NC 2007. \n\n18. Fulton TM, Chunwongse J and Tanksley SD: \n\nMicroprep protocol for extraction of DNA from \n\ntomato and other herbaceous plants. Plant \n\nMolecular Biological Reporter 1995,13:207-209. \n\n19. Wright V and Lincoln RE:Resistance to \n\ndefoliation diseases of tomato. Purdue \n\nAgricultural Experiment Station 1940, pp 42-43. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:40-47        Jhoshi et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   47              Nepjol.info/index.php/njb \n\n20. Czech AS, Szklarczyk M, Gajewsk Z, Zukowska \n\nE, Michalik B, Kobylko T and K \n\nStrzalka:Selection of tomato plants resistant to a \n\nlocal Polish isolate of tomato spotted wilt virus \n\n(TSWV). Journal of Applied Genetics 2003, 44:473-\n\n480. \n\n21. Williamson VM, Ho JY, Wu FF, Miller N and \n\nKaloshian I: A PCR-based marker tightly linked \n\nto the nematode resistance gene, Mi, in tomato. \n\nTheoretical and Applied Genetics 1994, 87:757-763.", "start_char_idx": 28051, "end_char_idx": 30034, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "71130145-75f6-4f3c-ad2c-eef6772671eb": {"__data__": {"id_": "71130145-75f6-4f3c-ad2c-eef6772671eb", "embedding": null, "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-36", "node_type": "4", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "ac51228a94975b02a2acc8a4693ab889e7b156e2f0c49679cb0483dcdffa2541", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "438bd900-40fc-4f5a-992a-3d246a8f0079", "node_type": "1", "metadata": {"identifier": "njb-35", "author": "Joshi, Bal K; Louws, Frank J; Yenco, G Craig; Sosinski, Byron R; Arellano, Consuelo; Panthee, Dilip R", "title": "Molecular Markers for Septoria Leaf Spot (Septoria lycopersicii Speg.) Resistance in Tomato (Solanum lycopersicum L.)", "date": "2015-12-30", "file": "njb-35.pdf"}, "hash": "fe4142f961527eb08ceb5b363740f380cee4cadfc1ab5519b88abf89f624701a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1a39b19b-1648-4dae-8c0b-f872cd9cf67a", "node_type": "1", "metadata": {}, "hash": "be0c67f95ddffcc7b16aa37e3058fbef1ab36dee9cc247b5080ffd7e1ac31a6a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:35-39  ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   35      Nepjol.info/index.php/njb \n\nMarker Assisted Screening of Nepalese Rice for Bacterial Leaf \nBlight (BLB) Resistance \n\nResham Babu Amgai1*, Raj Kumar Niroula1, Sumitra Pantha2, Shreya Singh Hamal1, Bishal Gole Tamang4, \n\nBindeshwar Prasad Sah1, Madan Raj Bhatta3 \n1Biotechnology Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal \n\n2Agriculture Botany Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal \n3National Plant Genetic Resource Centre, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal \n\n4Institute of Agriculture and Animal Science, Tribhuwan University, Rampur, Chitwan, Nepal \n\nAbstract \nBacterial Leaf Blight (BLB) is the most important yield limiting factor in Nepalese rice. BLB resistance \nrice varieties are highly demanding in the country. Breeding efforts for developing disease resistant \ndepends on availability and use of resistant gene donors. Nepalese rice landraces could be the source of \nresistant gene. Therefore, ninety six Nepalese rice accessions were screened using eight Simple Sequence \nRepeats (SSR) markers and one Sequence Tagged Sites (STS) marker for presence and absence of BLB \nresistance gene. We have detected BLB resistance gene Xa-10 on five accessions, Xa-13 on six accessions, \nXa-7 on 23 accessions, Xa-3 and Xa-4 on 52 accessions, Xa-5 on 25 accessions, Xa-8 on 30 rice accessions. \nNo any rice accessions tested have Xa-21. Similarly, 17 rice accessions showed three and more than three \nBLB resistance genes. Presence of Xa-13 on susceptible check variety CNTRL-85033 confirmed that this \nresistant gene is not working in Nepalese rice field. Therefore, Nepal need to pyramide the BLB resistant \ngenes for durable resistance. \n\nKeywords: Bacterial Leaf Blight, Simple Sequence Repeats, resistant gene, Nepalese rice, Marker \n\nAssisted Screening  \n\n*Correspondence Author  \n\nEmail: reshamamgain@yahoo.com \n\nIntroduction \nBacterial Leaf Blight (BLB) is one of the productions \n\nlimiting biotic stresses in rice. It is caused by \n\nXanthomonas oryzae pv. oryzae (Xoo). It can reduce \n\nthe yield up to 50% [1] and in Nepal; it reduced the \n\nyield from 5-60 % in Terai and mid-hills during hot \n\nand humid periods [2].  \n\nTwenty four genes conferring resistance to BLB have \n\nbeen identified through classical genetic analysis [3] \n\nand 10 out of 24 genes have been mapped using \n\nRestriction Fragment Length Polymorphism (RFLP), \n\nRapid Amplified Polymorphic DNA (RAPD) and \n\nmicrosatellite markers [4-7]. Among them, 6 genes are \n\nrecessive in nature [1]. Similarly, two new genes Xa-22 \n\nin rice variety Zha-Chang-long [8] and Xa-23 in Oryza \n\nrufipogon [9] were identified and mapped on different \n\nchromosomes. Most common BLB resistant gene used \n\nin rice breeding and BLB screening worldwide are Xa-\n\n1, Xa-2, Xa-3,  Xa-4, Xa-5, Xa-7, Xa-8, Xa-10, Xa-11, Xa-\n\n13, Xa-14 and Xa-21 [10]. \n\nBLB resistance rice varieties are highly demanded \n\nworldwide.", "start_char_idx": 47, "end_char_idx": 3204, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1a39b19b-1648-4dae-8c0b-f872cd9cf67a": {"__data__": {"id_": "1a39b19b-1648-4dae-8c0b-f872cd9cf67a", "embedding": null, "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-36", "node_type": "4", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "ac51228a94975b02a2acc8a4693ab889e7b156e2f0c49679cb0483dcdffa2541", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "71130145-75f6-4f3c-ad2c-eef6772671eb", "node_type": "1", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "f4e730e67afd242ccb726edc04e39906d763f5ef007a68faa1a2fabe7dd20fab", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "20762275-7095-41f0-9738-4aeef5d79da5", "node_type": "1", "metadata": {}, "hash": "b6e5de02f9138f319d06ebd9a0196fcb982d44fcedfe9f01d9c59fccea0db001", "class_name": "RelatedNodeInfo"}}, "text": "BLB resistance rice varieties are highly demanded \n\nworldwide. However, the continuous evolution of \n\npathogenic races leading to the breakdown of \n\nresistance in many improved varieties [9]. \n\n Thus, success of resistance breeding program \n\ndepends on the availability of the resistant donors. \n\nSimilarly, pyramiding different resistant genes in a \n\nsingle rice variety will increase the resistance. \n\nHowever, two or more resistance gene pyramiding in \n\na single variety is easy through molecular marker \n\nassisted selection  \n\n(MAS), and identification for the presence and absence \n\nof particular gene in a variety for MAS as donor and \n\nrecipient parent through molecular marker assisted \n\nscreening is very fast, reliable and cheaper. Therefore, \n\nthis study was carried out to identify the accessions \n\nwithin Nepalese gene pool with the potential of BLB \n\nresistance genes.", "start_char_idx": 3142, "end_char_idx": 4024, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "20762275-7095-41f0-9738-4aeef5d79da5": {"__data__": {"id_": "20762275-7095-41f0-9738-4aeef5d79da5", "embedding": null, "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-36", "node_type": "4", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "ac51228a94975b02a2acc8a4693ab889e7b156e2f0c49679cb0483dcdffa2541", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1a39b19b-1648-4dae-8c0b-f872cd9cf67a", "node_type": "1", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "e62f25018f85a96aba546982cec462849cc0e7104838315bdf9789edc4f909b0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7f7da86c-fc29-4751-a6d2-db40116c3c15", "node_type": "1", "metadata": {}, "hash": "0ae56f3fe7fc9b9d96b28539c298c264fe586958fd8c0511f607b497a269ee99", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:35-39        Amgai et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   36      Nepjol.info/index.php/njb \n\nMethods \nGermplasm Collection \nSeventy Nepalese rice accessions (NPGR No.s) \n\ncollected from Terai region of Nepal were obtained \n\nfrom National Plant Genetic Resources Centre \n\n(NPGRC); and five breeding lines (NR series) and \n\nreleased rice varieties (Chandannath-1, Chandannath-\n\n3, Chhomrong, Macchapuchre-3, Manjushree-2, \n\nPalung-2 and Taichung-176) from Agriculture Botany \n\nDivision of Nepal Agricultural Research Council \n\n(NARC). Similarly, rice varieties IR-64, Sabitri and \n\nMasuli, and breeding line CNTRL-85033; Terai rice \n\nlandraces Bagari, Bhatti, Karma, Lal Tenger and \n\nParewa Pankha; and Hill rice landraces Belkuti,  \n\nGerneli, Jumli Marshi and Seto Anadi were collected \n\nfrom different parts of Nepal (Table 1). \n\nMolecular Marker and check variety \nEight SSR markers and one STS marker (pTA248) for \n\nthe presence and absence of BLB resistance gene. \n\nMolecular markers are selected based on their linkage \n\nwith particular BLB resistance gene (Table 2). IR-64 \n\nand CNTRL-85033 were used as resistant and \n\nsusceptible check respectively. \n\nDNA extraction, PCR reaction and data \nanalysis \nGenomic DNA of rice accessions was prepared using \n\nmodified CTAB method as described by Sul and \n\nKorban [11]. Each PCR reaction was conducted \n\nwith100ng of genomic DNA, 1 \u00b5M of each primer and \n\n7.5 \u00b5l of 2x GoTaqGreen PCR Master Mix (Promega \n\nCorporation, Madison, WI, USA). PCR mixture was \n\namplified in MJ Research PTC-100TM Programmable \n\nThermal Controller (MJ Research, Inc, Watertown, \n\nMA, USA) with the following temperature regimes: \n\ninitial denaturation for 2 min at 95oC followed by 33 \n\ncycles of 95oC for 30 sec, annealing as per primer for 1 \n\nmin, extension at 72oC for 2 min and final extension at \n\n72oC for 7 min followed by holding at 4oC as \n\ndescribed on Table 2 and Gramene [12]. \n\nAmplified PCR products were separated in 2% \n\nanalytical grade agarose gel at 100V for 1H. Gels were \n\nstained with 0.1 \u00b5g/ml ethidium bromide (Promega \n\nCorporation, Madison, WI, USA) and then visualized \n\nunder UV transilluminator gel documentation system \n\n(Wilber Lourmat, Marne-La-Valleen, France) using 1 \n\n\u00b5g guide size DNA ladder (Genetix, Biotech Asia Pvt. \n\nLtd.). The presence and absence of particular band size \n\nwas scored for screening disease resistance genes.  \n\nResults and Discussion \nDifferent resistance genes were identified in Nepalese \n\nrice germplasm as defined by different molecular \n\nmarkers (Table 3). We identified BLB resistance gene \n\nXa-10 on two accessions, Xa-13 on six accessions, Xa-7 \n\non 23 accessions, Xa-10 on five accessions, Xa-3 and \n\nXa-4 on 52 rice accessions (Figure 2), Xa-5 on 25 \n\naccessions, Xa-8 on 30 rice accessions. No any rice \n\naccessions have Xa-21 (Figure 1). Similarly, 17 rice \n\naccessions showed three and more than three BLB \n\nresistance genes (Table 4).", "start_char_idx": 4029, "end_char_idx": 7043, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7f7da86c-fc29-4751-a6d2-db40116c3c15": {"__data__": {"id_": "7f7da86c-fc29-4751-a6d2-db40116c3c15", "embedding": null, "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-36", "node_type": "4", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "ac51228a94975b02a2acc8a4693ab889e7b156e2f0c49679cb0483dcdffa2541", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "20762275-7095-41f0-9738-4aeef5d79da5", "node_type": "1", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "701b059fdab427ac20af43aa565ec635565970f4338a703121c9072e78e9ab1a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ee006858-95ac-44d3-a465-2f1a8a152f66", "node_type": "1", "metadata": {}, "hash": "cfc7c5c786cef7991a244b1c1dcd95f41eb129d7eea4235153b86df18c8988b4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:35-39        Amgai et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   37      Nepjol.info/index.php/njb \n  \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:35-39        Amgai et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   38      Nepjol.info/index.php/njb \n\nNepalese rice accessions lack the Xa-21 genes which is \n\nmore important for controlling BLB epidemics \n\nthroughout the world. Kameshwara Rao [16] reviewed \n\nand noted that Xa-21 gene is transferred from O. \n\nlongistaminata and integrated into some IRRI \n\ndeveloped rice varieties. Lacks of such varieties in our \n\nstudy may be the result for this. Similarly, our \n\nsusceptible check \u2018CNTRL-85033\u2019 showed the \n\npresence of Xa-13 but Amgai [17] reported that it was \n\nheavily infected by BLB at Nepalese rice field. This \n\nmay be due to the difference on BLB isolated found in \n\nNepal which may break the resistance reaction \n\ndeveloped by Xa-13 gene. Resistant check IR-64 \n\nshowed the presence of Xa-5 indicating that it is \n\neffective for Nepalese BLB pathogen. \n\nConclusion \nNepalese rice landraces contains many marker alleles \n\nfor different BLB resistant genes. The rice landraces \n\nwith effective resistant gene can be used as donor \n\nparent for MAS. However, for the enhancement of \n\nresistance in Nepalese rice with absence and/or \n\nineffective resistance gene can be done by transferring \n\nbroad spectrum resistance gene like Xa-21. BLB \n\nresistance is also affected by multiple resistance genes \n\nand their interaction [17]. Therefore, pyramiding BLB \n\nresistance gene on Nepalese rice variety is most \n\nimportant for durable resistance with BLB. \n\nAcknowledgement \nThis work was conducted under Global Biodiversity \n\nTrust Grant No. GS10027. \n\nReferences \n1. Rao KK, Lakshminarasu M, Jena KK: DNA markers and \n\nmarker-assisted breeding for durable resistance to \n\nbacterial blight disease in rice. Biotech. Adv. 2002, 20: 33\u2013\n\n47. \n\n2. Burlakoti RR, Khatri-Chhetri GB: Bacterial Diseases of \n\nCrop Plants in Nepal-A Review. J. Inst. Agric. Anim. Sci., \n\n2005 25:1-10. \n\n3. Lee KS, Angeles ER, Khush GS: Inheritance of resistance \n\nto race 6 of bacterial blight in rice. Rice Genet News Letter \n\n2001, 17:73\u20134. \n\n4. McCouch, SR, Tanksley SD: Genetic and physical analysis \n\nof the rice bacterial blight disease resistance locus Xa 21. \n\nMol. Gene. Genet. 1992, 236: 113\u2013120. \n\n5. Ronald PC, Albano B, Tabien R, Abenes L, Wu K, McCouch \n\nSR, Tanksley SD: Genetic and physical analysis of the \n\nbacterial blight disease resistance locus, Xa-21. Mol. Gen. \n\nGenet. 1992, 236:113\u2013120. \n\n6. Borines LM, VeraCruz CM, Redona ED, Hernandez JF, \n\nNatural MP, Raymundo AD and Leung H: Marker-aided \n\npyramiding of bacterial blight resistance genes in \n\nmaintainer lines for hybrid rice production. IRRI \n\nConference Abstract 2000, 4(2):162. \n\n7. Lang NT, Buu BC: Molecular genetic analysis and marker \n\nassisted selection for restorer line and bacterial blight \n\nresistance in hybrid rice.", "start_char_idx": 7049, "end_char_idx": 10085, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ee006858-95ac-44d3-a465-2f1a8a152f66": {"__data__": {"id_": "ee006858-95ac-44d3-a465-2f1a8a152f66", "embedding": null, "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-36", "node_type": "4", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "ac51228a94975b02a2acc8a4693ab889e7b156e2f0c49679cb0483dcdffa2541", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7f7da86c-fc29-4751-a6d2-db40116c3c15", "node_type": "1", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "efb71833769f5df28d92b6d44bb2a6c5b1290ba8a2baf463c563261b68d022bb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1ca41f7b-2b08-489f-b66b-2cbe92b6d3a1", "node_type": "1", "metadata": {}, "hash": "49dabc24773e6e0b1f7318a76eb9ff36ccc9f43dd3aebe264f952497754c0c53", "class_name": "RelatedNodeInfo"}}, "text": "Sabrao 2004, 36 (2): 83-93. \n\n8. Gao HP, Lin XH, Yu GX, Zhang DP, Xie YF: Inheritance of \n\nresistance to bacterial blight of four Yunan rice varieties. \n\nRice Genet Newsl. 1999, 12: 233\u2013234. \n\n9. Phuc, NV, Lang NT, Buu BC: STS and microsatellite \n\nmarker-assisted selection for Bacterial Blight Resistance \n\nin rice Oryza sativa L. Omonrice 2005 13: 18-25. \n\n10. IRRI, 2012.  Disease Resistance BB Blast ver 5.0. Host Plant \n\nResistance to Diseases Group (HPR-D). Plant Breeding, \n\nGenetics and Biotechnology Division (PBGB). International \n\nRice Research Institute (IRRI), the Philippines. 2012 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:35-39        Amgai et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   39      Nepjol.info/index.php/njb \n\n11. Sul IW, Korban SS: A highly efficient method for \n\nisolating genomic DNA from plant tissues. Plant Tiss. \n\nCult. Biotech. 1996, 2: 113-116. \n\n12. Gramene. 2015. http://www.gramene.org/ (accessed 30 \n\nApril 2015). \n\n13. Davierwala  AP, Reddy APK, Lagu MD, Ranjekar PK, \n\nGupta VS: Marker assisted selection of bacterial blight \n\nresistance genes in rice. Biochemical Genetics 2001, 39: 261-\n\n278. \n\n14. Pha, NT, Lang NT: Marker assisted selection in rice \n\nbreeding for bacterial leaf blight. Omonrice 2004 12: 19-26. \n\n15. Blair MW, McCouch SR: Microsatellite and sequence-\n\ntagged site markers diagnostic for the rice bacterial leaf \n\nblight resistance gene Xa-5. Theoretical Applied Genetics \n\n1997, 95:174-184. \n\n16. Kameshwara Rao, K, Lakshminarasu M, Jena KK: DNA \n\nmarkers and marker-assisted breeding for durable \n\nresistance to bacterial blight disease in rice. \n\nBiotechnology Advances 2002, 20: 33-47. \n\n17. Amgai RB, Parajuli GP, Khan AH, Poudel B, GM PB, GC \nCB, Bhattarai EM, Pandey YR: Eco-friendly seed \n\ntreatment procedures in rice - identification and \n\ndissemination for western hill of Nepal. In \n\nIdentification and dissemination of eco-friendly seed \n\ntreatment procedures in rice. Final Technical Report for \n\nNARDF PP 408/2006/07. Kaski: Nepal Agriculture \n\nResearch Council Regional Agricultural Research Station \n\nLumle 2009: 2-11. \n\n18. Zhou Y, Cao Y, Huang Y, Xie W, Xu C, Li X, Wang S: \nMultiple gene loci affecting genetic background-\n\ncontrolled disease resistance conferred by R gene \n\nXa3/Xa26 in rice. Theoretical Applied Genetics 2009 < DOI \n\n10.1007/s00122-009-1164-5> \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n.", "start_char_idx": 10086, "end_char_idx": 12568, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1ca41f7b-2b08-489f-b66b-2cbe92b6d3a1": {"__data__": {"id_": "1ca41f7b-2b08-489f-b66b-2cbe92b6d3a1", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ee006858-95ac-44d3-a465-2f1a8a152f66", "node_type": "1", "metadata": {"identifier": "njb-36", "author": "Amgai, Resham Babu; Niroula, Raj Kumar; Pantha, Sumitra; Hamal, Shreya Singh; Tamang, Bishal Gole; Sah, Bindeshwar Prasad; Bhatta, Madan Raj", "title": "Marker Assisted Screening of Nepalese rice for bacterial leaf blight (BLB) resistance", "date": "2015-12-30", "file": "njb-36.pdf"}, "hash": "5c85adafcdb252b037a52ded68ba8f3c19e41c533cc33f5abbf23bd3df0ecb9d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a36e2d0d-7f5d-4400-a543-66f21749b26b", "node_type": "1", "metadata": {}, "hash": "e49a0971e45a79ab7e8538925bd244111a23cebd2bb090e366269225f8a14269", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   29    Nepjol.info/index.php/njb \n\nIsolation of Yeast from Soil and Different Food Samples and Its \nCharacterization Based on Fermentation \n\nSandeep Thapa*, Rajani Shrestha, Anjali Tibrewal, Arjun Sharma, Yuvraj K.C. \nCollege for Professional Studies, Department of Biotechnology, Kathmandu, Nepal \n\nAbstract \nYeasts are eukaryotic microorganisms which can also be used for bioethanol production. In this modern era of \n\nincreasing demand for energy and fuel, the microbial biosynthesis of ethanol has gained importance. In this study, the \n\npotential yeasts for ethanol production from pentose and hexose sugars were identified. Yeasts were isolated from soil \n\nand different food samples. They were identified and characterized based on cell morphology (e.g., mode of cell \n\ndivision and spore shape) and physiology (e.g., sugar fermentation tests). Furthermore, quantification of ethanol and \n\ncell concentration was performed throughout the fermentation. Spot plate count method was followed to determine \n\nthe viable yeast count whereas Potassium dichromate oxidation method was used for determining the ethanol \n\nconcentration. Six different species of yeasts were cultured in three sets of broth for 24, 48, 72, and 96 hours for \n\nbioethanol production. The yeasts isolated from black and green grapes relatively synthesized higher concentration of \n\nethanol.   \n\nKey words:  Yeast, Fermentation, Ethanol, Potassium Dichromate Oxidation Method, YEPDA. \n\n*Corresponding author:  \n\nEmail: sandipthapa_29@kathmandugenomics.com \n\nIntroduction \nYeasts are single-celled microorganisms that are \n\nclassified, along with molds and mushrooms, as \n\nmembers of the Kingdom Fungi [1, 2]. Although \n\nyeasts are unicellular organisms, they possess a \n\ncellular organization similar to that of higher \n\norganisms, including humans. Specifically, their \n\ngenetic content is contained within a nucleus. This \n\nclassifies them as eukaryotic organisms, unlike \n\ntheir single-celled counterparts, bacteria, which do \n\nnot have a nucleus and are considered \n\nprokaryotes [3, 4]. \n\nYeast is widely dispersed in nature with a wide \n\nvariety of habitats. They are commonly found on \n\nplant leaves, flowers, and fruits, as well as in soil. \n\nYeasts are also found on the surface of the skin \n\nand in the intestinal tracts of warm-blooded \n\nanimals, where they may live symbiotically or as \n\nparasites. Yeasts are responsible for several types \n\nof infections including oral thrush, vaginitis, \n\nurinary tract infection, endocarditis, respiratory \n\nsyndromes, meningitis, etc. The common \"yeast \n\ninfection\" is typically caused by Candida albicans.  \n\nBeside infections, yeast is very useful in \n\ncommercial application. Yeast has long been \n\nconsidered to be the organism of choice for the \n\nproduction of alcoholic beverages, bread, and a \n\nlarge variety of industrial products [1, 5, 6]. This is \n\nbased on the ease with which the metabolism of \n\nyeast can be manipulated using genetic \n\ntechniques, the speed with which it can be grown \n\nto high cell yields (biomass), the ease with which \n\nthis biomass can be separated from products and \n\nthe knowledge that it is generally recognized as \n\nsafe (GRAS). \n\nThe budding yeast, Saccharomyces cerevisiae and \n\nother yeast species have long been used to ferment \n\nthe sugars of rice, wheat, barley, and corn for the \n\nproduction of alcoholic beverages such as beer \n\nand wine [7,8]. There are two major types of \n\nbrewing yeast, top-fermenting ale yeast and \n\nbottom-fermenting lager yeast.", "start_char_idx": 47, "end_char_idx": 3762, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a36e2d0d-7f5d-4400-a543-66f21749b26b": {"__data__": {"id_": "a36e2d0d-7f5d-4400-a543-66f21749b26b", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1ca41f7b-2b08-489f-b66b-2cbe92b6d3a1", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "61c9ad056ecb214929289a9e62b91be613876cb6e8849a3323ef2817f1c0a98f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4cc74cef-02d1-4857-846b-cbe57940955b", "node_type": "1", "metadata": {}, "hash": "1701111d1e1c18e4708f6f3a83b301a6b6a0ceb52a02bc2f09db59ae75fb0467", "class_name": "RelatedNodeInfo"}}, "text": "Top-fermenting \n\nyeast such as S. cerevisiae rise to the surface during \n\nfermentation and are used to brew ales, porters, \n\nstouts and wheat beers. In contrast, S. pastorianus, \n\n(formerly known as S. carlsbergensis) is a bottom-\n\nfermenting yeast, used to make lager beer. Lager \n\nyeasts grow best at lower temperatures. As a \n\nresult they grow more slowly, produce less \n\nsurface foam, and therefore typically settle to the \n\nbottom of the fermenter [9, 10].  \n\nS. cerevisiae or baker\u2019s yeast has long been used as \n\na leavening agent in baking. Baker\u2019s yeast ferment \n\nsugars present in dough, producing carbon \n\ndioxide and ethanol. Murcha (locally available \n\nyeast) is the complex mixture of various types of \n\nmicroorganisms along with yeast. Murcha has \n\nbeen traditionally used in fermentation and \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34               Thapa et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   30    Nepjol.info/index.php/njb \n\ncommonly found in Nepal, Bhutan and even some \n\nparts of India. \n\nYeasts are being used in the petrochemical \n\nindustry where it has been engineered to produce \n\nbiofuels such as ethanol, diesel and jet fuel \n\nprecursor. They are also used in the production of \n\nenzymes, lubricants and detergents. Food \n\nadditives including colorants, antioxidants, and \n\nflavor enhancers can also be produced using the \n\nyeast cells. Likewise, it is used in the production of \n\npharmaceuticals including antiparasitics, \n\nanticancer compounds and bio-pharmaceuticals \n\nsuch as insulin, vaccines, and nutraceuticals.  \n\nAnother important characteristic of yeasts being \n\nused as model organisms is that they replicate \n\nquickly and are easy to manipulate genetically. \n\nThe doubling time for yeast is about 90 minutes, \n\nFurthermore, the study of genome and its \n\norganization have been completed [11,12].   \n\nMethodology \nSample Collection  \nFour samples were collected from local market \n\nand two from Department of Microbiology, \n\nPinnacle Academy, Lalitpur, Nepal and Central \n\nDepartment of Biotechnology, Tribhuvan \n\nUniversity (TU), Nepal. Collected samples and \n\nsubcultures were given different codes for the \n\nconvenience. A: Mango peel; B: Black Grapes \n\nsample; C: Banana Peel; D: Soil Sample; E: Green \n\ngrapessubculture from Pinnacle Academy); F: \n\nSubculture of TU. \n\n Sample Processing \nThe samples were washed with distilled water \n\nand were crushed using mortar and pestle. One \n\ngram of finely crushed sample was weighed and \n\nserially diluted up to 10-5. \n\nIsolation and Identification of Yeast  \nOne ml of sample from each dilution was spread \n\non YEPDA media to isolate yeast. Morphological \n\ncharacteristics were studied on the basis of colour, \n\ntexture, margin, elevation.  Simple staining was \n\nperformed to elucidate the morphology and \n\narrangement of yeast cells and budding. \n\nSubculture of isolated yeast \n\nSubculture of isolated yeast was done in the \n\nYEPDA plates for further study and preservation. \n\nSpot Plate Technique \nCounting of viable yeast was done by spot plate \n\nmethod. 5 \u00b5l of serially diluted samples was \n\ninoculated with the help of micropipette on the \n\nmarked area on YEPDA media. Plates were \n\nlabelled according to the dilution factor and \n\nincubated at 37\u00b0C for 24 hrs. Individual colonies in \n\nthe most dilute samples were counted and the \n\nnumber of viable cells in the original culture was \n\ncalculated. \n\n Preparation of Broth \nThree sets of broth (1-xylose, 2- glucose, 3- xylose \n\n+ glucose) were prepared for each sample and its \n\nfermentative characteristics were studied. \n\nInoculation in Broth \nAll isolated yeasts from each sample were \n\ninoculated in three series of broth and kept at \n\nshaker for 4-5 days with regular monitoring.", "start_char_idx": 3763, "end_char_idx": 7539, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4cc74cef-02d1-4857-846b-cbe57940955b": {"__data__": {"id_": "4cc74cef-02d1-4857-846b-cbe57940955b", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a36e2d0d-7f5d-4400-a543-66f21749b26b", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "c87fb716afa48c369705e7c1a02089771e850b9709cd4624487cf50767adf3da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d1a9ddc0-65a8-4f18-be37-b479400128a5", "node_type": "1", "metadata": {}, "hash": "4af69595564a57de208cdd79c1fa7cb443638cb775edaedcb3453c4dea0b1878", "class_name": "RelatedNodeInfo"}}, "text": "Absorbance was taken at 560nm at the interval of \n\n24 hours. Smell of the alcohol was also monitored \n\nat the same interval.  \n\nMeasurement of ethanol concentration \nusing solvent extraction and Potassium \ndichromate oxidation method \nPositive samples with characteristic alcoholic \n\nsmell were further monitored for estimation of \n\nmethanol concentration. 1ml of each positive \n\nsample was mixed with 1ml Tributyl phosphate \n\n(TBP) and was centrifuged at 10,000 rpm for 5 \n\nminutes and 950\u00b5l of upper TBP was transferred \n\nto new centrifuge tube. 950 \u00b5l of potassium \n\ndichromate was added, vortexed for 2-3 minutes \n\nand centrifuged at 10,000 rpm for 5 minutes. Then \n\nupper layer was discarded, lower layer was \n\npipetted out and its absorbance was taken at 595 \n\nnm using YEPD broth as blank [13, 14, 15]. \n\nPreparation of standard ethanol \nSolution \nStandard ethanol solution was prepared by \n\nmixing different aliquots of 75% ethanol from \n\nstock to make up to 5ml by mixing required \n\namount of ethanol and distilled H2O. \n\n \n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34               Thapa et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   31    Nepjol.info/index.php/njb \n\nTable 1:  Morphological characteristics of isolated Yeasts \n\nSample  Morphological characteristics \n\nColour Shape Margin Elevation Texture Consistency \n\nA Cream Oval Lobate Non Smooth Mucoid \nB White Round Entire Elevated Smooth Mucoid \nC White Oval Entire Non Smooth Mucoid \nD White Round Entire Non Smooth Mucoid \nE Light \u2013orange Oval Entire Elevated Smooth Mucoid \nF cream Oval Lobate Non Smooth Mucoid \n\n \n\nResults and discussion \nMorphological characteristics of isolated Yeasts \n\n The morphological characteristics were studied \n\nfrom the colonies isolated on YEPDA media.(Table \n\n1) \n\nSimple staining of yeast \n\nThe yeast appeared as round or oval cells that were \n\ndark purple in color and budding were also visible \n\nunder microscope. (Table 2) \nTable 2. showing the Color and shape of   the  isolated Yeasts \n\nCounting of viable yeast by Spot plate \nmethod \n Each row represents a dilution series from a \n\ndifferent yeast culture. The same volume of diluted \n\nculture is used for each spot. The dilution series \n\nwas designed so that the most dilute spots contain \n\na small number of individual colonies that can be \n\ndistinguished from one another, typically less than \n\n15. The colonies were counted respectively. (Table \n\n3) \n\nDetermination of growth curve of Yeasts \nAbsorbance of broth with inoculums added was \n\ntaken from day first to day fourth at the interval of \n\n24 hours at 565nm. All the readings of three sets of \n\nbroth of all series of samples with comparable chart \n\nare as follows: \n\n \n\nTable 3: Table showing the number of yeast by spot plate \n\ntechnique \n\nSample Dilution \nfactor \n\nColony \ncount \n\nNo of cells \n/100\u00b5l \n\nA 103 14 14 \u00d7 103 \n\nB 104 12 12 \u00d7104 \n\nC 105 8 8 \u00d7 105 \n\nD 104 10 10 \u00d7104 \n\nE 104 9 9 \u00d7104 \n\nF 105 6 6 \u00d7 105 \n\n Yeast Extract, Peptone and Xylose \n\n Yeast Extract, Peptone and Glucose \n\n Yeast Extract, Peptone, Xylose and Glucose \n\n \n\n \nFigure 1: Graph showing the Absorbance of different Samples in \n\nthe broth containing Peptone and Xylose from first day up to \n\nfourth day. \n\n \n\n \nFigure 2: Graph showing the Absorbance of different Samples in \n\nthe broth containing Peptone, and Glucose from first day up to \n\nfourth day. \n\nSample Staining  Characteristics \nShape Color \n\nA Oval shaped Purple in color \n\nB Oval and round Purple colored yeast with  \nbuds \n\nC Oval shaped Purple colored yeast with  \nbuds \n\nD Round shaped Purple in color \n\nE Oval shaped Light purple colored yeast \nwith  buds \n\nF Oval shaped Purple in color", "start_char_idx": 7542, "end_char_idx": 11237, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d1a9ddc0-65a8-4f18-be37-b479400128a5": {"__data__": {"id_": "d1a9ddc0-65a8-4f18-be37-b479400128a5", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4cc74cef-02d1-4857-846b-cbe57940955b", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "b3d01a2b0ff1512b3b9d6d0d9f662583816935c284da552e7203ff0b931927a7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d84cdc41-026d-4f29-ba55-a8589e177377", "node_type": "1", "metadata": {}, "hash": "564950e33e61760396aac0d48628b7d3802e9646f021ef4bcb864c3bc3a3e006", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34               Thapa et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   32    Nepjol.info/index.php/njb \n\n \nFigure 3: Graph showing the Absorbance of different Samples in \n\nthe broth containing Peptone, and xylose +Glucose from first \n\nday up to fourth day. \n\nMonitoring of Alcoholic smell  \n\nThe flasks were monitored for alcohol production \n\nby smelling. There was presence of characteristic \n\nalcoholic smell in all the sets of conical flask expect \n\n1 which contains xylose along with yeast extract \n\nand peptone (Table 4). \n\nTable 4:  Absorbance of each broth with respective sample and \n\ndetection of their characteristic alcoholic smell. \n\nSample Broth Absorbance at \n595nm \n\nA \n \n \n\n1 - \n2 - \n3 - \n\nB \n \n \n\n1 - \n2 0.841 \n3 0.629 \n\nC \n \n \n\n1 - \n2 0.714 \n3 0.542 \n\nD \n \n \n\n1 - \n2 0.621 \n3 0.428 \n\nE \n \n \n\n1 - \n2 0.913 \n3 0.624 \n\nF \n \n \n\n1 - \n2 0.357 \n3 0.601 \n\n \n\n \n\nFigure 4.  Absorbance of Standard ethanol solution \n\nPotassium dichromate testing \n\nAbsorbance of all the positive samples and \n\nstandard ethanol solution were taken at 595nm. \n\nAbsorbance were taken for those samples only \n\nwhich gave characteristic alcoholic smell. Since, \n\nthere was absence of alcoholic smell in sample A; \n\nits absorbance was not taken. \n\nComparison with the standard ethanol  \nBy comparing the absorbance of sample with \n\nstandard ethanol solution, it was found that all the \n\nisolated yeast were highly fermentative. \n\nDiscussion \nThe different fermentative yeasts were isolated \n\nfrom different samples. The maximum of Yeast \n\ncolonies were obtained from both grape samples, \n\nwhich is mostly used in fermentative purposes. \n\nIt is also found that when yeast is grown in liquid \n\nmedium, the culture follows a well established \n\npattern for microbial growth. Cultures are usually \n\nstarted by inoculating media with a small number \n\nof cells. A lag phase follows the inoculation, during \n\nwhich cells become acclimated to the new \n\nenvironment and begin to condition the media with \n\ntheir own metabolites. Lag phase is followed by an \n\nexponential, or log phase, when the number of cells \n\nincreases exponentially. During this phase, the cells \n\nare in actively growing stage with high metabolic \n\nactivities, making this phase a desirable stage for \n\nthe generation of primary metabolites including \n\nethanol. \n\nDuring the study, six carbon compound glucose, \n\nfive carbon compound xylose and their mixed \n\nculture has been used and were screened for their \n\nsuitability for fermentation. The broth containing \n\nonly five carbon compound or pentose sugar xylose \n\ndidn\u2019t show the fermentative characteristics, which \n\nwas confirmed by the absence of characteristic \n\nalcoholic smell. The broth containing glucose and \n\nthe other broth containing glucose and xylose has \n\nshown the fermentative characteristics.  \n\nSample E show growth in all the media but \n\nhowever show high growth in the broth containing \n\npeptone and glucose and hence it\u2019s highly \n\nfermentative. Thus, yeasts utilize glucose a major \n\ncarbon source for fermentation.   \n\nFrom the above graph, Sample B and C show \n\nrelatively similar growth in all the media but it \n\nshows high growth in medium containing peptone \n\nand glucose. Thus, Sample B and C prefer peptone \n\nand glucose as a major carbon source. \n\n Sample A and B utilize all carbon sources for its \n\nmetabolism. Sample F uses glucose as a major \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34               Thapa et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   33    Nepjol.info/index.php/njb \n\nsource. Thus, we can conclude, yeasts utilize six \n\ncarbon compounds as a fermentative source. \n\n The ethanol was extracted from the aqueous phase \n\nusing the solvent.", "start_char_idx": 11242, "end_char_idx": 15045, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d84cdc41-026d-4f29-ba55-a8589e177377": {"__data__": {"id_": "d84cdc41-026d-4f29-ba55-a8589e177377", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d1a9ddc0-65a8-4f18-be37-b479400128a5", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "61aa481856eb3afac56b390da313a05b69f6f59817135fc0e3d06d937d65e692", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "540bfe74-b38d-4375-a2da-28654ffb2202", "node_type": "1", "metadata": {}, "hash": "e1c8723c2b1b49c9670a4175b26060f2e70efbff7aeea91de8f816adbd6c5b23", "class_name": "RelatedNodeInfo"}}, "text": "The ethanol was extracted from the aqueous phase \n\nusing the solvent. The ethanol in the solvent phases \n\nmoved to an acidic aqueous phases and \n\nsubsequently reacted with dichromate. The ethanol \n\nconcentration was measured from the increase in \n\ngreen color from orange at 595nm against blank. \n\nThe solvent extraction in this ethanol measurement \n\nis a crucial step because glucose, yeast extract, \n\npeptone, and glycerol can cause a change in color \n\nthrough reaction with dichromate. In other words, \n\nwithout the ethanol extraction step, the measured \n\nethanol concentration in the culture medium may \n\nbe tainted by a direct reaction between the \n\ndichromate reagent and other component in the \n\nculture medium. \n\n Many solvents have previously been used for the \n\nselective extraction of ethanol, particularly for the \n\nethanol extraction fermentation. Primary aliphatic \n\nalcohol (eg. n-decanol, n- dodecanol) is a \n\nrepresentative solvent for extracting ethanol from a \n\nculture broth and benzyl alcohol has been used as \n\nan alternative to distillation. In this study, however, \n\nnon alcoholic solvents TBP was investigated in \n\norder to find better solvent for the extraction of \n\nethanol from a culture broth. Moreover, phase \n\nseparation after solvent extraction was inhibited \n\nwhen the culture medium was used to prepare the \n\nethanol standard solution. However, TBP made a \n\ndistinct interface between TBP and water and \n\nshowed a linear standard ethanol curve in various \n\nmedia [14, 15]. \n\n In the measurement of ethanol concentration of the \n\nyeast culture, the data from the solvent extraction-\n\ndichromate oxidation method were similar to those \n\nfrom gas chromatography [16, 17]. Therefore, this \n\nethanol assay format is practically useful for the \n\nselection of a strain having high productivity, the \n\ndevelopment of a bioethanol production process, \n\nand monitoring and control in alcoholic beverage \n\nproduction. \n\nFinally, sample E can be used as a yeasts source for \n\nfermentation. The further characterization of \n\nSample E is essential for the future use \n\nAcknowledgments \n\nThe authors are thankful to college for Professional \n\nStudies, Kathmandu, Nepal for providing financial \n\nassistance and encouraging research activities in \n\nDepartment of Biotechnology. The authors are also \n\nthankful to Pinnacle Academy, Lalitpur, Nepal and \n\nMr. Sushil Khanal from Central Department of \n\nBiotechnology, Trubhuvan University, Nepal for \n\nproviding us Yeasts samples. \n\nReferences \n\n1. Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea \n\nJL, Goma G, Benbadis L:  Improving ethanol \n\nproduction and viability of Saccharomyces cerevisiae \n\nby a vitamin feeding strategy during fed-batch \n\nprocess. Appl Microbiol Biotechnol, 2002. 60:67\u201372. \n\n2. Alfenore S, Cameleyre X, L Goma G, Molina-Jouve C, \n\nGuillouet SE: Aeration strategy:  A need for very high \n\nethanol performance in Saccharomyces cerevisiae \n\nfed-batch process. Appl Microbiol Biotechnol, 2004 \n\n63:537\u2013542.  \n\n3. Barnett JA, Payne RW, and Yarrow D: Yeasts: \n\nCharacteristics and Identification.   3rd edn. \n\nCambridge: Cambridge University Press. 2000 \n\n4. Boekhout T, Robert V, and Smith MT: Yeasts of the \n\nworld. Morphology, physiology, sequences and \n\nidentification. World Biodiversity Database CD-ROM \n\nSeries. ETI: University of Amsterdam. 2002 \n\n5. Abouzied M, Reddy CA:. Direct fermentation of \n\npotato starch to ethanol by coculture of Aspergillus \n\nniger and Saccharomyces cerevisiae. Appl. Environ. \n\nMicrobiol, 1986 52(5):1055-1057 \n\n6. Adesanya OA, Oluyemi KA, Josiah Adesanya RA, \n\nOfusori DA, Bankole MA. Ethanol production by \n\nSaccharomyces cerevisiae from cassava peels \n\nhydrolysate. Internet J. Microbiol.", "start_char_idx": 14976, "end_char_idx": 18695, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "540bfe74-b38d-4375-a2da-28654ffb2202": {"__data__": {"id_": "540bfe74-b38d-4375-a2da-28654ffb2202", "embedding": null, "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-37", "node_type": "4", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "4aab00bf3b3196ac93c4792f7a1cda511f68e851133ffbde39e385b7b38955b4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d84cdc41-026d-4f29-ba55-a8589e177377", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "66cec65d46e102adca71deb7527177c2358e3bde176c1ebfa673f6324e72f990", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1530e6f6-8c74-4193-b1d9-fe330c339676", "node_type": "1", "metadata": {}, "hash": "aa70990c4057318a3e84a1a2f3ef5408fcfd7d5967e766a2db51884ca732008a", "class_name": "RelatedNodeInfo"}}, "text": "Internet J. Microbiol. 5:1.  \n\n7. Barnett JA: The entry of D-ribose into some yeasts of \n\nthe genus Pichia. J. Gen. Microbiol. 1975 90(1):1-12. \n\n8. Boulton C and Quain D:  Brewing   Yeast and \n\nFermentation. Oxford: Blackwell Science Ltd. 2006 \n\n9. Kirsop B: Maintenance of yeast cultures\" in Yeast \n\nBiotechnology.   (Allen &mp; Umwin, Boston, \n\nMassachusetts. 1987 \n\n10. Chanchaichaovivat A, Ruenwongsa P, Panijpan B: \n\nScreening and identification of yeast strains from \n\nfruits and vegetables: Potential for biological control \n\nof postharvest chilli anthracnose (Colletotrichum \n\ncapsici). Biol. Contr. 2007 42:326\u2013335. \n\n11. M. Raines, Advanced Yeast Culturing Kit \n\nInstruction. Booklet (Brewers Resource, Camarillo, \n\nCalifornia,) 1992 \n\n12. Martini A: Biodiversity and conversation of yeasts. \n\nBiodiver. Conserv. 1992 1(4):324-333.  \n\n13. Senthilraja P, Kathiresan K, Saravanakumar K: \n\nComparative analysis of bioethanol production by \n\ndifferent strains of immobilized marine yeast. J. \n\nYeast Fungal Res. 2011 2(8):113\u2013116. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 29-34               Thapa et al.     \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal   34    Nepjol.info/index.php/njb \n\n14. Hyun-Beom Seo, Hyun-Joo Kim,  Oh-Kyu Lee, Ji-\n\nHye Ha,  Hyeon-Yong Lee,    Kyung-Hwan Jung: \n\nMeasurement of ethanol concentration using \n\nsolvent extraction and dichromate oxidation and its  \n\napplication to bioethanol production process.  J \n\nindust. microb. Biotech. 2009 36(2):285-292 \n\n15. Pilone GJ: Determination of ethanol in wine by \n\ntitrimetric and spectrophotometric dichromate \n\nmethods: collaborative study.  Association of official \n\nAnalytical chemists. 1985 68(2):188-90. \n\n16. Seo HB, Kim HJ, Lee OK, Ha JH, Lee HY,  Jung KH: \n\nMeasurement of ethanol concentration using \n\nsolvent extraction and dichromate oxidation and \n\nits application to bioethanol  production process. J \n\nInd Microbiol Biotechnol. 2009 36(2):285-292. Epub \n\n2008 Nov 7. \n\n17. Mart\u00ednez-Nieto P, Vanegas-Hoyos M, Zapata-\nPineda M, Robles-Camargo J:  Hydrolysis of \n\nEicchornia crassipes and Egeria densa for Ethanol \n\nProduction by Yeasts Isolated from Colombian \n\nLake F\u00faquene. International Journal of \n\nEnvironmental, Chemical, Ecological, Geological \n\nand Geophysical Engineering 2013 7(1) \n\n \n\n \n\n \n\n \n\n \n\nhttp://www.pubfacts.com/author/Hyun-Beom+Seo\nhttp://www.pubfacts.com/author/Hyun-Joo+Kim\nhttp://www.pubfacts.com/author/Oh-Kyu+Lee\nhttp://www.pubfacts.com/author/Ji-Hye+Ha\nhttp://www.pubfacts.com/author/Hyeon-Yong+Lee\nhttp://www.pubfacts.com/author/Kyung-Hwan+Jung", "start_char_idx": 18673, "end_char_idx": 21264, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1530e6f6-8c74-4193-b1d9-fe330c339676": {"__data__": {"id_": "1530e6f6-8c74-4193-b1d9-fe330c339676", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "540bfe74-b38d-4375-a2da-28654ffb2202", "node_type": "1", "metadata": {"identifier": "njb-37", "author": "Thapa, Sandeep; Shrestha, Rajani; Tirewal, Anjali; Sharma, Arjun; K.C., Yuvraj", "title": "Isolation of yeast from soil and different food samples and its characterization based on fermentation", "date": "2015-12-30", "file": "njb-37.pdf"}, "hash": "e6097d5dc153021bccabf7e6d46aabeefa58531368380df4b4e5a26a8e1eeadc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b8076169-85a2-4c99-a12b-0d72a6f216b4", "node_type": "1", "metadata": {}, "hash": "e8e4bf78108765eeb67126cadb088dfac0366fea5f839e49c2181e8cb1a139ff", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:22-28    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   22       Nepjol.info/index.php/njb \n\nHeat Shock Protein 70 (HSP70) Expression in Antimony \nSusceptible/Resistant Clinical Isolates of Leishmania donovani \n\nMahendra Maharjan1,2* and Rentala Madhubala2 \n1Central Department of Zoology, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n\n2 School of Life Sciences, Jawahar Lal Nehru University, New Delhi, India \n\nAbstract  \nPentavalent antimonials have long been the first line of defence against leishmaniasis, but resistance has been \nreported in different parts of the world. Pentavalent antimony is reduced into trivalent form in the cells and is \na potential inducer of HSP70 in L. donovani. Expression profile of HSP70 in antimony susceptible and resistant \nL. donovani isolates were characterized by Southern blot, Northern blot and western blot analysis. HSP70 gene \ncopy number, gene expression and HSP70 protein expression was found uniform in both antimony sensitive \nand resistant clinical isolates. In laboratory condition, Leishmania cells respond to antimonial drug stress by \nthree fold over expression of the HSP70 protein. The observed results indicated that HSP70 play important role \nin stress tolerance against antimonial drug without differential expression in antimony sensitive and resistance \nclinical isolates of L. donovani. \n\nKeywords: Pentavalent antimony; HSP70; visceral leishmaniasis; drug resistance; PFGE; hybridization \n\n*Corresponding Author \nEmail: mmaharjan@cdztu.edu.np \n\nIntroduction \nVisceral leishmaniasis is a protozoan parasitic disease \n\ncaused by Leishmania donovani. Leishmaniasis \n\nconstitutes a major public health problem with \n\nincreasing pattern of disease burden [1,2]. It is a \n\nneglected tropical disease (NTD) which affects mainly \n\nthe poor population groups, primarily in rural areas. \n\nUnfortunately, there is still lack of effective, affordable \n\nand easy-to-use drugs for leishmaniasis treatment. \n\nSince vaccine against leishmaniasis is still under \n\ndevelopment, the control lies solely on chemotherapy \n\n[3]. However, emergence of drug resistance in parasitic \n\nprotozoa is becoming a major public health problem. \n\nHeat shock proteins (HSPs) are highly conserved \n\nproteins found in both prokaryotic and eukaryotic \n\ncells. HSP70 is a molecular chaperone which plays an \n\nimportant role in protein folding and assembly of \n\npolypeptides within the cell. When cells are exposed to \n\nthe stressed condition, the proportion of misfolded \n\nproteins (MFPs) suddenly increases and the cell reacts \n\nby synthesizing HSPs to assist those proteins in \n\nrefolding. The stress response is controlled primarily at \n\nthe transcription level by a heat shock factor (HSF) [4].  \n\nIt has been shown that transcript level of HSP70 \n\nincreased in L. major and in L. infantum, in response to \n\nelevated temperature conditions [5, 6, 7]. Besides \n\nelevated temperature, metals are also known to be \n\nimportant stress inducers in the cells. Antimonial \n\ncompounds; Pentostam and Glucantime are still the \n\ndrug of choice in the treatment against all forms of \n\nLeishmania infections [8, 9]. Since, antimonial drug \n\nresistance is becoming a common problem in many \n\nleishmaniasis endemic regions, extensive studies have \n\nbeen carried out to investigate the mechanism of drug \n\nresistance in the laboratory mutants. One of the \n\nmechanisms of resistance is postulated to be HSP70. L. \n\ntarentolae cells transfected with HSP70 gene showed \n\nresistance to SbIII or arsenite [10]. This suggests that \n\nHSP70 has a role in antimony resistance mechanism.", "start_char_idx": 47, "end_char_idx": 3782, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b8076169-85a2-4c99-a12b-0d72a6f216b4": {"__data__": {"id_": "b8076169-85a2-4c99-a12b-0d72a6f216b4", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1530e6f6-8c74-4193-b1d9-fe330c339676", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "21083fa0c789a61f0bdc4ab4611f836c727e92b52e89162b31d13ab63d644e4d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e06fe881-a880-4354-8f48-8e5ea7ffc246", "node_type": "1", "metadata": {}, "hash": "4dc7608c92601f0ffda62247e01b5d2bcb3e144b7d383e5e02d88976e3771802", "class_name": "RelatedNodeInfo"}}, "text": "This suggests that \n\nHSP70 has a role in antimony resistance mechanism. In \n\nthe present study, we have characterized the \n\nexpression of Heat Shock Protein 70 in antimony \n\nsensitive and resistant clinical isolates of L. donovani. \n\nMaterials and Methods \nParasite and culture condition \n Promastigotes of Indian Leishmania donovani clones \n\nAG83-S (MHOM/IN/1983/AG83), GE1 (MHOM/IN \n\n/80/ GE1F8R) and three untyped strains 2001, NS2, \n\nand 41 were isolated from patients with visceral \n\nleishmaniasis (VL) and were routinely cultured at 22\uf0b0C \n\nin M-199 medium (Sigma, USA) supplemented with \n\n10% heat inactivated fetal bovine serum (FBS, \n\nGibco/BRL, Life Technologies, Scotland, UK) and 13 \n\nmg/ml penicillin and streptomycin (Sigma, USA) [11].", "start_char_idx": 3711, "end_char_idx": 4459, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e06fe881-a880-4354-8f48-8e5ea7ffc246": {"__data__": {"id_": "e06fe881-a880-4354-8f48-8e5ea7ffc246", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b8076169-85a2-4c99-a12b-0d72a6f216b4", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "f39d504d0e714f0045470bfaaf2e9f0bd4b913c90439f0903523e7f3cc8922fc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "68117860-844b-449c-9fb5-b9ec0c95f96a", "node_type": "1", "metadata": {}, "hash": "82591fef6cbbb120a03428ea2f56c24960f26139ffbaa80eee3e604f361860ff", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   23       Nepjol.info/index.php/njb \n\nClinical isolates obtained from VL patients who \n\nresponded to SAG chemotherapy were designated as \n\nSAG-S (SAG sensitive) whereas VL patients that did \n\nnot respond to SAG were designated as SAG-R (SAG \n\nresistant). Accordingly, SAG sensitive isolate used in \n\nthis study is 2001-S whereas the four SAG-R isolates \n\nare 41-R, NS2-R, and GE1-R. The SAG-resistant isolates \n\nwere maintained in the absence of drug pressure in \n\nvitro. The isolates have been passage through hamsters \n\nor BALB/c mice to retain their virulence and \n\nimportantly their chemo-sensitivity profiles have \n\nremained unchanged.  \n\nCloning of heat shock protein 70 (HSP70) \n\ngene from L. donovani. \n A 1962-bp DNA fragment was amplified from the \n\ngenomic DNA using a sense primer with a flanking \n\nBamHI site, 5\u2019-\n\nCGCGGATCCATGACATTCGACGGCGCCATC-3\u2019, at \n\nposition 1-21, and the antisense primer with a flanking \n\nHindIII site, 5\u2019 \u2013 \n\nCCCAAGCTTTTAGTCGACCTCCTCGACCTTGG \u2013 3\u2019, \n\nincluding the stop codon, at position 1939-1962. \n\nPolymerase chain reaction (PCR) was performed in 50 \n\n\u00b5l reaction volume containing 100 ng of genomic DNA, \n\n25 pmol each of gene-specific forward and reverse \n\nprimers, 200 \u00b5mol of each dNTP, 2 mM MgCl2, 5U Taq \n\nDNA polymerase (MBI Fermentas) and 5% DMSO. The \n\nPCR conditions were as follows: 940C for 10 min, 940 C \n\nfor 45 sec, 620C for 30 seconds, 720C for 2 min and 35 \n\ncycles. Final extension was carried for 10 min at 720. A \n\nsingle 1.9 kb PCR product was obtained and cloned \n\ninto the BamHI - HindIII site of pET-30a (Figure 1) \n\nvector (Novagen). The recombinant construct was \n\ntransformed into BL21 (DE3) strain (Figure 2) of \n\nEscherichia coli and subjected to automated sequencing..  \n\nExpression and purification of HSP70 \n\nprotein. \n\nExpression of HSP70 protein from the construct \n\npET30a - LdHSP70 was induced at OD of 0.7 with 50  \n\n\u00b5M, 100 \u00b5M and 500 \u00b5M isopropylthiogalactoside \n\n(IPTG) (Sigma) at 37\uf0b0C for different time periods. All \n\nthe steps of purification were carried out at 4\uf0b0C as \n\nmentioned in general materials and methods. \n\n \n\n \nFigure 1: Restriction map of vector pET-30a(+).The HSP70 \n\nPCR product (1.9 kb) was cloned into the BamHI and HindIII \n\nrestriction site of pET-30a vector. \n\n \nFigure 2 : Cloning strategy of heat shock protein (HSP70) in \n\npET-30a expression vector \n\nProtein determination \nProtein concentration was determined by Bradford\u2019s method \n\nusing bovine serum albumin (BSA) as standard [12]. \n\nAntibody production \nThe purified recombinant HSP70 protein (20 \u00b5g) was \n\nsubcutaneously injected in mice using Freund\u2019s complete \n\n\n\nNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   24       Nepjol.info/index.php/njb \n\nadjuvant followed by two booster doses of recombinant \n\nHSP70 protein (15 \u00b5g) with incomplete adjuvant at two-\n\nweek intervals to produce the polyclonal antibody against \n\nthe recombinant HSP70 protein. The mice were bled two \n\nweeks after the second booster dose and sera was collected \n\nfor Western blot analysis.", "start_char_idx": 4464, "end_char_idx": 7711, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "68117860-844b-449c-9fb5-b9ec0c95f96a": {"__data__": {"id_": "68117860-844b-449c-9fb5-b9ec0c95f96a", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e06fe881-a880-4354-8f48-8e5ea7ffc246", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "e18100ef527411ded68b57812aaf97371d139ef65fa94e999baa39c54cb86642", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "dc2c30b6-af4b-4174-aeda-1aeaf009f33d", "node_type": "1", "metadata": {}, "hash": "093fab2ca6672d292c85fee98ab706c0cda253d1b9c76db886d6f981e7e99ccd", "class_name": "RelatedNodeInfo"}}, "text": "Isolation of genomic DNA and total RNA. \n Genomic DNA was isolated from ~ 2 x 109 cells from \n\n10-15 ml culture (mid log phase promastigotes) as \n\ndescribed in general materials and methods.  5 \u00b5g of \n\ngenomic DNA was digested overnight with HindIII \n\nand subjected to electrophoresis in 0.8% agarose gels. \n\nThe gel was run at a constant voltage of 30V overnight. \n\nTotal RNA was isolated from 2 \uf0b4 108 promastigotes \n\nusing TRI reagentTM (Sigma) according to \n\nmanufacturer\u2019s instructions. The isolated RNA was \n\nstored in diethyl pyrocarbonate (DEPC) treated water \n\nin small aliquots at \u2013800C. For Northern blot analysis, \n\n15 \uf06dg of total RNA was fractionated by denaturing \n\nagarose gel electrophoresis and transferred to nylon \n\nmembrane following the standard procedure.  \n\nPulse Field Gel Electrophoresis (PFGE) \n\nLeishmania chromosomes were separated by PFGE. \n\nLow melting agarose blocks, containing embedded \n\ncells (108 log phase promastigotes/ml) were \n\nelectrophoresed in a contour clamped homogenous \n\nelectric field apparatus (CHEF DRIII, Bio-Rad) in 0.5 \uf0b4 \n\nTBE with buffer circulation at a constant temperature \n\nof 14\u00baC. Saccharomyces cerevisiae chromosomes were \n\nused as chromosomal size markers. Pulse field gel \n\nelectrophoresis (PFGE) running conditions were as \n\nfollows: initial switch time, 60 s; final switch time, 120 \n\ns; run time, 24 h; current 6V/cm; including angle 120\u00ba. \n\nThe transfer of PFGE separated chromosomes from \n\nagarose gels to nylon membrane was achieved by \n\ncapillary method as described by [13]. \n\nHybridization of Northern, Southern and PFGE blot \n\nPre-hybridization was done at 65\u00b0C for 4 hours in a \n\nbuffer containing 0.5 M sodium phosphate; 7% SDS; \n\n1mM EDTA (pH 8.0) and 100 \u00b5g/ml sheared \n\ndenatured salmon sperm DNA. The blots were \n\nhybridised with denatured \u03b1-[P32]-dCTP-labelled DNA \n\nprobe (PCR probe described for the L. donovani HSP70 \n\ncoding region) at 106cpm/ml. The probe was labeled \n\nby random priming (NEB Blot\u00aeKit, New England \n\nBiolabs, Inc.). Membranes were washed sequentially as \n\nfollows: 2\uf0b4 SSC, 0.1% SDS; 1\uf0b4 SSC, 0.1% SDS; 0.5\uf0b4 SSC, \n\n0.1% SDS; 0.2\uf0b4 SSC, 0.1% SDS for 10 min each at 65\u00b0C \n\nuntil the non-specific counts had substantially reduced. \n\nMembranes were air-dried and exposed to imaging \n\nplate. The image was developed by PhosphorImager \n\n(Fuji film FLA-5000, Japan) using Image Quant \n\nsoftware. \n\nWestern blot analysis \n\nLate log phase promastigotes (1 \uf0b4 108) were harvested \n\nand the resultant cell pellet was lysed by sonication \n\nand cell supernatants were prepared by centrifugation \n\nat 20,000 x g. 50 \u00b5g of protein from each cell line were \n\nfractionated by SDS-polyacrylamide gel \n\nelectrophoresis and blotted on nitrocellulose \n\nmembrane using electrophoretic transfer cell (BIO-\n\nRAD). Western Blot analysis was done using the ECL \n\nkit (Amersham Pharmacia Biotech) according to the \n\nmanufacturer\u2019s protocol. Polyclonal antibody to \n\npurified recombinant L. donovani HSP70 generated in \n\nmice was used for the Western blot analysis. \n\nAutoradiograms were analyzed by using model FLA \n\n5000 imaging densitometer (Fuji, Japan). The results \n\nshown are from a single experiment typical of at least \n\nthree giving identical results. \n\nResults \nHeat shock protein 70 (HSP70) sequence \nanalysis \nThe gene encoding the heat shock protein 70 kDa \n\n(HSP70) nucleotide sequence was retrieved from \n\nEMBL sequence data bank under the accession no.", "start_char_idx": 7715, "end_char_idx": 11152, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "dc2c30b6-af4b-4174-aeda-1aeaf009f33d": {"__data__": {"id_": "dc2c30b6-af4b-4174-aeda-1aeaf009f33d", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "68117860-844b-449c-9fb5-b9ec0c95f96a", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "cc6ef3b45791a0a0a5dcc75c82e9bbe72bbf2ff69b28dc9e5ce8d80b51844e05", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "76c773c1-fbef-45b0-9942-eb5c3deef43d", "node_type": "1", "metadata": {}, "hash": "1f792a5da3a42a04ca7b9aaf95b9dcdd57252ff243a45210c3461e744472ff44", "class_name": "RelatedNodeInfo"}}, "text": "X52314. In order to clone the full length gene encoding \n\nHSP70, polymerase chain reaction (PCR) was \n\nperformed using specific oligonucleotides. The sense \n\nprimer was 5'-CGCGGATCCATG \n\nACATTCGACGGCGCCATC-3\u2019 at position 1-21 and \n\nthe antisense primer with a flanking HindIII site, 5\u2019-\n\nCCCAAGCTTTTAGTCGACCTCCTCGACCTTGG- 3\u2019 \n\nincluding the stop codon at position 1939-1962. \n\nGenomic DNA from L. donovani AG83 \n\n(MHOM/IN/1983/AG83) promastigotes was used as \n\na template. A single full length 1962-bp PCR product \n\nwas obtained. The PCR fragment was purified from the \n\ngel and double digested with BamH1 and HindIII \n\nrestriction enzymes. The digested product was ligated \n\nwith pET30a cloning vector at 160C overnight. The \n\nligated product was transformed into DH5\u03b1 competent \n\ncells. The positive clones were confirmed by colony \n\nPCR. Further, the cloned HSP70 PCR fragment was \n\nconfirmed by sequencing. A single open reading frame \n\nconsisting of 1962-bp was isolated. No variation in the \n\ncoding sequence was found from the sequence \n\ndescribed earlier. \n\n\n\nNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   25       Nepjol.info/index.php/njb \n\nOver-expression and purification of full-\nlength L. donovani HSP70 in E. coli \n In order to overexpress and purify recombinant \n\nprotein, the encoding L. donovani HSP70 sequence was \n\ncloned inframe in pET-30a expression vector. The \n\nresultant pET-30a -LdHSP70 construct was \n\ntransformed into E. coli and protein over expression \n\nwas induced using IPTG (Figure 3A and 3B) as \n\ndescribed in general materials and method. A protein \n\nwith a molecular weight that matched the estimated 70 \n\nkDa according to amino acid composition of L. \n\ndonovani HSP70 with His6 tag and S-tag present at its \n\nN-terminal end was also induced. The recombinant \n\nprotein was purified by Ni2+-NTA affinity \n\nchromatography column  Purification of His-tagged L. \n\ndonovani HSP70 by metal affinity chromatography \n\n(Figure 3C) yielded ~200 \u00b5g of purified protein from 1-\n\nlitre bacterial culture. \n\n \nFigure 3: Overexpression and purification of L. donovani HSP70 \nprotein. (A) Coomassie blue staining of SDS\u2013PAGE showing \noverexpression of full-length L. donovani HSP70 protein in E. coli. The \npET-30a bacterial extract before induction (lane 2 and 6) and after \ninduction (lanes 3-5 and 7-9) of two positive clones at 1, 2, and 3 h, \nrespectively with 1 mM IPTG. Arrow shows the induced \nrecombinant HSP70 protein. Broad range protein MW marker (MBI-\nFermentas) was used to identify the size of the recombinant protein \n(Lane 1). ( B) The pET30a bacterial extract before induction (lane 1), \nmolecular marker (lane 3) and three hour after induction (lane 2, 4, 5 \n\nand 6) with 50 \uf06dM, 100 \uf06dM, 200 \uf06dM and 500 \uf06dM  IPTG respectively. \n(C) Purification profile of soluble form of HSP70 protein by Ni- NTA \naffinity bead affinity chromatography resin. Lane 1 flow through, \nlanes 2 and 3, washes with 20 mM and 60 mM immidazole, Lane 4-7 \nelutes using 100 mM, 150 mM, 200 mM, and 250 mM immidazole, \nLane 8 Ni- beads, lane 9- blank.", "start_char_idx": 11155, "end_char_idx": 14303, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "76c773c1-fbef-45b0-9942-eb5c3deef43d": {"__data__": {"id_": "76c773c1-fbef-45b0-9942-eb5c3deef43d", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "dc2c30b6-af4b-4174-aeda-1aeaf009f33d", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "ce3e56996a93a821ef90f27700e4bd775fac9b13332f0b76e471ac02d64c2435", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "da0c39f4-8a8c-4e8f-bf8f-1ed772cdcee6", "node_type": "1", "metadata": {}, "hash": "9cf8fe394a27307db6cad4049fe498d332f5d8e5e254e1f78b76c8c00b6170f0", "class_name": "RelatedNodeInfo"}}, "text": "All the purification fractions were \nrun along with the molecular weight protein marker (lane M). \n\nPolyclonal antibody production and \n\nWestern blotting \nWestern blot using size-fractionated parasite protein, \n\nantiserum could detect a band of anticipated L. \n\ndonovani HSP70 size ~70 kDa in promastigote extracts.  \n\nIn antimony resistant L. tarentolae mutants, HSP70 was \n\nreported to increase more than 4-fold compared to the \n\nwild-type cells [10]. The overexpression of HSP70 \n\nremained stable in L. tarentolae mutants after several \n\nhundreds of passages without drug [10]. To test the \n\nlink between HSP70 expression and resistant \n\nphenotype in antimony resistant L. donovani field \n\nisolates, we performed Western blot analysis using \n\npolyclonal antibody. A Western blot using equal \n\namount of parasite protein (50 \u00b5g each) extract from \n\nantimony sensitive (AG83-S and 2001-S) and resistant \n\n(GE1-R, 41-R and NS2-R) field isolate did not show any \n\ndifferential expression of HSP70 protein (Figure 4).  \n\n \nFigure 4. Western blotting using anti-His-HSP70 antibody raised \n\nagainst LdHSP70 in BALB/C mice. HSP70 protein in fractionated \nextracts obtained from SAG-S and SAG-R cultures harvested after 48 \n\nh of growth. The same blots were reprobed with antibody against \u03b2-\n\ntubulin protein to normalize the loading on to each lane of the gel.  \n\nSouthern blot hybridization of HSP70 gene \nThe copy number of HSP70 gene varies considerably \n\namong various Leishmania species [6, 10, 14]. To \n\ndetermine HSP70 gene copy number in L. donovani \n\nfield isolates, Southern blot analysis was performed \n\nusing 1.9 Kb HSP70 PCR product as a probe as \n\ndescribed under general materials and methods. \n\nHindIII which digests outside the gene producing a 10 \n\nkb restriction fragment (expected size) was used to \n\ncheck the copy number of the gene. The probe \n\nhybridized to a single fragment of 10 kb in all the field \n\nisolates (Figure 5) indicating that HSP70 exists as a \n\nsingle copy gene in these strains. Further, we checked \n\nthe chromosomal localization of HSP70 gene by \n\nSouthern blot hybridization of PFGE blot. The HSP70 \n\nprobe hybridized to a single chromosomal band of 1.2 \n\nMb region that corresponds to the chromosome \n\nnumber 28 of the L. infantum genome database (Figure \n\n6). \n\n\n\nNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   26       Nepjol.info/index.php/njb \n\n \nFigure 5. Southern blot analysis of HSP70 gene in SAG\u2013S and SAG\u2013R \nLeishmania donovani field isolates. Total genomic DNA of Leishmania \nstrains were isolated and digested with HindIII restriction enzyme. \nThe blot was probed with 1.9 Kb full length HSP70 gene. The blot \nwas reprobed with \u03b1-tubulin to monitor the amount of digested DNA \nlayered on the gel. \n \n\n  \nFigure 6. PFGE analysis of SAG-S and SAG-R L. donovani isolates \nindicating chromosomal localization of HSP70 gene. Chromosomes \nof L. donovani isolates were separated by CHEF and Southern blots \nwere hybridized with the HSP70 probe. The size of hybridizing band \nwas identified using chromosomes of S. Cerevisae as marker \n\n \nFigure 7. Expression analysis of HSP70 gene in L. donovani Northern \n\nblot analysis of mRNA from SAG-S and SAG-R isolates (log phase \n\nculture). 15 \u00b5g of total RNA was loaded per lane, transferred and \n\nhybridized with HSP70 probe. A \u03b1-tubulin probe and was used to \n\nmonitor the amount of RNA layerd on the gel. The rRNA stained \n\nwith ethidium bromide was used to normalize the RNA loading.", "start_char_idx": 14304, "end_char_idx": 17864, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "da0c39f4-8a8c-4e8f-bf8f-1ed772cdcee6": {"__data__": {"id_": "da0c39f4-8a8c-4e8f-bf8f-1ed772cdcee6", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "76c773c1-fbef-45b0-9942-eb5c3deef43d", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "338379ddb48b1968ad1b2bfdd15e634c194125d3fd7311f150e40fdae7e52d44", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "39893579-d8b3-45b3-8f37-284003cccdc1", "node_type": "1", "metadata": {}, "hash": "5b9fd1fa5ae4a317f72fe3b8707af704c043abfc6d04dbe5d9730d01dd4175cf", "class_name": "RelatedNodeInfo"}}, "text": "The rRNA stained \n\nwith ethidium bromide was used to normalize the RNA loading.  \n\nNorthern blot hybridization of HSP70 gene  \nSince, we couldn\u2019t find any difference in HSP70 protein \n\nexpression and copy number of gene in between SAG-\n\nS and SAG-R L. donovani field isolates, we checked \n\ntranscript level of HSP70 gene expression. Northern \n\nblotting of SAG-S and SAG-R L. donovani field isolates \n\nrevealed a single transcript of ~ 3.5 kb in all the strains \n\n(Figure 7). Densitometric analysis of hybridizing bands \n\ncorresponds to the internal control \u03b1-tubulin showed \n\nsimilar expression of HSP70 gene in between SAG \n\nsensitive and resistant field isolates. \n\nInduction of HSP70 by SbIII in promastigote \nof L. donovani field isolate \nLeishmania cells respond to SbIII exposure by \n\nincreasing HSP70 protein [10]. Induction of HSP70 was \n\nfound more than four (>4) fold in L. tarentolae and L. \n\ninfantum cells when incubated for 24 hours at IC50 \n\nconcentration of the drug [10]. To test whether strain \n\nwere incubated with 15 \u00b5M (IC50 concentration of \n\nAG83-S) SbIII for 24 hours. A time dependent increase \n\nof HSP70 protein expression in L. donovani field isolates \n\nwas observed. Protein expression increased by more \n\nthan > 1.7 fold at 12 hours and more than three (>3) \n\nfold after 24 hours compared to the cells incubated \n\nwithout drug (Figure 8A and 8B). \n\ntrivalent antimony could induce the expression of \n\nHSP70 protein in L. donovani field isolates, we used \n\npolyclonal antibody raised against HSP70 antigen in \n\nBALB/C mice. Promastigotes of L. donovani (AG83-S) \n\nDiscussion \nPhysiological role of the HSP70 as a molecular \n\nchaperon has been well described [15]. Induction of \n\nHSP70 can protect cells by binding to misfolded \n\nproteins in variety of adverse conditions [16]. Besides \n\nits physiological cytoprotective role an increased \n\nexpres \n\n  \nFigure 8. Effect of SbIII on HSP70 protein expression at different time \npoints. (A) Western blot analysis of HSP70 in L. donovani \npromastigotes after treatment with 15 \u00b5M SbIII for 0 h, 12 h and 24 h. \nThe same blot was reacted with antibody against \u03b2-tubulin protein to \nnormalize the loading on to each lane of the gel. (B) Densitometric \nscanning of the western blot in A. The bands were quantified by \nscanning on a densitometer and fold difference in signal intensities \nrelative to the zero control were plotted.  \n\n\n\nNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   27       Nepjol.info/index.php/njb \n\nexpression of HSP70 protein in antimony resistant L. \n\ntarentolae and L. infantum laboratory mutants and the \n\nincreased expression of HSP70 was stable after \n\nhundreds of passages without the drug [10]. If the \n\nlinkage between HSP70 overexpression and antimony \n\nresistant phenotype observed in those laboratory \n\nmutants is also true for L. donovani field isolates, HSP70 \n\ngene could be one of the important biomarker for \n\nmonitoring antimony resistance in the field conditions. \n\nWith this hypothesis we have cloned the HSP70 gene \n\nin bacterial expression vector, purified the recombinant \n\nprotein and raised the polyclonal antibody against \n\nHSP70 antigen in BALB/C mice. We checked the \n\nHSP70 protein expression in two antimony sensitive \n\n(AG83-S and 2001-S) and three natural antimony \n\nresistant (41-R, CK2-R and NS2-R) L. donovani clinical \n\nisolates using Western blotting. We found constitutive \n\nexpression of HSP70 protein in both SAG-S and SAG-R \n\nclinical isolates.", "start_char_idx": 17785, "end_char_idx": 21348, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "39893579-d8b3-45b3-8f37-284003cccdc1": {"__data__": {"id_": "39893579-d8b3-45b3-8f37-284003cccdc1", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "da0c39f4-8a8c-4e8f-bf8f-1ed772cdcee6", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "d8dc94f2a64cb14b93e92799a8d3d8a1c21a5fd8f06a3e9e625dfe3c8afb7d9e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "04ad943c-fc20-4af8-b1fa-e37e0359e42b", "node_type": "1", "metadata": {}, "hash": "d245101edddcdd6be0cd50cc9606615e64f6ed512964ce805af5a23e2141066a", "class_name": "RelatedNodeInfo"}}, "text": "No difference in HSP70 protein \n\nexpression was observed in antimony sensitive and \n\nresistant field isolates.  \n\nFurther, we characterized the HSP70 gene in SAG-S \n\nand SAG-R L. donovani Indian field isolates to check the \n\nchromosomal localization and copy number of the \n\ngene. The copy number of HSP70 gene has been \n\nreported to be considerably varied among different \n\ntrypanosomatids such as single copy in L. braziliensis, 2 \n\ncopies in L. tarentolae, 4 copies in L. major, 6 copies in L. \n\ninfantum, 7 copies in L. amazonensis and T. cruzi [5, 6, \n\n10, 14, 17, 18]. In L. donovani Indian field isolates, the \n\ncopy number of HSP70 gene was found to be single \n\ncopy. No difference in gene copy number was \n\nobserved in between SAG-S and SAG-R field isolates.  \n\nTo further verify the HSP70 gene amplification, we \n\nperformed Southern blot hybridization of PFGE blot. \n\nHSP70 probe hybridized to a highly amplified single \n\nband at the 1.2 Mb region which corresponds to the 28 \n\nchromosome of the L. infantum genome database.  \n\nTranscript level of the HSP70 gene has been shown to \n\nbe transiently induced by environmental stress such as \n\nexposure to heat shock or heavy metals [19]. During \n\nheat shock, HSP70 transcription rate increases rapidly \n\nand then decline slowly [20]. Increased HSP70 \n\ntranscript level has been shown in Leishmania when \n\ncells were exposed to heat [6]. The transcription \n\nmechanism has shown to be controlled at the \n\nposttranscriptional level specifically by the 3\u2019-\n\nuntranslated region of the gene in contrast to most \n\nother organisms [7, 21]. Similar regulation has also \n\nbeen described in the related parasite T. brucei [5]. \n\nIncreased expression of HSP70 has been reported in \n\nantimony resistant L. tarentolae and L. infantum mutants \n\ncompared to wild type cells [10]. The increased \n\nexpressions of HSP70 in laboratory mutant were found \n\nto be stable after hundreds of passages without the \n\ndrug [10]. Earlier studies indicated that HSP70 \n\noverexpression in antimony resistant mutants was the \n\nstable phenotype. To check whether similar \n\nmechanism is operating in field condition, we \n\nperformed Northern blot hybridization of HSP70 gene \n\nin between natural antimony resistant L. donovani \n\nclinical isolates to that of the antimony sensitive \n\nparasites. No difference in HSP70 transcript level was \n\nfound between SAG-S and SAG-R clinical isolates.  \n\nArsenite and Sb III have been described as the known \n\ninducer of the HSP70 in Leishmania [10, 22, 23]. By gene \n\ntransfection experiment, it has been shown that HSP70 \n\ncontribute first line of defense against Sb III [10] \n\nsuggesting the role of HSP70 in antimonial drug \n\nresistance. In the present study, we checked HSP70 \n\nprotein expression in promastigotes of L. donovani \n\n(AG83-S) in presence of Sb III. Interestingly, we could \n\nfind considerable over expression of HSP70 protein \n\nwhen cells were incubated in IC50 concentration of Sb \n\nIII. Protein over expression was about 1.7 fold at 12 \n\nhours that increased up to 3 fold at 24 hours time \n\nperiod. The observed results suggest that Leishmania \n\ncells respond to antimonial drug stress by over \n\nexpression of the HSP70 protein. \n\nAcknowledgements  \nThe authors thank University Grants Commission, \n\nNew Delhi, India for providing JRF/SRF to Mahendra \n\nMaharjan to conduct this study. \n\nReferences \n1. Desjeux P: The increase in risk factors for \n\nleishmaniasis worldwide. Trans R Soc Trop Med \n\nHyg 2001, 95: 239-243. \n\n2. Desjeux P: Leishmaniasis: current situation and \n\nnew perspectives. Comp Immunol Microbiol Infect \n\nDis 2004, 27: 305-318. \n\n3.", "start_char_idx": 21349, "end_char_idx": 24980, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "04ad943c-fc20-4af8-b1fa-e37e0359e42b": {"__data__": {"id_": "04ad943c-fc20-4af8-b1fa-e37e0359e42b", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "39893579-d8b3-45b3-8f37-284003cccdc1", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "192e7cddc3c6801515b0eb00b95553fc450023d96dc0d968019e591eefd0d877", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5e57c7de-205a-4967-91a8-360ba9079b8b", "node_type": "1", "metadata": {}, "hash": "9320aedf3140f5ca066875d0b98550cc90f0091e87ff8275b6716643c8c5a384", "class_name": "RelatedNodeInfo"}}, "text": "3. Brandonisio O and Spinelli R: Immune response to \n\nparasitic infections--an introduction. Curr Drug \n\nTarget. Immune Endocr Metabol Disord 2002, 2: 193-\n\n199. \n\n4. Pockley AG: Heat shock proteins in health and \n\ndisease: therapeutic targets or therapeutic agents? \n\nExpert Rev Mol Med 2001, 3: 1-21. \n\n\n\nNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1:22-28       Maharjan and Madhubala   \n\n\u00a9NJB, Biotechnology Society of Nepal   28       Nepjol.info/index.php/njb \n\n5. Lee MG, Atkinson BL, Giannini SH, Van der Ploeg \n\nLH: Structure and expression of the hsp 70 gene \n\nfamily of Leishmania major. Nucleic Acids Research, \n\n1988, 16(20):9567-9585. \n\n6. Quijada L, Soto M, Alonso C, Requena JM: \n\nAnalysis of post-transcriptional regulation \n\noperating on transcription products of the \n\ntandemly linked Leishmania infantum hsp70 \n\ngenes.  J  Biol Chem 1997, 272(7):4493-4499.  \n\n7. Quijada L, Soto M, Alonso C Requena JM: \n\nIdentification of a putative regulatory element in \n\nthe 3'-untranslated region that controls expression \n\nof HSP70 in Leishmania infantum. Mol Biochem \n\nParasitol. 2000, 110(1):79-91. \n\n8. Guerin PJ,  Olliaro P,   Sundar S,  Boelaert M,  Croft \n\nSL,  Desjeux P,  Wasunna MK, Bryceson AD: \n\nVisceral leishmaniasis: current status of control, \n\ndiagnosis, and treatment, and a proposed research \n\nand development agenda.  Lancet Infectious \n\nDiseases, 2002, 2(8):494-501. \n\n9. Haimeur A, Brochu C, Genest P,  Papadopoulou B,  \n\nOuellette M: Amplification of the ABC transporter \n\ngene PGPA and increased trypanothione levels in \n\npotassium antimonyl tartrate (SbIII) resistant \n\nLeishmania tarentolae. Mol Biochem Parasitol 2000, \n\n108(1):131-135.  \n\n10. Brochu C, Haimeur A, Ouellette M: The heat shock \n\nprotein HSP70 and heat shock cognate protein \n\nHSC70 contribute to antimony tolerance in the \n\nprotozoan parasite Leishmania. Cell Stress \n\nChaperones 2004, 9(3):294-303. \n\n11. Bhattacharjee H, Carbrey J, Rosen BP,  \n\nMukhopadhyay R: Drug uptake and \n\npharmacological modulation of drug sensitivity in \n\nleukemia by AQP9. Biochemical and Biophysical \n\nResearch Communications 2004, 322(3):836-841. \n\n12. Bradford MM: A rapid and sensitive method for \n\nthe quantitation of microgram quantities of \n\nprotein utilizing the principle of protein-dye \n\nbinding. Analytical Biochemistry, 1976, 72:248-25,. \n\n13. Sambrook J, Fritsch EF, Maniatis T: Molecular \n\ncloning: A laboratory manual, Cold Spring \n\nHarbour Press, 1989. \n\n14. Zurita AI,  Rodriguez J,  Pinero JE, Pacheco R,  \n\nCarmelo E,  del CA,  Valladares B: Cloning and \n\ncharacterization of the Leishmania (viannia) \n\nbraziliensis Hsp70 gene, Diagnostic use of the C-\n\nterminal fragment rLb70(513-663). Journal of \n\nParasitology 2003, 89(2):372-378. \n\n15. Hartl FU, Hayer-Hartl M: Molecular chaperones in \n\nthe cytosol: from nascent chain to folded protein.", "start_char_idx": 24978, "end_char_idx": 27828, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5e57c7de-205a-4967-91a8-360ba9079b8b": {"__data__": {"id_": "5e57c7de-205a-4967-91a8-360ba9079b8b", "embedding": null, "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-38", "node_type": "4", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "2be9ae6aaeded6f5088633da298a6246465a135ca1a2167568ada2f8b1ce7065", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "04ad943c-fc20-4af8-b1fa-e37e0359e42b", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "408f56abd4d1fd062c7c850106cbee70a5d00d775c62096910352be7f75219ed", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "37755ab8-b635-40c7-a935-4c5cdb54d2e3", "node_type": "1", "metadata": {}, "hash": "d06599feaf4fae217d8635a3b9894bb9c06827070e5829d4e1bce9415802f148", "class_name": "RelatedNodeInfo"}}, "text": "Science 2002, 295(5561):1852-1858. \n\n16. Morimoto RI, Santoro MG: Stress-inducible \n\nresponses and heat shock proteins: new \n\npharmacologic targets for cytoprotection.  Nature \n\nBiotechnology 1998, 16(9):833-838. \n\n17. Bock JH, Langer PJ: Sequence and genomic \n\norganization of the hsp70 genes of Leishmania \n\namazonensis. Molecular and Biochemical Parasitology \n\n1993, 62, no. 2, pp. 187-197. \n\n18. Requena JM,  Lopez MC,  Jimenez-Ruiz A,  de la \n\nTorre JC, Alonso C: A head-to-tail tandem \n\norganization of hsp70 genes in Trypanosoma \n\ncruzi. Nucleic Acids Research, 1988, 16:1393-1406. \n\n19. Milarski KL, Morimoto RI: Expression of human \n\nHSP70 during the synthetic phase of the cell cycle. \n\nProceedings of the National Academy of Sciences 1986, \n\n83(24):9517-9521. \n\n20. Theodorakis NG, Morimoto RI: Posttranscriptional \n\nregulation of hsp70 expression in human cells: \n\neffects of heat shock, inhibition of protein \n\nsynthesis, and adenovirus infection on translation \n\nand mRNA stability. Molecular and Cellular Biology, \n\n1987, 7(7):4357-4368. \n\n21. Brandau S, Dresel A, Clos J: High constitutive \n\nlevels of heat-shock proteins in human-\n\npathogenic parasites of the genus Leishmania. \n\nBiochemical Journal, 1995, 10:225-232. \n\n22. Del Razo LM, Quintanilla-Vega B, Brambila-\n\nColombres E, Calderon-Aranda ES,  Manno M, \n\nAlbores A: Stress proteins induced by arsenic. \n\nToxicology and Applied Pharmacology, 2001 \n\n177(2):132-148. \n\n23. Lawrence F, Robert-Gero M: Induction of heat \nshock and stress proteins in promastigotes of \n\nthree Leishmania species. Proceedings of the National \n\nAcademy of Sciences, 1985, 82(13):4414-4417.", "start_char_idx": 27831, "end_char_idx": 29471, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "37755ab8-b635-40c7-a935-4c5cdb54d2e3": {"__data__": {"id_": "37755ab8-b635-40c7-a935-4c5cdb54d2e3", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5e57c7de-205a-4967-91a8-360ba9079b8b", "node_type": "1", "metadata": {"identifier": "njb-38", "author": "Maharjan, Mahendra; Madhubala, Rentala", "title": "Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani", "date": "2015-12-30", "file": "njb-38.pdf"}, "hash": "ee5092b2791daf9eaeb49df1b78edbafee3ad8781b022c6f6e2725285e6c012e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c8ff4beb-a4d3-46e2-bef5-53aab6c6b463", "node_type": "1", "metadata": {}, "hash": "21688b1bc8332cf943aa0b018f325e65336c89b85d2be66abf15c4dcadec6b5e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21   ISSN 2091-1130 (Print) /ISSN 2467-9319 (online) \n\n \n\nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   15    Nepjol.info/index.php/njb \n\nEffect of Lyophilization on Infectivity and Viral Load of \nAdenovirus \n\nBimlesh Kumar Jha1*, Birendra Prasad Gupta 2, Prashanna Maharjan3, Somila kakshapati3,  \n\nNabin Narayan Munankarmi,3 \n1National Public Health Laboratory, Teku, Kathmandu, Nepal \n\n2Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n3Biotechnology Society of Nepal (BSN), Kathmandu, Nepal \n\nAbstract \nFreeze drying (Lyophilization) performed at temperature and pressure below the triple point is being practiced for the \n\npreservation of virus stocks for longer periods. The present study is aimed to lyophilize adenovirus strain to study its effects \n\non infectivity and viral load. In-house adenovirus reference strain (stock virus) was propagated in Hep-2 cell line in 25cm2 \n\ncell culture flasks. In 24-well plates the serial dilutions of stock virus from 10-1 to 10-7 (100\u00b5l inoculum) was inoculated in \n\neach well with Hep-2 cells for TCID50 titer and viral DNA was extracted separately to determine viral load by Taqman Real \n\nTime PCR.  Stock virus was lyophilized in 3 lots and stored at RT (25\u00b12\u00b0C) and 4\u00b0C separately for 1, 4 and 6 months and \n\nsubjected to TCID50 (for viral infectivity) and viral load assay (for total viral genome copies). Following lyophilisation and \n\nstorage of adenoviral strains at RT and 4\u00b0C separately did not affect significantly on the viral stability, infectivity as well as \n\nviral copy number till 4 months. However, storage at RT for 6 months resulted in 1 log reduction in viral copy number. \n\nThus, storage of even lyophilized virus stock would necessitate a temperature of at least 4\u00b0C for prolonged periods. The \n\npresent study could successfully lyophilize adenovirus and retain its infectivity over a period of 6 months when stored at \n\nRT and 4\u00b0C. No significant difference in the infectivity or TCID50 titer was observed in the lyophilized virus as compared to \n\nthe stock virus. However, the viral load was observed to increase with lyophilization of the virus over 6 months when \n\nstored at 4\u00b0C which possibly is due to the concentration of the virus on freeze-drying. \n\nKeywords: Adenovirus, lyophilisation, Physical stability, Formulation \n\n*Corresponding Author \n\n Email: jhabimlesh7@gmail.com \n\nIntroduction \nThe structure and function of organisms change and \n\nget lost with time, as in laboratory cultures. Attempts to \n\nstop the biological clock have been conjured by ancient \n\nand modern minds; and the heart of many such \n\nschemes has been experiments with temperature and \n\nwater content. Whereas refrigeration technology \n\nprovides a means of slowing the rate of deterioration of \n\nperishable goods, the use of much lower temperatures \n\nhas proved a means of storing living organisms in a \n\nstate of suspended animation for extended periods. \n\nAdenoviruses (AdV), belonging to family \n\nAdenoviridae, are double stranded Deoxyribonucleic \n\nacid (DNA) viruses that carry DNA insert of size 7kb. \n\n52 subtypes of adenoviruses are known to infect \n\nhumans [1]. Adenoviruses are frequent cause of human \n\nmucosal surface, infections particularly in pediatric \n\npopulation and can be responsible for ocular and \n\ngastrointestinal illnesses in humans. To preserve \n\nmaximum infectivity for long periods, cell culture-\n\ngrown adenovirus must be stored frozen at very low \n\ntemperatures [2].  However, this makes its handling \n\ndifficult.", "start_char_idx": 47, "end_char_idx": 3673, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c8ff4beb-a4d3-46e2-bef5-53aab6c6b463": {"__data__": {"id_": "c8ff4beb-a4d3-46e2-bef5-53aab6c6b463", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "37755ab8-b635-40c7-a935-4c5cdb54d2e3", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "55b2d3f9d5d9091325f0e0d118c5f45d8f30e3437c7975c32e5e2a0ada33c258", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "95da32fc-83db-4ad5-bd0f-73c132f364f8", "node_type": "1", "metadata": {}, "hash": "3a627d27344bad7bfc83a8a37086fd4b239958c61b77f86426289a03531f4b64", "class_name": "RelatedNodeInfo"}}, "text": "However, this makes its handling \n\ndifficult. The removal of water from viable biological \n\nmaterial in the frozen state (freeze-drying) provides \n\nanother means of arresting the biological clock by \n\nwithholding water, and commencing again by its \n\naddition. Lyophilization or freeze-drying is a \n\ncontrollable method of dehydrating labile products by \n\nvacuum desiccation [3]. It is considered advantageous \n\nto freeze dry viruses and vaccines wherever possible in \n\norder to reduce their volume for storage in cold, to \n\nenable easy handling and transport, and to enhance \n\ntheir keeping quality [4]. \n\nMaterials and Methods \nCell Line and Virus Stock Preparation  \nA confluent monolayer (90%) of the Hep-2 cell lines \n\nwere grown in 25mm\u00b2 presterile disposable NUNC \n\ntissue culture flask using (full form?)MEM with \n\nantibiotics (penicillin, streptomycin) and 10% fetal calf \n\nserum (FCS). Adenovirus reference strain isolated from \n\nDepartment of virology was propagated for virus stock \n\npreparation in Hep2cell line and used for TCID50 assay \n\nand viral load. \n\nVirus Titration by Tissue culture infectious \n\ndose 50 (TCID50) Assay \nHep2 cell monolayer was prepared and virus infection \n\nwas performed at different dilutions in 10ml sterile \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   16    Nepjol.info/index.php/njb \n\ntissue culture tubes. Cell monolayer was observed daily \n\ntill the cytopathic effect (CPE) appeared, along with the \n\ncontrol wells (MEM inoculated) kept under the same \n\ncondition. TCID50 titer was be calculated by using Reed \n\nand Muench [5].  \n\nExtraction of viral DNA \nViral DNA was be extracted from 200\u00b5l of tissue \n\nculture lysate using RNeasy Minikit (QIAGEN, GmbH, \n\nHilden, Germany) according to the manufacturer\u2019s \n\ninstructions. In brief, 200\u00b5l of AVL Buffer containing \n\n20\u00b5l of carrier RNA were dispensed in 1.5 ml of micro \n\ncentrifuge tube. 200\u00b5l of tissue culture fluid was added \n\nto the Buffer AVL containing proteinase K into micro \n\ncentrifuge tube and is mixed by vortexing for 15 sec. \n\nThe suspension was incubated in the dry temperature \n\nbath (56\u00b0C) for 10 minutes followed by the \n\ncentrifugation.  After adding 200\u00b5l of absolute ethanol, \n\nthe sample was vortexed for 15sec and centrifuged \n\nbriefly. Carefully 620\u00b5l of the solution was be applied \n\nto the QIAamp Mini column with 2ml of collecting tube \n\nwithout wetting the rim. The cap is closed and \n\ncentrifuged at 6000g (8000rpm) for 1 min. The QIAamp \n\nMini column is placed into the 2ml of clean collecting \n\ntube and the tube containing filtrate were discarded. \n\nThe QIAamp Mini column were opened carefully and   \n\n500\u00b5l of AW1 Buffer were added to the QIAamp Mini \n\ncolumn and centrifuged at 10000g for 1 minute. \n\nFollowed by the replacement of new 2ml of collection \n\ntube and the tube containing filtrate were discarded. \n\n500\u00b5l of the AW2 Buffer was added to the column and \n\ncentrifuged at 20000g for 3 minutes. Followed by the \n\nreplacement of new 2ml of collection tube and the tube \n\ncontaining filtrate were discarded and centrifuged at \n\nfull speed for 1 minute. Finally the QIA amp mini \n\ncolumn was placed in a Sterile, DNAase, RNAase, free \n\n1.5ml of the micro centrifuge tube. The old collecting \n\ntube containing the filtrate was discarded. 50\u00b5l of AVE \n\nBuffer was added to the QIAamp Mini column and \n\nwere equilibrated to room temperature for 1 min \n\nfollowed by centrifugation at 8000g for 1 min.", "start_char_idx": 3628, "end_char_idx": 7168, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "95da32fc-83db-4ad5-bd0f-73c132f364f8": {"__data__": {"id_": "95da32fc-83db-4ad5-bd0f-73c132f364f8", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c8ff4beb-a4d3-46e2-bef5-53aab6c6b463", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "9dc1fdfec627d52b673be7218854811872e7286968354e5ab38faed21b16e7d6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e616c400-c297-4713-a3b0-4fa757cad70a", "node_type": "1", "metadata": {}, "hash": "44a68920382303a31f418c04f9b08546b83b2dfd2a38ad6791ffd98e6ae48580", "class_name": "RelatedNodeInfo"}}, "text": "Competent cells preparation, Ligation \n\nreaction and Plasmid isolation \nDH5-\u03b1, a non-pathogenic strain of E.coli was used for \n\nthe purpose. It were grown overnight in 100ml conical \n\nflask containing Luria broth (LB). The overnight \n\nbacterial culture was diluted 1/200 to 25 ml of SOC \n\nmedia in 10X culture volume flasks (25 ml in 250 ml \n\nflask). Flask containing bacteria was grown to early log \n\nphase in shaker incubator. The growth of the culture \n\nwas measured at an interval of 30 minutes until an OD \n\nof 0.4 at 600nm were obtained. The cells were collected \n\nby centrifugation at 5000g for 5 min in cold centrifuge \n\n(4\u00b0C) and were kept on ice in all the further steps. The \n\ncells were re suspended in 12 ml of 0.1M ice cold CaCl2 \n\nand placed on ice for minimum 30 minutes. The cells \n\nwere centrifuged at 5000g for 10 min and the pellet was \n\ncollected. The pellet was further re suspended in 1/10 \n\nvolume of 0.1M CaCl2. Cells were ready to use as \n\ncompetent cells for the transformation experiment. Ice \n\ncold sterile glycerol was added to final concentration of \n\n10% (v/v) with a gentle mixing. Aliquots of 100\u00b5l of \n\ncells were made and stored at -70\u00b0C until further use. \n\nThe tube containing the pGEM-T easy vector and the \n\ncontrol insert DNA tubes were centrifuged briefly to \n\ncollect the content at the bottom of the tube. The 2X \n\nrapid ligation buffer was vigorously vortexed before \n\nuse.  Ligation reaction was prepared and reaction \n\nmixture was mixed properly by pipetting and was \n\nincubated for 30 min at 4\u00b0C for optimal ligation.  Two \n\n\u00b5l of the ligation reaction were added to the eppendorf \n\ntube containing 100 \u00b5l of the competent cells. The \n\ntransformation tube containing ligation reaction \n\nmixture and competent cells were given heat shock at \n\n42\u00b0C for 40-50 second and was immediately returned to \n\nthe ice bucket. 950\u00b5l of the SOC media containing \n\nantibiotic was added to the above tube and were \n\nincubated in a shaking incubator for 1.5 hrs at 37\u00b0C \n\nwith shaking (~150 rpm). 100 \u00b5l of the transformation \n\nculture was plated onto duplicate \n\nLB/ampicillin/IPTG/X-Gal plates.  The plates were \n\nincubated overnight at 37\u00b0C and were screened for blue \n\nand white colonies. The white colonies were the desired \n\ninsert. To facilitate the blue color development, plates \n\nwere stored at 4\u00b0C (after 37\u00b0C overnight incubation). \n\nFinally the white colonies, supposed to contain the \n\nvector with the desired insert were selected and were \n\ngrown in 10 ml of LB containing 100 \u00b5g/ml Ampicillin, \n\novernight in a shaker incubator at 37\u00b0C at speed of \n\n250rpm. The bacterial cells grown in the LB medium by \n\naddition of antibiotic were harvested by centrifugation \n\nat 6000g for 15 min at 4\u00b0C. The cell pellet was re-\n\nsuspended in 0.3 ml of buffer P1. 300\u00b5l of buffer P2 was \n\nadded and mixed thoroughly and vigorously and were \n\nincubated at room temperature (15-25\u00b0C) for 5 min.  \n\n300\u00b5l of chilled buffer P3 was added followed by \n\nmixing vigorously inverting the tube (8-10 times) and \n\nwere incubated on ice for 5 min. Mixture was \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   17    Nepjol.info/index.php/njb \n\ncentrifuged at 10,000g in a micro centrifuge for 10 min. \n\nThe supernatant containing the plasmid DNA were \n\naspirated out carefully.", "start_char_idx": 7172, "end_char_idx": 10573, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e616c400-c297-4713-a3b0-4fa757cad70a": {"__data__": {"id_": "e616c400-c297-4713-a3b0-4fa757cad70a", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "95da32fc-83db-4ad5-bd0f-73c132f364f8", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "2693a87ed08fc1cab96b2c27dc5d5945944834db31e6d79375b58358bfea330d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "30f4cd29-5e18-4b66-98be-b4ff650cd217", "node_type": "1", "metadata": {}, "hash": "7d737174f56b2742672ebd8ff5dcda564a250b10f916b6db1b9f0530e3119e13", "class_name": "RelatedNodeInfo"}}, "text": "The supernatant containing the plasmid DNA were \n\naspirated out carefully. During the mean time \n\nQIAGEN-tip 20 was equilibrated by applying 1ml of \n\nthe buffer QBT, and was allowed to empty the column \n\nby gravity flow. The supernatant obtained after \n\ncentrifugation was applied to the QIAGEN-tip 20 and \n\nallowed to enter the resin by gravity flow. The \n\nQIAGEN-tip 20 were washed with 2x2 ml of buffer QC, \n\nFollowed by elution of DNA with 0.8ml of buffer QF. \n\nThe eluted DNA was precipitated by adding \n\nisopropanol at room temperature. (0.56ml per 0.8ml of \n\nelution volume). The mixture was be then mixed and \n\ncentrifuged immediately at \u2265 10,000rpm for 30 min in a \n\nmicro centrifuge at 4\u00b0C and the supernatant was be \n\ndecanted carefully. The residual DNA pellet were \n\nwashed with 1ml of 70% ethanol at least thrice by \n\ncentrifuging the pellet at 10,000rpm for 10 min each. \n\nThe supernatant was aspirated carefully without \n\ndisturbing the pellet. The pellet was air dried for 5-10 \n\nmin, and was re-dissolved in 100\u00b5l of TE buffer, pH 8.0. \n\nVirus revival, titration and Viral load in \n\nlyophilized adenovirus \nLyophilized virus was revived in sterile conditions \n\nusing sterile double-distilled water. The virus ampoule \n\nwas broken with a clean metal rod in one strike at the \n\nneck region of the ampoule in the biosafety cabinet. 1ml \n\nsterile water were added to the dried virus and mixed \n\nby pipetting atleast 20 times. The revived virus was \n\nkept at 4\u00b0C for 2h so as to acclimatize virus. The virus \n\nwas aliquoted and further stored at -70\u00b0C. To \n\ndetermine the effect of lyophilization on virus titer the \n\nlyophilized virus after revival was titrated in Hep-2 cell \n\nline and virus titer was determined by TCID50 and PFU \n\nfollowing the same procedure as described earlier for \n\nthe stock virus. To determine the effect of \n\nlyophilization on adenoviral load the lyophilized \n\nadenovirus was be subjected to Real Time PCR as \n\ndescribed earlier and the Ct values obtained was \n\nextrapolated with standard curve to estimate the viral \n\nload. \n\nStatistical Analysis  \nStatistical analysis of significance was undertaken by a \n\npaired sample t-test using SPSS/ QQ Graph Pad Prism \n\nsoftware with a value of P<0.05 for significance. \n\nResult \nConfirmation of Stock Virus \nAdenovirus reference strain (kindly obtained from \n\nDepartment of Virology, PGIMER, Chandigarh) was \n\nsuccessfully propagated in (Hep-2) cells. Cytopathic \n\neffect (CPE) positive cell culture bottles for adenovirus \n\ninfected cells showed cell grapening, clustering, \n\nrounding, clumping and focal dislodging of cells in \n\ncomparison to control mock infected cells were seen on \n\nthird to fourth day post inoculation. CPE positive cells \n\nwere scrapped with a sterile cell scrapper and subjected \n\nto Immunofluorescence using polyclonal antisera for \n\nAdV. It showed the characteristic intra nuclear brilliant \n\napple green fluorescence in adeno infected cells. \n\nAdenovirus PCR was done in virus infected cell line \n\nwhere a fragment of 161bp was amplified from hexon \n\ngene of AdV genome and visualized by 2% agarose gel \n\nelectrophoresis (Figure 1). \n\n \nFigure 1: Agarose gel analysis of adenovirus PCR; lane1: \n\n100bp Molecular marker (Fermentas, USA), lane2: negative \n\ncontrol, lane3: AdV \n\nThese culture bottles were preserved at -80\u00baC deep \n\nfreezer and subjected to repeated freeze and thaw in \n\nthree occasions for cell lysis. The supernatant was \n\ntransferred to a sterile vial and centrifuged aseptically. \n\nThe clear supernatant occasions for cell lysis.", "start_char_idx": 10499, "end_char_idx": 14059, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "30f4cd29-5e18-4b66-98be-b4ff650cd217": {"__data__": {"id_": "30f4cd29-5e18-4b66-98be-b4ff650cd217", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e616c400-c297-4713-a3b0-4fa757cad70a", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "137ee1767115aa55d1e25a73ff1b76a5260a90b4cd02ae2ba572a5c35c677203", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4abb4281-eb69-4c90-b5e4-b1bf90a83d72", "node_type": "1", "metadata": {}, "hash": "c3d1349ed93eea8a5a718ea2cc6ab39550c82466af68b7431d486bb6a8902f62", "class_name": "RelatedNodeInfo"}}, "text": "The clear supernatant occasions for cell lysis. The \n\nsupernatant was transferred to a sterile vial and \n\ncentrifuged aseptically. The clear supernatant \n\ncontaining the virus particles was used as positive \n\ncontrol for qualitative RT-PCR as well as preparation of \n\nstandards in Quantitative Real Time RT-PCR. \n\nComparison of TCID50 titer of lyophilized \n\nvirus stored for different time intervals \nThe lyophilized virus was stored at room temperature \n\n(RT) and 4\u00b0C for 1month, 4 months and 6 months. The \n\nvirus was revived after these fixed intervals of time and \n\ninfected in Hep-2 to check the infectivity of virus and \n\nchange in the Tissue culture infectious dose 50 (TCID50) \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   18    Nepjol.info/index.php/njb \n\ntiter. It was observed that lyophilized virus was \n\ninfective till 6 months and there was no change in the \n\nTCID50 titer of the lyophilized AdV revived after \n\n1month to 6 months of storage at 4\u00b0C. In case of \n\nlyophilized AdV stored at RT the TCID50 titer was \n\nobserved to reduce from 10-4.5 at 1 month to 10-3.22 at 4 \n\nmonths but it remained constant to 10-3.22 after 6 \n\nmonths of storage (Figure 2 and 3).  \n\n \nFigure 2: TCID50 of lyophilized virus stored at RT and 4\u00b0C \n\nand revived after 1 month, 4 and 6 months. The value of \n\nTCID50 on Y- axis corresponds to 10-y of different sample of \n\nadenovirus. \n\n \nFigure 3: Comparison of TCID50 in stock virus and \nlyophilized virus revived at different time intervals. The \nvalue of TCID50 on Y- axis corresponds to 10-y of different \nsample of adenovirus \n\nComparison of TCID50 titer of lyophilized \n\nvirus stored for different time intervals and \n\nstock virus \nThe TCID50 titer of the lyophilized adenovirus stored at \n\nRT and 4\u00b0C for 1, 4 and 6 months was compared with \n\nthat of the stock virus. The TCID50 titer of lyophilized \n\nvirus is summarized in Table 1 whereas the TCID50 \n\ntiter of stock virus was observed to be 10-3.  \n\nTable 1: Arrangement of data in calculation of TCID50 titer \nby Reed and Muench formula \nVirus \ndilution \n\nInfected Non-\nInfected \n\nAccumulative Values \n\nInfected Non\u2013\ninfected \n\n          Mortality \n\nRatio Percent \n\n10-1 4 0 14 0 14/14 100 \n\n10-2 4 0 10 0 10/10 100 \n\n10-3 3 1 6 1 6/7 85.71 \n10-4 2 2 3 3 3/6 50 \n10-5 1 3 1 6 1/7 14.28 \n10-6 0 4 o 10 0/10 0 \n\n \n\nNo significant difference (p, 0.342) was observed in the \n\nTCID50 titer or the infectivity of the lyophilized virus \n\nstored at 4\u00b0C for 1, 4 and 6 months as well as AdV \n\nstored at RT for 4 and 6 months as compared to the \n\nstock virus. The adenoviral load in stock and \n\nlyophilized adenovirus was correlated with TCID50 titer \n\nas described in the Table 2 using Spearman correlation \n\ncoefficient. However, no statistical correlation was \n\nobserved in the viral load and TCID50 titer.", "start_char_idx": 14012, "end_char_idx": 16921, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4abb4281-eb69-4c90-b5e4-b1bf90a83d72": {"__data__": {"id_": "4abb4281-eb69-4c90-b5e4-b1bf90a83d72", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "30f4cd29-5e18-4b66-98be-b4ff650cd217", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "16937cfae0900e8edf877dc523786c2fda08fab439252e1ea841904d1055167c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f0f75531-c570-482e-afc8-fd0f9cf3bf9c", "node_type": "1", "metadata": {}, "hash": "79795429e9cd2607826add8657d888c7d61e8925197d87c45c1c34856f8a9972", "class_name": "RelatedNodeInfo"}}, "text": "Table 2: Correlation of TCID50 and Viral load in lyophilized and \nstock virus \n\nConfirmation of successful cloning  \nHexon region of the viral genome was cloned in pGEM-\n\nT Easy vector (chloramphenicol resistance) and \n\ntransformed in E coli DH5\u03b1. Cloned plasmid \n\ncontaining the AdV hexon insert had a defective LacZ \n\ngene and those cells are transformed with this vector \n\nformed white colony in ampicillin agar plate using JH7 \n\nand KW53 primers (Figure 4).  \n\n \nFigure 4: Plate showing Blue (vectors without inserts) and \n\nwhite (vectors with insert) colonies. \n\nStock virus TCID50 Viral load by Real-Time \nPCR(Copies/ml) in 104 \n\n 10-3 1590 \n\n \nStore\nd at \n4\u00b0C \n\nStored \nat RT \n\nStored \nat 4\u00b0C \n\nStored \nat RT \n\nLyophilized AdV \nrevived after 1 \nmonth \n\n10-4.5 10-3.7 2920 6810 \n\nLyophilized AdV \nrevived after 4 \nmonths \n\n10-3.22 10-4.5 2220 7200 \n\nLyophilized AdV \nrevived after 6 \nmonths \n\n10-3.22 10-3.7 672 2650 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   19    Nepjol.info/index.php/njb \n\nWhite colony from the plate, theoretically containing \n\nthe desired inserts were picked and subjected to \n\nplasmid extraction. The plasmids isolated from the \n\nwhite colonies were subjected to PCR to yield 161bp \n\nbands in agarose gel. Polymerase Chain reaction (PCR) \n\nresulted in 161 bp amplicon with hexon region specific \n\nprimer pair (Figure 5). \n\nFigure 5: Conformation of successful cloning; lane1: 100 Base \n\npair molecular marker (Fermentas, USA), lane 2: NC and 3: \n\nPC & lane 4: 161 bp band using hexon specific primer in AdV \n\nclone. \n\nComparison of Viral load of lyophilized virus \n\nstored for different time intervals \nThe effect of lyophilization on viral load of AdV over a \n\nperiod of time was studied. No significant difference \n\nwas observed in viral load of AdV stored at 4\u00b0C for 1, 4 \n\nand 6 months. \n\nHowever, in lyophilized virus stored at RT a significant \n\ndifference in viral load was observed between the virus  \n\nstored for 1month and that stored for 6 months at RT \n\n(P<0.05) but no difference was observed in viral load of \n\nAdV stored at RT for 1 and 4 months (Figure 6). \n\n \nFigure 6: Viral load of lyophilized virus stored at RT and 4\u00b0C \n\nfor 1 to 6 months \n\nDiscussion  \nFreeze-drying will not reverse the damage incurred \n\nprior to formulation and care must be exercised when \n\nselecting an appropriate cell type or technique used to \n\nculture or purify the cell or its extracts prior to freeze-\n\ndrying. To sustain freeze-drying it is necessary to \n\nestablish a pressure gradient from a sample (highest \n\npressure), to condenser, and finally vacuum pump \n\n(lowest pressure) so that water migrates from the \n\nsample as drying progresses. In the present study \n\nadenovirus was successfully propagated in Hep-2 cells. \n\nThe lyophilization cycle was standardized and the \n\nvirus suspension was lyophilized at temperature below \n\n-40\u00b0C and under 50m Torr vacuum. The essence of the \n\nformulation exercise should be to minimize freeze-\n\ndrying damage, loss of viability, or activity. This study \n\nconsiders the stability of adenovirus after freeze-drying \n\nand storage and also determines its titer and load. The \n\ninfectivity of the virus was retained when stored for \n\nover 6 months at 4\u00b0C or RT.", "start_char_idx": 16926, "end_char_idx": 20263, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f0f75531-c570-482e-afc8-fd0f9cf3bf9c": {"__data__": {"id_": "f0f75531-c570-482e-afc8-fd0f9cf3bf9c", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4abb4281-eb69-4c90-b5e4-b1bf90a83d72", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "c717ef8f97e63a10e107bd527ed3586c4f5595f789faff7a4c111d77ba850681", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e2002cc2-1b08-4f03-ac5c-6c97bfa68506", "node_type": "1", "metadata": {}, "hash": "65d0c7ded60b7e9843fd2be0b816955fb1e9a4e10950e2346cb8abec12aafba4", "class_name": "RelatedNodeInfo"}}, "text": "We determined the TCID50 \n\ntiter of the adenovirus lyophilized for 1-6 months and \n\nobserved no significant difference in the infectivity \n\nwhen stored at 4\u00b0C or RT. Thus, the cell culture-grown \n\nadenovirus could withstand the freeze-drying process, \n\nand it should be stored at 4\u00b0C and RT to retain \n\nmaximum infectivity. Many culture collections and \n\ngene banks insist on high recovery values prior to a \n\nprotocol being adopted for regular use; 50% viability \n\npost thaw has been accepted in some culture collections \n\nas a nominal cutoff for adopting maintenance by \n\ncryopreservation alone (McClure et al., 2011). The ability \n\nto determine viral titer rapidly and at high accuracy is \n\none of the most important tools desired when working \n\nwith viruses in the research laboratory [7, 8]. Real-time \n\nPCR has been shown to be more sensitive than cell \n\nculture based techniques for detection of viruses in \n\nclinical specimens; it was therefore interesting to \n\ninvestigate whether real-time PCR technology could \n\nalso be an important tool for rapid and efficient \n\nestimation of viral titer [9]. In the present study the viral \n\nload of stock and lyophilized adenovirus was \n\ndetermined and compared with each other. The viral \n\nload of the lyophilized virus stored at 4\u00b0C was \n\nobserved to increase as compared to the stock virus \n\nwhereas that stored at RT for 1 and 4 months the viral \n\nload increased slightly but at 6 months the viral load \n\ndecreased as compared to the stock virus. There is \n\nevidence that a cryopreservation method yielding high \n\ninitial recovery values, maintains viability at that level \n\non prolonged storage. Titer determined using either by \n\nplaque forming unit (PFU) or endpoint dilution \n\n(TCID50) assays, methods that are time-consuming and \n\nlabor intensive [10]. As a complement to these \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   20    Nepjol.info/index.php/njb \n\nestablished methods of determination of virus titers, we \n\nhere describe a Real-Time Quantitative PCR assay that \n\nenables rapid recording of viral load values that \n\ncorresponds to titer measurements of viruses. \n\nHowever, the results presented here showed no \n\nsignificant relationship between Real Time PCR v/s \n\nTCID50. Even though the TCID50 technique is time \n\nconsuming, need expertise but is capable of dealing \n\nwith the infective/live virus which has the great \n\nimportance in viral isolation, manufacturing live-\n\nattenuated vaccines, viral antigen and viral standards. \n\nReal Time PCR which detects both the infective and \n\nnon-infective viral particles is highly sensitive and \n\nrapid, used most popularly in the viral diagnostic labs \n\nand has great importance in the early diagnosis and \n\nmanagement by of the patient [9]. It should, however, \n\nbe taken into consideration that viruses causing \n\npersistent infection that consequently would give a \n\npositive signal in real-time PCR cannot be converted to \n\nviral titer, as this would give a false impression that the \n\nvirus is able to cause cytopathic effect. Our data also \n\ndemonstrated that using real-time PCR technology is a \n\nreliable, rapid and robust method that corresponds to \n\nstandard quantification methods used to measure viral \n\ntiters. Thus, highly reproducible over a dynamic range \n\nof viral concentrations commonly used in research \n\nlaboratories and could therefore save time in \n\ncomparison with the traditional cell culture-based viral \n\ntitration methods. The reproducibility of quantitative \n\nreal-time PCR was higher than for TCID50, which could \n\nbe partially explained by the fact that real-time PCR \n\ndoes not require a manual estimation based on visual \n\nobservations. \n\nConclusion \nThe present study could successfully lyophilize \n\nadenovirus and retain its infectivity over a period of 6 \n\nmonths when stored at RT and 4\u00b0C. No significant \n\ndifference in the infectivity or TCID50 titer was \n\nobserved in the lyophilized virus as compared to the \n\nstock virus.", "start_char_idx": 20264, "end_char_idx": 24343, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e2002cc2-1b08-4f03-ac5c-6c97bfa68506": {"__data__": {"id_": "e2002cc2-1b08-4f03-ac5c-6c97bfa68506", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f0f75531-c570-482e-afc8-fd0f9cf3bf9c", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "4d22ace8691534ca2df56782a581b2459a296bffaf2e357fd8c3d81fb551d317", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a5afb757-002d-4ce0-9ffd-dd161883b69a", "node_type": "1", "metadata": {}, "hash": "4665bf1f7a3c1caf851f8f6380cb043c98566309119910a831c7787367b1002d", "class_name": "RelatedNodeInfo"}}, "text": "However, the viral load was observed to \n\nincrease with lyophilization of the virus over 6 months \n\nwhen stored at 4\u00b0C which possibly is due to the \n\nconcentration of the virus on freeze-drying. \n\nCompeting Interest \nThe authors declare no competing interest.  \n\nAuthors Contribution \nBimlesh Kumar Jha designed the experiment. Bimlesh \n\nKumar Jha, Birendra Prasad Gupta, Prashanna \n\nMaharjan, Somila kakshapati and Nabin Narayan \n\nMunankarmi performed the cloning and transfection \n\nexperiment. Bimlesh Kumar Jha, Prashanna Maharjan, \n\nSomila kakshapati, Nabin Narayan Munankarmi and \n\nBirendra Prasad Gupta performed the virus titration \n\nexperiment and data analysis. Bimlesh Kumar Jha, \n\nPrashanna Maharjan, Somila kakshapati Nabin \n\nNarayan Munankarmi and Birendra Prasad Gupta \n\nwrote the manuscript.  \n\nAcknowledgement \nWe would like to thank National Institute of Virology \n\n(NIV) Pune for providing us viral stock.  \n\nReference \n1. Walls T, Shankar AG, Shingadia D: Adenovirus: an \n\nincreasingly important pathogen in paediatric bone \n\nmarrow transplant patients. Lancet Infect Dis 2003, \n\n3(2):79-86. \n\n2. Croyle MA, Cheng X, Wilson JM: Development of \n\nformulations that enhance physical stability of viral \n\nvectors for gene therapy. Gene Ther 2001, 8(17):1281-\n\n1290. \n\n3. Nail SL, Jiang S, Chongprasert S, Knopp SA: \n\nFundamentals of freeze-drying. Pharm Biotechnol \n\n2002, 14:281-360. \n\n4. Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, \n\nWinter G: Systematic investigation of the effect of \n\nlyophilizate collapse on pharmaceutically relevant \n\nproteins I: stability after freeze-drying. J Pharm Sci \n\n2010, 99(5):2256-2278. \n\n5. Chen S, Guo D, Guo B, Liu J, Shen Y, Xu X, Huang W, \n\nGuo S: Investigation on formulation and preparation \n\nof adenovirus encoding human endostatin \n\nlyophilized powders. Int J Pharm 2012, 427(2):145-152. \n\n6. McClure C, Cole KL, Wulff P, Klugmann M, Murray \n\nAJ: Production and titering of recombinant adeno-\n\nassociated viral vectors. J Vis Exp 2011(57):e3348. \n\n7. Xiao X, Li J, Samulski RJ: Production of high-titer \n\nrecombinant adeno-associated virus vectors in the \n\nabsence of helper adenovirus. J Virol 1998, 72(3):2224-\n\n2232. \n\n8. Aurnhammer C, Haase M, Muether N, Hausl M, \n\nRauschhuber C, Huber I, Nitschko H, Busch U, Sing \n\nA, Ehrhardt A et al: Universal real-time PCR for the \n\ndetection and quantification of adeno-associated", "start_char_idx": 24344, "end_char_idx": 26739, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a5afb757-002d-4ce0-9ffd-dd161883b69a": {"__data__": {"id_": "a5afb757-002d-4ce0-9ffd-dd161883b69a", "embedding": null, "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-39", "node_type": "4", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "032c8b6e8ac1e9e01d32671c466391c0e59951b10fcb453097ffc32e069f474e", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e2002cc2-1b08-4f03-ac5c-6c97bfa68506", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "2e1d549f33a8cfb38fbfe94fbf487a6183e6d3404a6ca6a1bd24d46858ea4ecb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "05cdd3c4-49ac-4b8f-82a8-8790381e0bc0", "node_type": "1", "metadata": {}, "hash": "5de92121c9f8e05a6d81b5ca111006b7f74916491fba11f17bbb94875ec76b88", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:15-21                   Jha et \n\nal. \n     \n\n\u00a9NJB, Biotechnology Society of Nepal   21    Nepjol.info/index.php/njb \n\nvirus serotype 2-derived inverted terminal repeat \n\nsequences. Hum Gene Ther Methods 2012, 23(1):18-28. \n\n9. Grigorov B, Rabilloud J, Lawrence P, Gerlier D: Rapid \n\ntitration of measles and other viruses: optimization \n\nwith determination of replication cycle length. PLoS \n\nOne 2011, 6(9):e24135. \n\n10. Biacchesi S, Skiadopoulos MH, Yang L, Murphy BR, \n\nCollins PL, Buchholz UJ: Rapid human \n\nmetapneumovirus microneutralization assay based \n\non green fluorescent protein expression. J Virol \n\nMethods 2005, 128(1-2):192-197.", "start_char_idx": 26744, "end_char_idx": 27452, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "05cdd3c4-49ac-4b8f-82a8-8790381e0bc0": {"__data__": {"id_": "05cdd3c4-49ac-4b8f-82a8-8790381e0bc0", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a5afb757-002d-4ce0-9ffd-dd161883b69a", "node_type": "1", "metadata": {"identifier": "njb-39", "author": "Jha, Bimlesh Kumar; Gupta, Birendra Prasad; Maharjan, Prashanna; Kakshapati, Somila; Munankarmi, Nabin Narayan", "title": "Effect of lyophilization on infectivity and viral load of Adenovirus", "date": "2015-12-30", "file": "njb-39.pdf"}, "hash": "fd97d9042ba106b072412e3c237cfa41430696cd2b0952e7131014b9612351b1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "addedf29-2342-407a-a2e0-e408f14112c7", "node_type": "1", "metadata": {}, "hash": "ea13c9c641bd547cb79ae8aae8540d8cef42d93a5f0a7f1ad37894698f0d7c8f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  D e c . 9 (2): 21-28 Research article DOI: https://www.doi.org/10.54796/njb.v9i2.41910 \n\n\u00a9NJB, BSN 21 \n\nPotential surface active agent production using very low grade and cheap \nsubstrate by Bacillus subtilis as microbial cell factory \nNiranjan Koirala1,2\u2e38 , Sareeta Khanal1,3\u2e38, Sujan Chaudhary3,4 , Sagar Gautam5, Shiv Nandan Sah3 , Prince \n\nSubba3, Najat Marraiki6, Gaber El-Saber Batiha7\n\n1Department of Natural Products Research, Dr. Koirala Research Institute for Biotechnology and Biodiversity, \n\nKathmandu 44600, Nepal \n2Laboratory of Biotechnology, Faculty of Science and Technology, University of Macau, Macau SAR 999078, China \n3Department of Microbiology, Central Campus Technology, Dharan 56700, Tribhuvan University, Nepal \n4Department of Botany, Amrit Science Campus, Kathmandu 44600, Tribhuvan University, Nepal \n5Department of Microbiology. Trichandra Multiple Campus,Ghantaghar, Kathmandu 44600, Tribhuvan University, \n\nNepal \n6Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia \n7Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour \n\n22511, AlBeheira, Egypt \n\u2e38Both the authors contributed equally to this work and share the first authorship \n\nReceived: 15th Oct 2020; Revised: 19th Nov 2021; Accepted: 20th Dec 2021; Published online: 31st Dec 2021 \n\nAbstract \nBio-surfactants are surface-active molecules which are produced by the wide range of microbes including bacteria, fungi, \nmoulds, and yeast. This study was conducted to identify bio-surfactants by Bacillus subtilis combined with use of cheap \nsubstrates and industrial wastes (Mustard cake, Whey and Soya cake) which are found locally in Nepal. Bacillus subtilis, one \nof the most potential bio-surfactants producer; was isolated from soil sample of hydrocarbon contaminated site. Isolates were \ngrown in a Minimal Salt Media (MSM) with 10% (v/v) mustard oil cake, whey and soya cake separately. The presence and \npotential of surfactant was determined by the oil spreading technique, emulsification index (%E24) and surface tension \nmeasurement. It was revealed that the surface tensions of cell free extract were 54.41, 60.02 and 56.64 mN/m for from mustard \ncake, whey and soya cake respectively as compared to distilled water (72.09) at 25oC. The emulsification index values was \nfound to be highest in engine oil from the bio-surfactant extracted from mustard cake, soya cake and whey respectively. \nSimilarly, mustard oil showed the lowest value of emulsification index. The highest emulsification activity was shown in \nmustard oil i.e. 1.13 from the cell free extract from mustard oil and lowest in engine oil i.e., 0.07, by the extract from soya cake \nmedium, when measured in spectrophotometer at 540 nm. In conclusion, strain of Bacillus subtilis was found to be the potential \nsurface active agent producers on the mustard oil cake, which can be useful medium for various environmental, food, \nmedicinal and industrial processes. \n\nKeywords: Bacillus subtilis; Bio-surfactants; Emulsification index; Hydrocarbons; Surface tension\n Corresponding author, email: koirala.biochem@gmail.com \n\nIntroduction \nSurfactants are the compounds capable of reducing \n\nsurface tension between any two substances of same or \n\ndifferent phase.", "start_char_idx": 48, "end_char_idx": 3408, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "addedf29-2342-407a-a2e0-e408f14112c7": {"__data__": {"id_": "addedf29-2342-407a-a2e0-e408f14112c7", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "05cdd3c4-49ac-4b8f-82a8-8790381e0bc0", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "4b9518c5c7ddce6282b778d33fd2ddf65d6676baa6d67be6ef6695a77261769a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "708ba53a-1c45-47db-9412-8ca09f296d5c", "node_type": "1", "metadata": {}, "hash": "7e39bf73cf851f2e1e86f08928ed4ffa0d1d03eda1ecaf0f83e8de81f10a7547", "class_name": "RelatedNodeInfo"}}, "text": "Surfactants can be either chemically \n\nsynthesized or obtained biologically. When surfactants \n\nare produced on living surfaces, mostly microbial cell \n\nsurfaces or excreted extracellularly, they are termed as \n\nbio-surfactants. Such bio-surfactants generally contain \n\nhydrophobic and hydrophilic moieties[1]. Surfactants are \n\ncharacterized by their capacity to alter the surface and \n\ninterfacial properties of a liquid, which allows the \n\nformation of micro emulsions, which further allows oils \n\nand related substances to become solubilized in water or \n\nvice-versa [2]. Such properties of surfactant enable a wide \n\nrange of industrial applications including emulsification, \n\ndetergency, foaming capacity, lubrication, moisture \n\nretention, solubilization, and phase dispersion [3]. \n\nIn recent years, there has been a steady increase in the \n\ninterest in bio-surfactants as they have numerous \n\nadvantages over the chemical surfactants. Few of such \n\nadvantages include lower toxicity, higher \n\nbiodegradability, higher foaming, better environmental \n\ncompatibility and effective properties even at extreme \n\ncondition of temperature, pH and salinity[4]. In light of \n\nsuch advantages, bio-surfactants can be used for oil \n\nrecovery, treatment of oil spillage and bioremediation, in \n\nfoods, cosmetics and pharmaceuticals. Moreover, due to \n\ntheir biodegradability they can safely be used in the \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\nhttps://orcid.org/0000-0002-7777-1191\nmailto:koirala.biochem@gmail.com\nhttps://orcid.org/0000-0002-8300-4570\nhttps://orcid.org/0000-0003-3300-8191\nhttps://orcid.org/0000-0002-7817-425X\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 22 \n\nenvironment without the risks observed of some of their \n\nchemically synthesized counterparts. As a result, bio-\n\nsurfactant occupies an advantageous position both for \n\nresearch and industrial production [5].The most \n\nprevalent bacterial species capable of  producing  \n\nsurfactant belong to the genera are Pseudomonas, , \n\nMicrococcus Flavobacterium,  Bacillus, Acinetobacter, \n\nAchromobacter, Arthrobacter, Klebsiella, Aeromonas, \n\nAlkaligenes, Streptococcus sp, Corynebacteriumsp, Moraxella, \n\nand Proteobacteria [6]. Microorganism  utilize the \n\nsubstrate i.e. hydrocarbons to produce bio-surfactant and \n\noften minimalize them and produce  ecologically \n\nharmless products [7]. Bacillus is one of such genus of \n\nbacteria associated with the production of vital bio-\n\nsurfactant. They are gram positive rod shaped aerobic \n\nspore former bacterium commonly found in soil. \n\nMembers of the genus are considered as a suitable group \n\nfor research as well as synthesis of bio-surfactants \n\nindustrially mainly due to their capacity to produce \n\nvarious metabolites including surface active ones [8]. \n\nThey not only produce good bio-surfactants, but are also \n\ncapable of growing under facultative or anaerobic \n\nconditions, and have also been reported to be non-\n\npathogenic, which permits their use in wide range \n\nindustries, apart from environmental applications [8]. \n\nAmong the many species, Bacillus subtilis is one of the \n\nmost potential species capable of producing a wide range \n\nof extracellular metabolites including surfactant.", "start_char_idx": 3409, "end_char_idx": 6800, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "708ba53a-1c45-47db-9412-8ca09f296d5c": {"__data__": {"id_": "708ba53a-1c45-47db-9412-8ca09f296d5c", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "addedf29-2342-407a-a2e0-e408f14112c7", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "2e21e533cdfb7ab01b0dcf0baec6e39dd9f15cf9a4fb884d5293e121f7be6163", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5a9ea8e4-4f06-4d0d-b2d3-2485070166cd", "node_type": "1", "metadata": {}, "hash": "e1a4367511bda7e0acb57a5bacf1255ed6f1df87ecb0a6e41c946ef68db5d998", "class_name": "RelatedNodeInfo"}}, "text": "Surfactin, a lipopeptide bio-surfactant  produced from B. \n\nsubtilis, is  able to reduce the surface tension of water to \n\n25 mN/m and interfacial tension of water/hexadecane \n\nup to <1 mN/m which is among the best result given by \n\nany class of bio-surfactants. Most bio-surfactant can \n\nlower surface tension of water from 72 to35 mN/m and \n\nthe interfacial tension of water/ hexadecane from 40 to 1 \n\nmN/m [9]. The cosmopolitan distribution of B. subtilis, \n\nability to form resistant spores and extreme tolerance \n\nability of the bacteria makes it a suitable candidate for \n\nfurther research and industrial application for the \n\npurpose.  \n\nHydrocarbons are commonly used as the substrate for \n\nthe production of bio-surfactants. It has been postulated \n\nthat the biological function of surface-active compounds \n\nis related to hydrocarbon uptake, and therefore a \n\nspontaneous release occurs with these substrates[10, 11]. \n\nA wide range of hydrocarbon rich substrate has been \n\nexperimented and studied for the commercial application \n\nfor bio-surfactant production. However, there have been \n\ndifficulties in industrial production and \n\ncommercialization of the product due to the higher \n\nmanufacturing cost. The cost of substrate account for 10-\n\n30 % of total production cost of bio-surfactant which \n\nmakes the overall production cost higher that chemical \n\nsurfactants [12].This is mainly due the use of different \n\nchemically synthesized media for production. \n\nConsequently, chemical surfactant holds a higher \n\nposition in the market. Thus, the practical and possible \n\nway to win over the market of chemical surfactant is to \n\nreduce the manufacturing cost of bio-surfactant which \n\ncan then be made available in the market at lower cost. \n\nReduction in cost of substrate by using lower grade and \n\ncheap substrate and using more efficient microbes could \n\nsignificantly reduce the manufacturing cost [13]. \n\nPresent study aims for the production of bio-surfactant \n\nby B. subtilis on the industrial wastes, cheaper and low \n\ngrade substrates (mustard oil cake, soya cake and whey) \n\nwhich are found locally and abundantly in different areas \n\nof Nepal. \n\nMaterials and Methods \nThis work was conducted at the Central Campus of \n\nTechnology, Hattisar, Dharan. The eight soil samples \n\nfrom auto-mobile workshop (garage) were collected form \n\nDharan-8 by using simple random sampling method. All \n\nsamples were collected in aseptically dried and clean \n\nplastic bags and were transported to laboratory as soon \n\nas possible. \n\nBacterial isolation \nThe test organism (Bacillus subtilis) was isolated form a \n\nsoil sample, by heating test samples at 80o C/ 10 min by \n\nusing water bath to destroy all the vegetative cells.  Then \n\n1g of soil was weighed and was suspended in 9 ml sterile \n\ndistilled water. Serial dilution of soil sample was done up \n\nto104. Nutrient agar with 1% soluble starch was prepared \n\nonto which an aliquot (0.1 ml) of 102 to 104 dilutions were \n\ninoculated and spread plate method was followed. The \n\nplates were incubated aerobically at 37o C for 48 hours \n\n[14]. \n\nColonies that showed big, creamy, wrinkle, and \n\nspreading colonies were picked and sub cultured on \n\nnutrient agar broth and on nutrient agar slant. Gram \n\nstaining and endo-spore staining were carried out for the \n\npresumptive identification of the isolates[14]. \n\nIdentification \nThe test isolate was identified by using standard \n\nmicrobiological techniques as described in Bergey\u2019s \n\nManual of Systematic Bacteriology (1986). Different \n\nbiochemical tests viz; catalase, citrate, urease, indole, \n\nstarch hydrolysis, methyl red voges-proskauer, sugar \n\nfermentation test (Lactose, Mannitol, Sucrose), Triple \n\n\n\nNepal J Biotechnol.", "start_char_idx": 6803, "end_char_idx": 10546, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5a9ea8e4-4f06-4d0d-b2d3-2485070166cd": {"__data__": {"id_": "5a9ea8e4-4f06-4d0d-b2d3-2485070166cd", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "708ba53a-1c45-47db-9412-8ca09f296d5c", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "4b296ca9606749bd89768d643b53f10b3d359dcc6ca7566dd07292c997779978", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ed551080-6183-4e80-abc1-1002210357a8", "node_type": "1", "metadata": {}, "hash": "ae3606cd09a3a013dab343211ae05713f6c6bab1f1b5c95fd552f14eec2fd4cd", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 23 \n\nSugar Iron (TSI) and SIM were carried out to identify the \n\nisolates. \n\nSubstrate Preparation \nThe medium used for the experiment was a Minimal Salt \n\nMedium supplemented with 10% substrate (Mustard oil \n\ncake, Soya cake and Whey). It was prepared by \n\ndissolving 1.73 g dipotassium phosphate, 0.68g \n\npotassium dihydrogen phosphate, 0.1 g magnesium \n\nsulphateheptahydrade, 0.33 g ferrussulphate, 4 g sodium \n\nchloride, 1 g ammonium nitrate, 0.02 g calcium chloride \n\nand 5 g glucose[15]. \n\nInoculation and incubation \nOne ml of 24 hours broth culture of the isolate was \n\npipetted into each flask. All six flasks were plugged with \n\ncotton and allowed to stand in water bath shaker for 5 \n\ndays. Temperature of the water bath shaker was \n\nmaintained at 37o C. After 5 days all flask were taken out \n\nfrom shaker and centrifugation was done to obtain cell \n\nfree supernatant [15]. \n\nOil spreading technique \nThe oil displacement test was performed according to the \n\nprotocol described by Walter (2010). A Petri-dish (150 \n\nmm diameter) was filled with 40 ml of distilled water. 15 \n\nml of weathered crude oil was added. The crude oil will \n\nform a thin oil layer on the water surface. Then, 10 ml of \n\nfree cell culture supernatant was carefully placed on the \n\ncenter of the oil film. If there were microbial surfactants \n\npresent in the supernatant, the oil was displaced and a \n\nclearing zone was formed [16]. \n\nDetermination of emulsification activity \n0.5 ml of the extracted supernatant was added to 7.5 ml \n\nof 1M tris-HCl buffer and 0.1 ml of oil (kerosene, mustard \n\noil, sunflower oil and engine oil). The mixture was \n\nvigorously vortexed and allowed to stand for 1 hour. \n\nAbsorbance was measured at 540 nm 5 times at an \n\ninterval of 1 hour. After obtaining the absorbance \n\nemulsification activity was calculated by calibrated \n\ngraph [17]. \n\nMeasurement of Emulsification Index (E24) \nThe bacterial broth was centrifuged and was studied for \n\nits emulsifying ability by a modified method of [18]. Two \n\nml Cell-free broth was pipetted into the screw cap test \n\ntube, and 3 ml of oil (Kerosene, Mustard, Engine and \n\nSunflower) was then added. The mixture was vortexed at \n\nhigh speed for 2 min and left at room temperature. The \n\nresult was observed after 24 h for the stability of \n\nemulsion. Photograph 4 shows the test tubes left for the \n\ncalculation of emulsification index.  The total volume of \n\nthe mixture, volume of emulsified, and volume of non-\n\nemulsified phase was observed [3]. The emulsification \n\nindex (E24) was calculated by the equation: \n\nE24 =  \nHeightofemulsionlayer\n\nTotalHeight\n x100 \n\nThe method was followed at the end of each day to obtain \n\nreading for 5 days. \n\nSurface tension measurement \nSurface Tension was determined at room temperature i.e. \n\n30oC by Drop Number Method by using \n\nStalagmometer[19]. Basically, the weight of a drop of a \n\nliquid falling after passing through a capillary tube of \n\nuniform radius is approximately proportional to the \n\nsurface tension of the liquid.", "start_char_idx": 10527, "end_char_idx": 13638, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ed551080-6183-4e80-abc1-1002210357a8": {"__data__": {"id_": "ed551080-6183-4e80-abc1-1002210357a8", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5a9ea8e4-4f06-4d0d-b2d3-2485070166cd", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "89bd17d936657dda3f84497f16c2522b08376e342b1b29e1d2c8d941e54d5b40", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f2b24b67-b5e3-4b6e-a230-dfd85bc71d27", "node_type": "1", "metadata": {}, "hash": "4d2af60a088c7ab9a825446bc4ef42f3e8a8a908dc6484b3f3594cba3c318203", "class_name": "RelatedNodeInfo"}}, "text": "For two liquids of surface \n\ntension \u03d21 and \u03d22, d1 and d2 are the densities and n1 and \n\nn2 are the number of drops made by the same volume of \n\nliquids then the surface tension is calculated as \n\n\u03d21\n\n\u03d22\n=\n\n\ud835\udc511\ud835\udc4b\ud835\udc5b2\n\n\ud835\udc5b1\ud835\udc4b\ud835\udc512 \nPyknometer was used to determine the density of a liquid \n\nand Stalagmometer was used to determine the number of \n\ncomplete drops made by a liquid [20]. \n\nResults \nBacterial Isolation and identification \nThe test organism (Bacillus subtilis) was isolated from a \n\nsoil sample of automobile workshop. Presence of Bacillus \n\nsubtilis was confirmed based on the macroscopic \n\nexamination of colonies, microscopic examination and \n\nthe different chemical tests which were performed as \n\ngiven in Table 1. The microscopic examination of the \n\nisolates after Gram staining is shown in Photograph 1 \n\nand the biochemical test for the conformation of the \n\nisolates is shown in Photograph 2. \n\nTable 1. Biochemical tests for confirmation of Bacillus subtilis \n\nBiochemical test Bacillus subtilis properties \n\nCatalase Positive \nOxidase Positive \nCitrate  Positive \nIndole Negative \nMR (Methyl Red) Negative \nVP (VogesProskauer) Positive \nUrease Negative \nStarch hydrolysis Positive \nGelatin hydrolysis Positive \nSucrose Acid production \nLactose Negative \nGram staining Positive \nEndospore staining Positive (Central spore) \nNitrate reduction Positive \nArabinose Positive \nArabitol Positive \nGlucose Positive \nGlycerol Positive \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 24 \n\nEmulsification activity (EA) \n\nFigure 1. The emulsification activity of cell free broth \n\nextracted from mustard cake \n\nFigure 2. The emulsification activity of cell free broth extracted \n\nfrom whey \n\nFigure 3. The emulsification activity of cell free broth extracted \n\nfrom soya cake \n\nThis study recorded the emulsification activity was \n\nobserved highest in mustard oil with the cell free extract \n\nfrom mustard oil cake whereas engine oil showed the \n\nlowest (Figure. 1). Emulsification activity in mustard oil \n\nfrom extract from mustard oil cake was found to be 1.13, \n\n0.99, 0.81, 0.95 and 0.76 at the time interval of 1, 2, 3, 4 and \n\n5 hour respectively. However, EA of engine oil was found \n\nto be 0.13, 0.06, 0.08, 0.07 and 0.07 at the time span of 1, 2, \n\n3, 4 and 5 hour respectively (Figure 1). Similarly, EA by \n\nextract from whey was also found to be higher of mustard \n\noil. Emulsification value of mustard oil was found to be \n\n0.69, 0.58, 0.46, 0.53 and 0.42 at the time span of 1, 2, 3, 4 \n\nand 5 hour respectively (Figure 2). The EA by the extract \n\nfrom whey was followed by sunflower oil, kerosene oil \n\nand engine oil with the lowest emulsification activity \n\n(Figure 2). EA in mustard oil was again found \n\ndominating in the extract from the soya cake followed EA \n\nin sunflower oil, kerosene oil and engine oil (Figure 3) \n\nrespectively. The emulsification activity of cell free broth \n\nextracted from mustard cake, Whey and Soya cake are \n\nshown in Figure 1, 2 and 3 respectively. \n\nEmulsification index (E24) \nThe emulsification index revealed highest in engine oil by \n\nthe extract from mustard oil cake whereas mustard oil \n\nshowed the lowest values (Table. 1).", "start_char_idx": 13639, "end_char_idx": 16851, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f2b24b67-b5e3-4b6e-a230-dfd85bc71d27": {"__data__": {"id_": "f2b24b67-b5e3-4b6e-a230-dfd85bc71d27", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ed551080-6183-4e80-abc1-1002210357a8", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "474ccde524e822cd4c44a6371cef8c72d7713d825bcd6435949bdd89b6641223", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9ed46c53-e17a-48f7-bcdf-3f54aea4e53e", "node_type": "1", "metadata": {}, "hash": "c0a5455bedff4766063f96f308a57bc98ab96bdbcb5cb79594073538382155e3", "class_name": "RelatedNodeInfo"}}, "text": "1). Corresponding E24 \n\nin Engine oil by extract form mustard oil cake substrate \n\nwas found to be 22.09, 28.55, 29.96, 32.85 and 38.82 in the \n\ntime span of 1, 2, 3, 4, and 5 day respectively while E24 in \n\nmustard oil was 1.43, 5.57, 7.27, 9.32 and 12.21 in 1-5 day \n\nrespectively. Similarly, E24 in engine oil with extract from \n\nwhey was found to be highest with 11.40, 14.48, 23.60, \n\n33.45 and 36.30 in the time span of 1, 2, 3, 4 and 5 day \n\nrespectively. Emulsification index of extract from the \n\nwhey in different medium was followed by sunflower, \n\nkerosene and mustard oil. Similar result was found with \n\nthe extract from soya cake. Emulsification index of bio-\n\nsurfactants obtained from Mustard oil, Whey and Soya \n\ncake are listed in Table 1, 2 and 3 respectively. \n\nTable 2. Emulsification Index of bio-surfactants \nobtained from Mustard oil cake \n\nTime \n(Day) \n\nKerosene \noil \n\nSunflower \noil \n\nMustard \noil \n\nEngine \noil \n\n1 4.44 19.28 1.43 22.09 \n\n2 5.80 20.01 5.57 28.55 \n\n3 9.26 20.81 7.27 29.96 \n\n4 12.50 22.74 9.32 32.85 \n\n5 17.86 23.26 12.21 38.82 \n\nTable 3. Emulsification Index of bio-surfactants obtained \nfrom Whey. \n\nTime \n(Day) \n\nKerosene \noil \n\nSunflower \noil \n\nMustard \noil \n\nEngine \noil \n\n1 0.85 16.40 2.79 11.40 \n\n2 3.27 18.38 5.43 14.48 \n\n3 6.69 19.62 6.63 23.60 \n\n4 9.36 20.08 8.48 33.45 \n\n5 11.61 21.29 10.33 36.30 \n\n0.00\n\n0.20\n\n0.40\n\n0.60\n\n0.80\n\n1.00\n\n1.20\n\n1 2 3 4 5\n\nA\nb\n\nso\nrb\n\na\nn\n\nce\n (\n\n5\n4\n\n0\nn\n\nm\n)\n\nTime (hrs)\n\nKerosine Oil\nEngine Oil\nSunflower Oil\nMustard Oil\n\n0.00\n\n0.10\n\n0.20\n\n0.30\n\n0.40\n\n0.50\n\n0.60\n\n0.70\n\n0.80\n\n1 2 3 4 5\n\nA\nb\n\nso\nrb\n\na\nn\n\nce\n (\n\n5\n4\n\n0\nn\n\nm\n)\n\nTime (hrs)\n\nMustard oil\nKerosine Oil\nEngine Oil\nSunflower Oil\n\n0.00\n\n0.10\n\n0.20\n\n0.30\n\n0.40\n\n0.50\n\n0.60\n\n0.70\n\n0.80\n\n1 2 3 4 5\n\nA\nb\n\nso\nrb\n\na\nn\n\nce\n (\n\n5\n4\n\n0\nn\n\nm\n)\n\nTime (hrs)\n\nMustard oil\nKerosine Oil\nEngine Oil\nSunflower Oil", "start_char_idx": 16848, "end_char_idx": 18690, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9ed46c53-e17a-48f7-bcdf-3f54aea4e53e": {"__data__": {"id_": "9ed46c53-e17a-48f7-bcdf-3f54aea4e53e", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f2b24b67-b5e3-4b6e-a230-dfd85bc71d27", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "5c7393289203b97b193462c4470c0bd3153277b2b67515caab6f825c4ddb04fd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6c7c27e0-b49a-417f-91e2-786ea4d3f8dd", "node_type": "1", "metadata": {}, "hash": "31afc7ef046c812eb6f9046de38a16ee88bb953a751087a7c8e851af7ac6376e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 25 \n\nTable 4. Emulsification Index of bio-surfactants obtained \nfrom Soya cake. \n\nTime \n(Day) \n\nKerosene \noil \n\nSunflower \noil \n\nMustard \noil \n\nEngine \noil \n\n1 1.77 16.79 3.73 17.76 \n2 4.90 18.68 4.74 22.01 \n3 8.54 20.50 6.31 25.80 \n4 9.08 21.64 7.57 32.70 \n\n5 12.84 22.20 8.06 37.05 \n\nThe emulsification index was found to be increasing \n\nevery day and the maximum result was obtained at day \n\n5. The comparative study of the emulsification index of\n\nthree substrates with different oils is given in Figure 4. \n\nFigure 4. Bar diagram for comparative study of emulsification \n\nindex of three substrates with different oil at maximum level (5 \n\ndays). \n\nSurface tension \nThe surface tension of water at 30oC was 71.34 dyne/cm. \n\nThe result calculated from cell free broth of mustard oil \n\ncake, whey and soya cake were found to be 54.41, 60.02 \n\nand 56.64 dyne/cm respectively (Table 4). Photograph 3 \n\nshows the cell free broth extracted using different \n\nsubstrates. \n\nTable 5. The surface tension of cell free broths \n\nCell free broth Surface tension (dyne/cm) \n\nMustard Cake 54.41 \nWhey 60.02 \n\nSoya cake 56.64 \n\nPhotograph 1. Gram stain of B. subtilis \n\nPhotograph 2. Biochemical test (citrate) \n\nPhotograph 3. Bio-surfactants extraction (cell free broth) \n\nPhotograph 4. Emulsification index of Hydrocarbons. \n\nDiscussion \nBio-surfactants, compounds with such wonderful \n\nadvantages over chemical ones, have not yet been \n\ncommercialized significantly as a result of high \n\nproduction cost. Since the cost of substrates, chemical \n\nones used more widely, account for 10-20% of the \n\n0.00\n\n5.00\n\n10.00\n\n15.00\n\n20.00\n\n25.00\n\n30.00\n\n35.00\n\n40.00\n\n45.00\n\nMustard cake Whey Soya cake\n\nE\nm\n\nu\nls\n\nif\nic\n\na\nti\n\no\nn\n\n i\nn\n\nd\ne\n\nx\n \n\nKerosene oil Engine oil\nmustard oil Sunflower oil\n\ni) Sunflower oil ii) Engine oil    iii) Kerosene oil\n\ni) Mustard ii) Whey iii) Soya\n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 26 \n\nproduction cost of bio-surfactants, the production cost is \n\nhigher [12]. The possible measure to minimize the cost is \n\nto reduce the cost of substrate i.e. use of cheap substrates \n\npreferably natural substrates like mustard oil cake, soya \n\ncake and whey. Present study was performed with the \n\nobjective of identifying the utility of the substrates \n\nmentioned above for production of bio-surfactants using \n\nbacterial species isolated from oil contaminated soil. \n\nFrom the total 4 soil samples, one isolate of Bacillus \n\nsubtillis was isolated based on its morphological and bio \n\nchemical characteristics based on the study of Banat et al. \n\n[21] and used for the production of bio-surfactant. \n\nHowever, the study couldn\u2019t identify the exact strain as a \n\nresult of less resources for genetic analysis. The isolate \n\nwas then accessed for its capacity to produce bio-\n\nsurfactant in mustard oil cake, soya cake and whey. \n\nLuckily it was able to grow on all the substrate used.", "start_char_idx": 18694, "end_char_idx": 21696, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6c7c27e0-b49a-417f-91e2-786ea4d3f8dd": {"__data__": {"id_": "6c7c27e0-b49a-417f-91e2-786ea4d3f8dd", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9ed46c53-e17a-48f7-bcdf-3f54aea4e53e", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "583658aa57ff6f74f5279d4b612b3efb8f9ceb9be05d26ef2ad8d196fc56c017", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1b1652d4-6cf0-42ec-86ea-11e4dd202d01", "node_type": "1", "metadata": {}, "hash": "3f807ce05c55eff3cd4e3029cb192e10a6bd244aa353cfd6efec6a4ddc27d7fc", "class_name": "RelatedNodeInfo"}}, "text": "Luckily it was able to grow on all the substrate used. The \n\noil spreading technique proved that the isolate has the \n\nability to produce the surfactin as it spread the oil when \n\ncell free broth was poured on it. The result is comparable \n\nto the study of Banat et al.[22]. \n\nThe produced extracellular metabolites were then \n\ncompared by calculation of emulsification activity and \n\nemulsification index on 4 different oil medium; Kerosene \n\noil, Mustard oil, Engine oil and Sunflower oil. The \n\nemulsification activity was found to be highest for \n\nmustard oil while lowest for engine oil. This shows that \n\nthe bio-surfactants extracted possessed lower ability to \n\nemulsify the mustard oil but has higher to engine oil. \n\nSince, the habitat of microbial used in the study was from \n\nautomobile workshop, the organism showed higher \n\nability to emulsify the engine oil as the organism might \n\nhad adapted to the oil contaminated soil. \n\nThis study revealed that the emulsification index on all \n\ntested oils mediums was found a little higher by the bio-\n\nsurfactant produced in mustard oil cake as compared to \n\nsoya cake and whey. This might be due to the ability of \n\nB. subtilis to utilize nitrogen from various sources for cell \n\nmultiplication and biosynthetic pathway [23]. This point \n\nwas also supported by Patel and Desai [24] where \n\noptimum C/N ratio would be a favorable factor in \n\nmustard oil cake for the production of surfactants by B. \n\nsubtilis. \n\nBio-surfactant produced from the respective substrate \n\nhas the highest emulsification index value of 38.82, 37.05 \n\nand 36.05% (Table 1, 2 and 3) respectively in engine oil \n\nwhile they have the lowest emulsification index value of \n\n12.21, 10.33 and 8.06% (Table 1, 2 and 3) respectively in \n\nmustard oil. Similarly, the emulsification index in \n\nsunflower was also found higher value than in the \n\nmustard oil. This difference of emulsification index in \n\ndifferent medium can be attributed to biochemical \n\nproperties of the medium. Sunflower oil contains the long \n\nchain of mono and polyunsaturated fatty acids (91.49 \u00b1 \n\n1.91) compared to mustard oil (86.80 \u00b1 3.07) [25]. A \n\nsimilar prediction based on the length of hydrocarbon \n\nchain can be made about the higher value of \n\nemulsification index in engine oil. However, any solid \n\nresearch is lacking regarding the topic and is open for \n\nspeculations. \n\nThe Emulsification index in each of the oil medium is \n\nfound to be increasing gradually with increase in time in \n\ncase of each of the bio-surfactant extracted. The fifth day \n\nshowed the maximum value of emulsification index for \n\neach of the cell free extracts, which is quiet obvious as the \n\nindex is found to increase with time in most of the \n\nprevious similar studies. With increase in time, the \n\nemulsion gradually gets back together forming a separate \n\nlayer as before being vortexed. This study has also \n\nrevealed that the surface tension of the extracted bio-\n\nsurfactants from the substrate mustard cake, whey and \n\nsoya cake was found to lie within the normal range as \n\nprescribed by [26, 27] \n\nMany similar studies have identified the bacterial strain \n\nas well as the possible substrates of bio-surfactant \n\nproduction. Makkar and Cameotra[28] have reported the \n\nstudies on bio-surfactant production by Bacillus strains \n\nunder thermophilic conditions on sucrose and molasses \n\nas substrate. Al-Bahry et al.  has reported production of \n\nbio-surfactant by Bacillus subtilis B20 using date molasses \n\nand its possible application in enhanced oil recovery [29]. \n\nNevertheless, the mustard oil cake, an agro-industrial \n\nwaste, could be the potential substrate for the commercial \n\nbio-surfactant production as suggested by present study.", "start_char_idx": 21642, "end_char_idx": 25385, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1b1652d4-6cf0-42ec-86ea-11e4dd202d01": {"__data__": {"id_": "1b1652d4-6cf0-42ec-86ea-11e4dd202d01", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6c7c27e0-b49a-417f-91e2-786ea4d3f8dd", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "decd6bbf37e2ed944e74afbfadffa147a4e5aed19d82ce5374402f40e58f0340", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3058fa36-c5d7-4014-a431-428d9abb6518", "node_type": "1", "metadata": {}, "hash": "3dccfad5c14fb41e1ff5e77460e5d3cafb2dc10f806444cc6c25481deb76480b", "class_name": "RelatedNodeInfo"}}, "text": "Although the potential of the crude bio-surfactant \n\nproduced is lower than expected, it cannot be denied that \n\nhigher potential could have been achieved with better \n\nresources. Moreover, the extracted crude surfactant was \n\nalso able to give satisfying result despite of being crude. \n\nHowever, the study could have been considered more \n\nachieving if the purity of the crude extract could have \n\nbeen increased.  \n\nConclusion \nThe result of this study showed that the strain of Bacillus \n\nsubtilis isolated from Mustard cake, Whey and Soya cake \n\nwas found to be the potential surface active agent \n\nproducers which are useful tools for various \n\nenvironmental, food and industrial processes. The \n\norganism isolated from oil contaminated area showed \n\ngreater ability to produce bio-surfactants. Furthermore, \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 27 \n\neach of the substrates obtained from industrial and \n\nhousehold waste were found to show great potential as \n\nsubstrate for the production of bio-surfactant. Among \n\nthem, the mustard oil cake substrate is cheap and best \n\namong the three studied substrate for bio-surfactant \n\nproduction. \n\nAuthor\u2019s Contribution \nConception, data acquisition, analysis and drafting were \n\ndone by SK, SC, PS, and SG. Experimental work was \n\nperformed by SK, SG, SC, and PS. Writing and \n\npreparation of manuscript was performed by SC, SG, and \n\nSK. Analysis and interpretation of data and critical \n\nrevision of manuscript was done by PS, SNS, GE-SB and \n\nNM. Supervision by NK and Funding acquisition was \n\ndone by GE-SB, NM and NK. Final approval of \n\nmanuscript was done by all the authors. \n\nAcknowledgement \nWe want to express our sincere gratitude to the helping \n\nhand members/Staffs of laboratory for making our work \n\nmore convenient. The authors also appreciate the \n\nresearchers\u2019 supporting project number RSP-2020/201 \n\nfrom King Saud University, Riyadh, Saudi Arabia. We \n\nwant to thank all the researchers who have previously \n\nworked in the realm of this topic. \n\nConflict of Interest \nThe authors declare that there is no conflict of interest \n\nwith present publication. \n\nDeclaration \nWe hereby declare that this study is our own work and \n\nthat to the best of our knowledge and belief. It contains \n\nno matter and data previously published or produced by \n\nanother party.  \n\nReferences \n1. Karanth, NGK., Deo P, Adi V, Microbial production of \n\nbiosurfactants and their importance. Current Science, 1999; 77. \n2. Dubey KV, Charde PN, Meshram SU, Shendre LP, Dubey VS, \n\nJuwarkar AA. Surface-active potential of biosurfactants produced \nin curd whey by Pseudomonas aeruginosa strain-PP2 and Kocuria \nturfanesis strain-J at extreme environmental conditions. \nBioresource technology. 2012 Dec 1;126:368-74. \nhttps://doi.org/10.1016/j.biortech.2012.05.024\n\n3. Walter V, Syldatk C, Hausmann R. Screening concepts for the \nisolation of biosurfactant producing microorganisms. \nBiosurfactants. 2010:1-3. https://doi.org/10.1007/978-1-4419-\n5979-9_1 \n\n4. Cho W, Lee EH, Shim EH, Kim J, Ryu HW, Cho KS. Bacterial \ncommunities of biofilms sampled from seepage groundwater \ncontaminated with petroleum oil. Journal of microbiology and \nbiotechnology. 2005;15(5):952-64. \n\n5. Arima K, Kakinuma A, Tamura G, Surfactin, a crystaline \npeptidelipid surfactant produced by Bacillus subtilis: Isolation, \ncharacterization and its inhibition of fibrin clot formation. 1968. 31: \n488-494. \n\n6.", "start_char_idx": 25388, "end_char_idx": 28859, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3058fa36-c5d7-4014-a431-428d9abb6518": {"__data__": {"id_": "3058fa36-c5d7-4014-a431-428d9abb6518", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1b1652d4-6cf0-42ec-86ea-11e4dd202d01", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "e5f253420b80838193687e9a2f3e4bccefb615bcb659a89ec8b3b758737c72cd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f51bdcf5-5282-46ca-916c-695ab925db12", "node_type": "1", "metadata": {}, "hash": "114a28fa1e01cfa3afd5e1ff10a1cfcfd58cea6a5de2488b513182d92a88cc06", "class_name": "RelatedNodeInfo"}}, "text": "1968. 31: \n488-494. \n\n6. Kowall M, Vater J, Kluge B, Stein T, Franke P, Ziessow D. \nSeparation and characterization of Surfactin isoforms produced \nbyBacillus subtilisOKB 105. Journal of colloid and interface \nscience. 1998 Aug 1;204(1):1-8. https://doi.org/ \n10.1006/jcis.1998.5558 \n\n7. Chen SY, Wei YH, Chang JS. Repeated pH-stat fed-batch \nfermentation for rhamnolipid production with indigenous \nPseudomonas aeruginosa S2. Applied microbiology and \nbiotechnology. 2007 Aug;76(1):67-74. https://doi.org/10.1007/ \ns00253-007-0980-2 \n\n8. Das Neves LC, De Oliveira KS, Kobayashi MJ, Penna TC, Converti \nA. Biosurfactant production by cultivation of Bacillus atrophaeus \nATCC 9372 in semidefined glucose/casein-based media. \nInApplied Biochemistry and Biotecnology 2007 (pp. 539-554). \nHumana Press. https://doi.org/10.1007/978-1-60327-181-3_45 \n\n9. Mulligan CN. Environmental applications for biosurfactants. \nEnvironmental pollution. 2005 Jan 1;133(2):183-98. \nhttps://doi.org/10.1016/j.envpol.2004.06.009\n\n10. Hisatsuka K, Nakahara T, Minoda Y, Yamada K. Formation of \nprotein-like activator for n-alkane oxidation and its properties. \nAgricultural and Biological Chemistry. 1977;41(3):445-50. \nhttps://doi.org/10.1080/00021369.1977.10862518\n\n11. Holdom RS, Turner AG. Growth of Mycobacterium rhodochrous \non n\u2010Decane: a New Growth Factor and Emulsifying Agent. \nJournal of Applied Bacteriology. 1969 Dec;32(4):448-56. \nhttps://doi.org/10.1111/j.1365-2672.1969.tb00997.x \n\n12. Sobrinho HB, Luna JM, Rufino RD, Porto AL, Sarubbo LA. \nBiosurfactants: classification, properties and environmental \napplications. Recent developments in biotechnology. \n2013;11(14):1-29. \n\n13. Deleu M, Paquot M. From renewable vegetables resources to \nmicroorganisms: new trends in surfactants. Comptes Rendus \nChimie. 2004 Jun 1;7(6-7):641-6. https://doi.org/10.1016/ \nj.crci.2004.04.002 \n\n14. Aneja KR, Experiments in microbiology, plant pathology and \nbiotechnology. 2012; New Age International Limited, New Delhi\n\n15. Raza ZA, Khan MS, Khalid ZM, Rehman A. Production kinetics \nand tensioactive characteristics of biosurfactant from a \nPseudomonas aeruginosa mutant grown on waste frying oils. \nBiotechnology letters. 2006 Oct;28(20):1623-31. https://doi.org/ \n10.1007/s10529-006-9134-3. \n\n16. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, \nFracchia L, Smyth TJ, Marchant R. Microbial biosurfactants \nproduction, applications and future potential. Applied \nmicrobiology and biotechnology. 2010 Jun;87(2):427-44. \nhttps://doi.org/10.1007/s00253-010-2589-0 \n\n17.", "start_char_idx": 28835, "end_char_idx": 31396, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f51bdcf5-5282-46ca-916c-695ab925db12": {"__data__": {"id_": "f51bdcf5-5282-46ca-916c-695ab925db12", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3058fa36-c5d7-4014-a431-428d9abb6518", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "c51a66f56ce133ddab7ac6bb21b51f993f24441c9af44528205b6cfce9de6777", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9286a4d2-99a6-4b26-8dc3-a7ef335f982f", "node_type": "1", "metadata": {}, "hash": "1415b46a4285c8e77a6149dcdf2b6721ca4e4ded38e7b441549e748dc99b0858", "class_name": "RelatedNodeInfo"}}, "text": "Nakano MM, Marahiel MA, Zuber P. Identification of a genetic \nlocus required for biosynthesis of the lipopeptide antibiotic \nsurfactin in Bacillus subtilis. Journal of Bacteriology. 1988 \nDec;170(12):5662-8. https://doi.org/ 10.1128/ jb.170.12. 5662-\n5668.1988 \n\n18. Cameron DR, Cooper DG, Neufeld RJ. The mannoprotein of \nSaccharomyces cerevisiae is an effective bioemulsifier. Applied \nand Environmental Microbiology. 1988 Jun;54(6):1420-5. \nhttps://doi.org/10.1128/aem.54.6.1420-1425.1988 \n\n19. Khadka NM, Gautam SD, Yadav DPN. Surface Tension, in a core \nexperimental chemistry. Benchmark Education Supporters Pvt. \nLtd.: Kathmandu; 2009: 150-156. \n\n20. Morikawa M, Hirata Y, Imanaka T. A study on the structure\u2013\nfunction relationship of lipopeptide biosurfactants. Biochimica et \nBiophysica Acta (BBA)-Molecular and Cell Biology of Lipids. 2000 \nNov 15;1488(3):211-8. https://doi.org/10.1016/S1388-\n1981(00)00124-4 \n\n21. Banat IM, Makkar RS, Cameotra SS. Potential commercial \napplications of microbial surfactants. Applied microbiology and \nbiotechnology. 2000 May; 53(5):495-508. https://doi.org/10.1007 \n/s002530051648 \n\n22. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, \nFracchia L, Smyth TJ, Marchant R. Microbial biosurfactants \nproduction, applications and future potential. Applied \nmicrobiology and biotechnology. 2010 Jun;87(2):427-44. \nhttps://doi.org/10.1007/s00253-010-2589-0 \n\nhttps://doi.org/10.1016/j.biortech.2012.05.024\nhttps://doi.org/10.1007/978-1-4419-5979-9_1\nhttps://doi.org/10.1007/978-1-4419-5979-9_1\nhttps://doi.org/10.1007/s00253-007-0980-2\nhttps://doi.org/10.1007/s00253-007-0980-2\nhttps://doi.org/10.1007/978-1-60327-181-3_45\nhttps://doi.org/10.1016/j.envpol.2004.06.009\nhttps://doi.org/10.1080/00021369.1977.10862518\nhttps://doi.org/10.1111/j.1365-2672.1969.tb00997.x\nhttps://doi.org/10.1007/s00253-010-2589-0\nhttps://doi.org/10.1128/aem.54.6.1420-1425.1988\nhttps://doi.org/10.1016/S1388-1981(00)00124-4\nhttps://doi.org/10.1016/S1388-1981(00)00124-4\nhttps://doi.org/10.1007/s002530051648\nhttps://doi.org/10.1007/s002530051648\nhttps://doi.org/10.1007/s00253-010-2589-0", "start_char_idx": 31397, "end_char_idx": 33515, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9286a4d2-99a6-4b26-8dc3-a7ef335f982f": {"__data__": {"id_": "9286a4d2-99a6-4b26-8dc3-a7ef335f982f", "embedding": null, "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-4", "node_type": "4", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "ecde5e6291587be800de6fd9f35a6ec9f8b77884cbe60330a923a67f382449bd", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f51bdcf5-5282-46ca-916c-695ab925db12", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "e8d3145124257d16caa2546abeee73caadb05d774317f43250415c495fa0f2b5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70a3f6af-9ff1-428d-8971-374e4f27de0a", "node_type": "1", "metadata": {}, "hash": "678f28d9dfa20342535da3cb67d18c93052da2b41009a3f66beb2fc55b90365f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2021 Dec.9 (2): 21-28  Koirala et al.   \n\n\u00a9NJB, BSN 28 \n\n23. Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. \nOverview of the antimicrobial compounds produced by members \nof the Bacillus subtilis group. Frontiers in microbiology. 2019 Feb \n26;10:302. https://doi.org/10.3389/fmicb.2019.00302\n\n24. Patel RM, Desai AJ. Biosurfactant production by Pseudomonas \naeruginosaGS3 from molasses. Letters in Applied Microbiology. \n1997 Aug;25(2):91-4. \n\n25. Chowdhury K, Banu LA, Khan S, Latif A. Studies on the fatty acid \ncomposition of edible oil. Bangladesh Journal of Scientific and \nIndustrial Research. 2007;42(3):311-6. https://doi.org/10.3329/ \nbjsir.v42i3.669\n\n26. Cooper DG, Goldenberg BG. Surface-active agents from two \nBacillus species. Applied and environmental microbiology. 1987 \nFeb;53(2):224-9. https://doi.org/10.1128/aem.53.2.224-229.1987 \n\n27. Das K, Mukherjee AK. Crude petroleum-oil biodegradation \nefficiency of Bacillus subtilis and Pseudomonas aeruginosa strains \nisolated from a petroleum-oil contaminated soil from North-East \nIndia. Bioresource technology. 2007 May 1;98(7):1339-45. \nhttps://doi.org/10.1016/j.biortech.2006.05.032\n\n28. Makkar RS, Cameotra SS. Biosurfactant production by a \nthermophilic Bacillus subtilis strain. Journal of Industrial \nMicrobiology and Biotechnology. 1997 Jan 1;18(1):37-42. \nhttps://doi.org/10.1038/sj.jim.2900349\n\n29. Al-Bahry SN, Al-Wahaibi YM, Elshafie AE, Al-Bemani AS, Joshi \nSJ, Al-Makhmari HS, Al-Sulaimani HS. Biosurfactant production \nby Bacillus subtilis B20 using date molasses and its possible \napplication in enhanced oil recovery. International \nBiodeterioration & Biodegradation. 2013 Jul 1;81:141-6. \nhttps://doi.org/10.1016/j.ibiod.2012.01.006\n\nhttps://doi.org/10.3389/fmicb.2019.00302\nhttps://doi.org/10.1016/j.biortech.2006.05.032\nhttps://doi.org/10.1038/sj.jim.2900349\nhttps://doi.org/10.1016/j.ibiod.2012.01.006", "start_char_idx": 33518, "end_char_idx": 35444, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70a3f6af-9ff1-428d-8971-374e4f27de0a": {"__data__": {"id_": "70a3f6af-9ff1-428d-8971-374e4f27de0a", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9286a4d2-99a6-4b26-8dc3-a7ef335f982f", "node_type": "1", "metadata": {"identifier": "njb-4", "author": "Koirala, Niranjan; Khanal, Sareeta; Chaudhary, Sujan; Gautam, Sagar; Sah, Shiv Nandan; Subba, Prince; Marraiki, Najat; Batiha, Gaber El-Saber", "title": "Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory", "date": "2021-12-30", "file": "njb-4.pdf"}, "hash": "82f97983fba62fba6e7f41c65a04b0c26f5e0cb8163291d3c1dce8790c81eedc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "08bb07a4-90d4-432b-be1e-fc6ecb856872", "node_type": "1", "metadata": {}, "hash": "bb23e051e7f2e9b005801eb4b2082a3bd57a5e31ad5908b33881290ae18fbdaf", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:10-14 ISSN 2091-1130 (Print) /ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal    10    Nepjol.info/index.php/njb \n\nDoxorubicin Induced Histomorphometric Changes in Testes of \n\nAlbino Rats \nSurendra Kumar Sah1, Saroj Khatiwada2*, Deepak Chaudhary1, Chandra Bhushan Jha3, \n\n Soumya Bhattacharya3 \n1Department of Anatomy, Nobel Medical College, Biratnagar, Nepal \n\n2Department of Biochemistry, Modern Technical College, Lalitpur, Nepal \n3Department of Human Anatomy, B P Koirala Institute of Health Sciences, Dharan, Nepal \n\nAbstract  \nAnticancer drugs like doxorubicin have been found to affect male gonads thereby leading to infertility. This study \n\nwas conducted to evaluate the effects of doxorubicin over short, mid and long term on testes of male albino rats. \n\nSixty male albino rats aged 6-8 weeks were taken for study. The rats were randomly divided into 3 groups of \n\nexperimental (each group containing 10 rats) and 3 groups of control (each group containing 10 rats). The \n\nexperimental groups were given a single dose of doxorubicin i.e. 10 mg/kg body weight intra-peritoneally and \n\nsacrificed after 3 different duration for each group (second week, eighth week and sixteenth week). All rats under 3 \n\ncontrol groups were given a single intra-peritoneal dose of 2.5 ml/kg body weight normal saline and sacrificed with \n\ntheir respective experimental groups. Significant difference in diameters (p=0.029) and cross-sectional area (p=0.028) \n\nof seminiferous tubules was observed between short term experimental and short term control rats. For both \n\nbetween midterm experimental and midterm control group, and between long term experimental and long term \n\ncontrol group, a significant difference in right testis weight (p<0.001 for both), left testis weight (p<0.001 for both), \n\nvolume of testis (p<0.001 and p=0.038), diameter (p<0.001 for both) and area (p<0.001 for both) of seminiferous \n\ntubules was observed. As compared to short term experimental group, midterm experimental group and long term \n\nexperimental group had significantly lower right testis weight (p<0.001 for both), left testis weight (p<0.001 for both), \n\ndiameter of seminiferous tubule (p<0.001 for both) and cross-sectional area of seminiferous tubule (p<0.001 both). \n\nCross-sections of the seminiferous tubules of all the control groups had normal architecture. However, there was \n\nprogressive destruction of seminiferous tubules structure across the experimental groups. Doxorubicin has \n\ndeleterious effect on seminiferous tubules of albino rat testis. \n\nKey words: Doxorubicin, Histomorphometry, Seminiferous tubule, albino rat \n\n*Corresponding author  \n\nEmail: khatiwadasaroj22@gmail.com  \n\nIntroduction \nDoxorubicin, also known as hydroxyd-aunorubicin, \n\nis a drug used in cancer chemotherapy. It is highly \n\neffective in many human tumors and is currently the \n\nfirst line anti-cancer drug in many chemoresponsive \n\ntumors such as ovarian cancers, breast cancers and \n\nlymphomas [1]. The clinical use of doxorubicin can \n\nbe viewed as double edged sword. On one hand, \n\ndoxorubicin plays an undisputed key role in the \n\ntreatment of many neoplastic diseases; on the other \n\nhand, chronic administration of doxorubicin induces \n\norgan toxicity particularly testicular injury [2].", "start_char_idx": 47, "end_char_idx": 3432, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "08bb07a4-90d4-432b-be1e-fc6ecb856872": {"__data__": {"id_": "08bb07a4-90d4-432b-be1e-fc6ecb856872", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70a3f6af-9ff1-428d-8971-374e4f27de0a", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "b42612e698e54d7dd74c66cdeeb3e34ce9596eb00d0532ec856dc2a817d717a6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5ce8707b-d666-4ccd-8701-eb5602b53578", "node_type": "1", "metadata": {}, "hash": "d6c7d60726fd4141ffa38dab7afbb2228b1717d9bacf0d60dd9ff25ef20ddf68", "class_name": "RelatedNodeInfo"}}, "text": "Previous investigations indicated that doxorubicin \n\nhas the ability to induce mutations and \n\nchromosomal aberrations in normal and malignant \n\ncells in addition to a wide variety of toxic side effects \n\non organs like testes [3]. Doxorubicin is said to alter \n\nsperm development, production, structural integrity \n\nand motility rates in association with increased \n\ncellular apoptosis in spermatogonia and spermocyte \n\n[4]. The reduction of oxidative DNA damage by \n\nantioxidants has been evaluated as a \n\nchemotherapeutic approach for reducing damage \n\ncaused by chemotherapy agents such as doxorubicin \n\n[5].  \n\nDamage to the testicular germinal epithelium is a \n\npotential side effect of cancer therapy, and is of \n\nparticular concern in case of men of reproductive \n\nage having tumors with high cure rates [6]. Thus it \n\nseems important to assess the effects of doxorubicin \n\non various components of male reproductive system \n\nand particularly in testis where sperm production \n\ntakes place. Different studies have been carried out \n\nto investigate the effects of doxorubicin on testis, \n\nhowever there are only few studies utilizing \n\nstereological tool to elucidate histomophometric \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:10-14     Sah et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    11    Nepjol.info/index.php/njb \n\nevidences of doxorubicin mediated derangement of \n\nthe testis and evaluate midterm and long term \n\neffects of doxorubicin [7]. Considering this we \n\nconducted the present study among albino rats to \n\nfind short, mid and long term effects of doxorubicin \n\non histomorphometric characters of testis as \n\ncompared to controls. \n\nMethods \nThe study was conducted at the department of \n\nanatomy of B P Koirala Institute of Health Sciences, \n\nDharan, Nepal to find the short, mid and long term \n\neffects of doxorubicin on testis of albino rat as \n\ncompared to controls. Sixty healthy albino rats aged \n\n6-8 weeks and weighing 150-200 gm were obtained \n\nfrom the animal house of anatomy department and \n\nused in study. They were housed in well ventilated \n\nroom at controlled ambient temperature (25\u00b15 0C) \n\nwith a 12 hour alternating light-dark cycle and \n\nprovided standard rodent diet and water \n\nthroughout the study period. The experimental \n\nwork was carried out as per research and ethical \n\nguidelines of Nepal Health Research Council \n\n(NHRC) for the care and use of animals in health \n\nresearch in Nepal and the research protocol was \n\napproved by the institute review board of B P \n\nKoirala Institute of Health Sciences. \n\nThe rats were randomly divided into 3 groups of \n\nexperimental (each group containing 10 rats) and 3 \n\ngroups of control (each group containing 10 rats), \n\nsuch that weight do not vary by more than 10% of \n\naverage weight of study population. The \n\nexperimental groups were given a single dose of \n\ndoxorubicin i.e. 10 mg/kg body weight intra-\n\nperitoneally and sacrificed after 3 different duration \n\nfor each group. Group 1 animals, which were \n\ninvestigated for short term effect, were sacrificed on \n\n14th day (2nd week). Group 2 animals (studied for \n\nmidterm effects) were sacrificed on 56th day (8th \n\nweek), and group 3 animals (studied for long term \n\neffects) on 112th day (16th week). All rats under 3 \n\ncontrol groups (control 1, control 2 and control 3) \n\nwere given a single intra-peritoneal dose of 2.5 \n\nml/kg body weight normal saline and sacrificed \n\nwith their respective experimental groups (at \n\nsecond, eighth and sixteen weeks respectively). \n\nComparison was done among 3 groups of \n\nexperimental rats in terms of effect of doxorubicin, \n\nand as well as between each matched experimental \n\nand control group. \n\nAll rats were weighed at the time of euthanasia. \n\nAnimals were anesthetized with ether soaked in \n\ncotton and their testes were fixed by in vivo \n\nperfusion technique as mentioned below.", "start_char_idx": 3435, "end_char_idx": 7344, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5ce8707b-d666-4ccd-8701-eb5602b53578": {"__data__": {"id_": "5ce8707b-d666-4ccd-8701-eb5602b53578", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "08bb07a4-90d4-432b-be1e-fc6ecb856872", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "58ec500a0eba533aa539710efdb105c367307f2b8b293e24afc6aac9b36d9445", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8abe9866-0f19-4930-928f-db139f803c7d", "node_type": "1", "metadata": {}, "hash": "82ab913429da1a9546d33464126a5b6cafc735fe4378650478647d2a4497d781", "class_name": "RelatedNodeInfo"}}, "text": "Rats were \n\nkept on the dissecting tray with their ventral surface \n\nfacing upward and all four limbs pinned. The \n\nabdomen was opened to expose the abdominal aorta \n\nand inferior vena cava. A 18 guage needle was \n\ninserted on the abdominal aorta and tied with \n\nthread to keep it in constant position. The needle \n\nwas attached to clean flask tube, connected with two \n\nbottles containing bouin\u2019s fluid and physiological \n\nsaline separately. Then the chest was opened and \n\nthe right atrium was nicked by a scissor or knife \n\n(scalpel) to permit the drainage of blood. Thereafter, \n\nthe physiological saline was perfused to flush out \n\nthe blood. The fixative bouin\u2019s fluid was then \n\ntransfused with the help of three ways stopcock. \n\nApproximately 200 ml of each fluid was perfused. \n\nPerfusion was stopped when clear fixative drops \n\nstarted oozing out from the snout of the animal. \n\nAfter completion of perfusion, the testes were \n\nisolated from the scrotum with the help of scalpel \n\nand forceps and weight was measured followed by \n\nvolume measurement by the application of water \n\ndisplacement method and post fixed for 24 hours by \n\nthe same fixative. \n\nAfter 18 hour fixation in bouin\u2019s fluid, the tissue was \n\nprocessed and histological slides were prepared \n\nfrom vertical sections from the polar and the \n\nequatorial regions of each testis. Micrometer was \n\nused for quantitative measurement of parameter; \n\ndiameter of seminiferous tubules, and interstitial \n\nspaces, germ cells, sertoli cells and leydig cells were \n\nobserved qualitatively in histological slides. \n\nDiameter (D) of each seminiferous tubule was \n\nmeasured by two directions and mean of two was \n\ntaken as a diameter of that seminiferous tubule. The \n\ncross-sectional area of the seminiferous tubules were \n\ndetermined by formula; Area (A)=\u03c0D2/4. Two slides \n\nper rat were observed for both control and \n\nexperimental groups. \n\nStatistical analysis was done in SPSS version 19. All \n\ndata were expressed as mean \u00b1SD. Independent t \n\ntest and one way ANOVA was applied to test for \n\nstatistical significance at 95% confidence interval. A", "start_char_idx": 7345, "end_char_idx": 9462, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8abe9866-0f19-4930-928f-db139f803c7d": {"__data__": {"id_": "8abe9866-0f19-4930-928f-db139f803c7d", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5ce8707b-d666-4ccd-8701-eb5602b53578", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "161ac3e31297332e8a9980601854b171ee5d75308555b0866ae01c48ac5b6d6d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2dd0787f-c387-4bf3-afcd-9ce1d176f769", "node_type": "1", "metadata": {}, "hash": "7dfd630c7e06ec4e5aa5561b8e346229a8223153011cfda37f392a6de3b1ab26", "class_name": "RelatedNodeInfo"}}, "text": "A \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:10-14     Sah et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    12    Nepjol.info/index.php/njb \n\nvalue of p<0.05 was considered statistically \n\nsignificant. \n\nResults  \nIt was observed that mean body weight consistently \n\ndecreased among the short term and long term rats. \n\nOn the other hand, there was increase in body \n\nweight among the midterm rats in comparison to its \n\ninitial body weight, as well as in controls. Various \n\nanatomical parameters in experimental groups and \n\ncontrols are shown in table 1. \n\nNo significant difference in right testis weight \n\n(p=0.184), left testis weight (p=0.179), and testicular \n\nvolume (p=0.388), but significant difference in \n\ndiameters (p=0.029) and cross-sectional area \n\n(p=0.028) of seminiferous tubules, were observed \n\nbetween short term experimental and short term \n\ncontrol rats. However, a significant difference in \n\nright testis weight (p<0.001), left testis weight \n\n(p<0.001), volume of testis (p<0.001), diameter \n\n(p<0.001) and area (p<0.001) of seminiferous tubules \n\nwas observed between midterm experimental and \n\nmidterm control group. Similarly significant \n\ndifference in right testis weight (p<0.001), left testis \n\nweight (p<0.001), volume of testes (p=0.038), and \n\ndiameter (p<0.001) and area (p<0.001) of \n\nseminiferous tubules was observed between long \n\nterm experimental and long term control group. \n\nAs compared to short term experimental group, \n\nmidterm experimental group and long term \n\nexperimental group had significantly lower right \n\ntestis weight (p<0.001 for both), left testis weight \n\n(p<0.001 for both), diameter of seminiferous tubule \n\n(p<0.001 for both) and cross-sectional area of \n\nseminiferous tubule (p<0.001 both). However, no \n\nsignificant difference were observed among \n\nmidterm and long term experimental group in terms \n\nof testis weight, volume, diameter and cross-\n\nsectional area of seminiferous tubules.  \n\nFigure 1 (A, B, C and D) shows the cross-section of \n\nseminiferous tubules of control and various \n\nexperimental groups. It was observed that the cross-\n\nsections of the seminiferous tubules of all the control \n\ngroups were moderately circular or oval in outline \n\nwith normal seminiferous epithelium and numerous \n\nspermatozoans within their lumen. The interstitial \n\ncells were also normal and prominent. Rats that \n\nwere given doxorubicin showed progressive \n\ndegenerations in their seminiferous tubules lumen. \n\nGroup 1 rats showed a little reduction in the basal \n\nepithelial layer resulting in the apparent detachment \n\nof the basement membrane. The rats of group 2 \n\n(midterm experimental group) showed \n\nvacuolization of the cells, reduction in the number of \n\nspermatozoa. Lumen of the seminiferous tubules \n\nwas significantly vacant. The interstitial spaces were \n\ndistinct. Group 3 rats (long term experimental \n\ngroup) showed marked variation in its architecture. \n\nThere was much testicular atrophy and most of the \n\nspaces were occupied by the interstitial cells. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:10-14     Sah et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    13    Nepjol.info/index.php/njb \n\n \nFigure 1. A, B, C and D show the cross-section of seminiferous \ntubules of control (all control groups), short term experimental \ngroup, midterm experimental group and long term experimental \ngroup respectively.  \n\nDiscussion  \nDoxorubicin is one of the most widely used \n\nanticancer agents in the clinic despite its dose\u2013\n\nlimiting side-effects.", "start_char_idx": 9461, "end_char_idx": 13045, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2dd0787f-c387-4bf3-afcd-9ce1d176f769": {"__data__": {"id_": "2dd0787f-c387-4bf3-afcd-9ce1d176f769", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8abe9866-0f19-4930-928f-db139f803c7d", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "49a2875e7e7b98f2e02ef0ad0f2890df4efba8dee51ba5cc35b210d0249ccd94", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c6c34579-7aa6-4f32-8022-c40404b483a4", "node_type": "1", "metadata": {}, "hash": "d032618629cd39a88c61953ed22be462af4975e75f0b5cf6a905c74cc37b4a12", "class_name": "RelatedNodeInfo"}}, "text": "In the present study, we \n\nassessed the effects of a single dose (i.e. 10mg/kg \n\nbody weight of animal) of the drug on male albino \n\nrats. Present study shows that weight of the rats that \n\nwere given doxorubicin decreases in comparison to \n\nthe control groups. The decrease in body weight \n\nmay be due to loss of appetite in experimental rats. \n\nWe however observed that, the midterm \n\nexperimental rats showed increase in body weight. \n\nThe increase in body weight in midterm might be \n\ndue to the reversible metabolic changes caused by \n\nthe drug. After some period of time the body regains \n\nits metabolic function and then it again decreases \n\ndue to the long term effect of the drugs. \n\nIn the present study significant difference in \n\ntesticular weight among the midterm experimental \n\nand the midterm control groups, and among the \n\nlong term experimental and the long term control \n\ngroups was observed, which is similar to the \n\nfindings of Saalu et al [7]. We also observed \n\nprogressive decrease in testis weight from short \n\nterm to long term experimental group. Previous \n\nstudy done by Howell et al. also reported that \n\nadministration of doxorubicin can decrease the \n\ntesticular weight of the rats. This could be due to the \n\nsevere parenchymal atrophy in the seminiferous \n\ntubules following the doxorubicin administration \n\n[8]. However study by Prahalathan et al. did not \n\nreport any significant testicular weight changes with \n\ndoxorubicin treatment [9]. \n\nIn the present study, we found that the diameter and \n\ncross-sectional area of seminiferous tubules \n\ndecreases with the time in the rats given \n\ndoxorubicin. As compared to short term \n\nexperimental group, midterm experimental group \n\nand long term experimental group have decreased \n\ndiameter and cross-sectional area of the \n\nseminiferous tubules, which may be due to more \n\neffect of the drugs on exposure to long time. Our \n\nfindings are in accord to previous study by Saalu et \n\nal [7]. Study by Saalu et al. reported that doxorubicin \n\nchemotherapy induces morphological and \n\nmorphometric impairments of testes of rats and \n\nprogressive worsening of testicular derangement \n\nwith time following a single dose of doxorubicin \n\ntreatment [7]. Recent study done in India showed \n\nthat treatment with doxorubicin alone caused \n\ndecrease in body weight, sperm count and serum \n\ntestosterone and increase in serum level of lactate \n\ndehydrogenase (LDH), creatine phosphokinase \n\n(CK), and glutamic oxaloacetate transaminase (GOT) \n\n[10]. \n\nIn the present study, we observed deterioration in \n\nseminiferous tubules architecture in experimental \n\ngroup, and the architecture was more impaired in \n\nlong term experimental group than midterm and \n\nshort term experimental rats. The study also \n\ndemonstrates that testicular cytotoxicity caused by \n\ndoxorubicin provokes germ cell depletion in the \n\nseminiferous epithelium of the rats. Saalu et al. \n\nshowed that even a single dose of doxorubicin \n\nprogressively causes testicular derangement which \n\nmay be due to its capacity to generate intracellular \n\nfree radicals and reactive oxygen species [7]. Single \n\nhigher doses or multiple lower doses of doxorubicin \n\nhave been referred to be toxic to the stem \n\nspermatogonia, diminishing their survival [11]. The \n\nalteration of the measurements of testicular volume \n\nand weight, tubular diameter, seminiferous \n\nepithelium height and volume densities of tubular \n\nlumen and seminiferous epithelium can give \n\ninformation about the testicular damage degree as a \n\nconsequence of germ cell death [12]. Franca et al. \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:10-14     Sah et al. \n\n  \n\n\u00a9NJB, Biotechnology Society of Nepal    14    Nepjol.info/index.php/njb \n\nreported that when a massive germ cell loss occurs, \n\nit is followed by a sharp decline in testicular \n\nmorphometric parameters [13].", "start_char_idx": 13046, "end_char_idx": 16941, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c6c34579-7aa6-4f32-8022-c40404b483a4": {"__data__": {"id_": "c6c34579-7aa6-4f32-8022-c40404b483a4", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2dd0787f-c387-4bf3-afcd-9ce1d176f769", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "ea70336492f15e64ada23262ca0099734ebc579a83cadc81caaaf475056db50b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "42f5a84f-731a-4f49-ba5f-4662489a4ff3", "node_type": "1", "metadata": {}, "hash": "addeb6caab6b063c4e931db075289910d27ea5dcdf7e1b8f62e191d4e6121bb0", "class_name": "RelatedNodeInfo"}}, "text": "In general, germ cell \n\ndeath caused by anticancer drugs, including \n\ndoxorubicin, culminates with a reduction of \n\nmorphometric parameters [14]. Many of doxorubicin \n\ndose-limiting toxicities occur due to its generation of \n\ntoxic oxygen species, resulting in oxidative stress. \n\nIn summary, present study reveals deteriorative \n\neffect of doxorubicin on seminiferous tubules of rats. \n\nThe study has however, several limitations. First, we \n\ndid not assess the ultrastructural changes in testis \n\ncaused by doxorubicin. Also, sperm count was not \n\ndone in this study. The body weight of the \n\nexperimental rats fluctuated with the duration. The \n\nexact causes for the fluctuation of body weight were \n\nnot identified in this study. \n\nCompeting interests \nNone  \n\nAuthor\u2019s contribution \nSKS, SK, DC, CBJ and SB designed the study. SKS, \n\nSK and DC conducted the experiments. SKS and SK \n\nwrote the manuscript. DC, CBJ and SB reviewed \n\nmanuscript. All authors read and approved the final \n\nversion of manuscript. \n\nAcknowledgements \nWe kindly acknowledge B P Koirala Institute of \n\nHealth Sciences for the support in the study.  \n\nReferences  \n1. Atessahin A, Karahan I, Turk G, Gur S, Yilmaz S, \n\nCeribasi AO: Protective role of lycopene on cisplatin \n\ninduced changes in sperm characteristics, testicular \n\ndamage and oxidative stress in rats. Reprod Taxicol. \n\n2006 21(1):42-7. \n\n2. Rubin P, Louis SC, Lawrence BM, Paul O. Late effect of \n\ncancer treatment on normal tissues. Springer Verlog, \n\nBerlin, Heidelberg. 2008 1:109-30. \n\n3. Yagmurcaa M, Erdoganb H, Irazc M, Songurd A, Ucare \n\nM, Fadillioglu E: Caffeic acid phenethyl ester as a \n\nprotective agent against doxorubicin nephrotoxicity in \n\nrats. Clin Chim Acta. 2004 348(1-2):27-34. \n\n4. Sawada T, Tamada H, Mori J: Secretion of testosterone \n\nand epidermal growth factor in mice with \n\noligozospermia caused by doxorubicin hydrochloride. \n\nAndrologia.1994 26(3):151-3. \n\n5. Quiles JL, Huertas JR, Battino M, Mataix J, Ram\u00edrez-\n\nTortosa MC: Antioxidants nutrients and adriamycin \n\ntoxicity. Toxicology. 2002 180(1):79-95. \n\n6. Kobayashi H, Urashima M, Hoshi Y, Uchiyama H, \n\nFujisawa K, Akatsuka J: Testicular morphological \n\nchanges in children with acute lymphoblastic \n\nleukemia following chemotherapy. Pediatrics \n\nInternational. 1996 38:640-3. \n\n7. Saalu LC, Enye LA, Osinubi AA: An assessment of the \n\nhistomorphometric evidences of doxorubicin-induced \n\ntesticular cytotoxicity in Wistar rats. Int J Med Med Sci. \n\n2009 1(9):370-4. \n\n8. Howell ST, Shaler SM: Testicular function following \n\nchemotherapy. Hum Reprod. 2001 7:363-9. \n\n9. Prahalathan C, Selvakumar E, Varalakshmi P: Lipoic \n\nacid ameliorates adriamycin- induced testicular \n\nmitochondriopathy. Reprod Toxicol. 2005 20:111-6. \n\n10. Patil L, R. Balaraman R: Effect of Melatonin on \n\nDoxorubicin induced testicular damage in rats. 2009 \n\n1(3):879-84. \n\n11.", "start_char_idx": 16942, "end_char_idx": 19829, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "42f5a84f-731a-4f49-ba5f-4662489a4ff3": {"__data__": {"id_": "42f5a84f-731a-4f49-ba5f-4662489a4ff3", "embedding": null, "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-40", "node_type": "4", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "bed2156e9c57d5e608cfb989c50ec2d73b7dfe4998c98f3332502acafae5b2b1", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c6c34579-7aa6-4f32-8022-c40404b483a4", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "a34d0f10c837dae706424c7a6c39578289f6f3b84020a92646a106dc6151c9ab", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9f273ac9-7086-4a58-9508-4e2d6ea06835", "node_type": "1", "metadata": {}, "hash": "e86d558bb0e4e3a308cbe3be2eaf109f3ade1a8ac6e31cc1edae1fc38db30280", "class_name": "RelatedNodeInfo"}}, "text": "2009 \n\n1(3):879-84. \n\n11. Jahnukainen K, Hou M, Parvinen M, Eksborg S, S\u00f6der \n\nO: Stage-specific inhibition of deoxyribonucleic acid \n\nsynthesis and induction of apoptosis by antracyclines \n\nin cultured rat spermatogenic cells. Biol Reprod. 2000 \n\n63(2):482-7. \n\n12. Vendramini V, Sasso-Cerri E, Miraglia SM: Amifostine \n\nreduces the seminiferous epithelium damage in \n\ndoxorubicin-treated prepubertal rats without \n\nimproving the fertility status. Reprod Biol Endocrinol. \n\n2010 8:3. \n\n13. Franca LR, Russel LD: The testis of domestic animals. \n\nIn: Mart\u00ednez-Garcia F, Regadera J, eds. Male \n\nreproduction: a multidisciplinary overview. Churchill \n\nCommunications, Madrid. 1998 :198-219. \n\n14. Stumpp T, Sasso-Cerri E, Freymuller E, Miraglia SM: \n\nApoptosis and testicular alterations in albino rats \n\ntreated with etoposide during the prepubertal phase. \n\nAnat Rec. 2004 279:611-22.", "start_char_idx": 19804, "end_char_idx": 20688, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9f273ac9-7086-4a58-9508-4e2d6ea06835": {"__data__": {"id_": "9f273ac9-7086-4a58-9508-4e2d6ea06835", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "42f5a84f-731a-4f49-ba5f-4662489a4ff3", "node_type": "1", "metadata": {"identifier": "njb-40", "author": "Sah, Surendra Kumar; Khatiwada, Saroj; Chaudhary, Deepak; Jha, Chandra Bhushan; Bhattacharya, Soumya", "title": "Doxorubicin induced histomorphometric changes in testes of albino rat", "date": "2015-12-30", "file": "njb-40.pdf"}, "hash": "04a3a93daf5bf12f0e74b80e1b18603a3cdacb43f83722ea3b6c629063f16fec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c96947df-d578-4758-bda5-99cfe40044a0", "node_type": "1", "metadata": {}, "hash": "fbe5734503fef26bd924579bc602bfb88b5d44889c9a819ea1bdcb45c38746bc", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:6-9    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n\u00a9NJB, Biotechnology Society of Nepal   6        Nepjol.info/index.php/njb \n\nDetection and Quantitation of Aflatoxin f or the Diagnosis of \n\nAspergillus flavus  \nGeeta Rajbhandari Shrestha1* and Amin Udhin Mridha2  \n\n1Amrit Campus, Tribhuvan University, Kathmandu, Nepal. \n2University of Chittagong, Chittagong, Bangladesh \n\nAbstract \nAflatoxins are the potent mycotoxins produced by Aspergillus flavus, which is hepatotoxic causing \nhepatocellular carcinoma. A. flavus produces sufficient amount of Aflatoxin B1 under favourable \nenvironments.   Inhalation of spores and use of Aflatoxin B1, contaminated food by Aspergillus spp., \ncould transfuse the toxins in the blood streams. The presence of these toxins in body fluid can be \ndetected by immunological assays and which provides an effective technique for the diagnosis of the \ndisease caused by A. flavus. Aflatoxins producing strain of A.  flavus were screened in Aflatoxin \nProducing Medium. Production of Aflatoxin B1 by A. flavus was studied in different parameters such \nas incubation periods, temperatures, pH variations, sucrose concentration in Yeast Extract Sucrose \nmedium and different natural media such as par-boiled rice, corn and groundnuts. The detection of \ntoxins was done by TLC using silica gel (Merk) coated plates and confirmative test was done by \nAssociation of Official Analytical Chemists (AOAC) method. Presence and quantization was done by \nEnzyme Linked Immunosorbent Assay (ELISA) technique. Highest amount of Aflatoxin B1 was \nreported 68.56 ng/ml by ELISA in synthetic medium (Yeast Extract Sucrose) with 2% sucrose, pH 5.5, \non 14th days of incubation, at 28\u00b110C (p-value 0.05). Similarly, highest amount was recorded in \ngroundnuts (121.20ng/g) by ELISA and (500ng/kg) by TLC methods.  ELISA is one of the most \nefficient methods used for detection and diagnosis of human diseases cause due to exposure of \nAflatoxin B1 and A. flavus.  \n\nKey words: Aflatoxin, Aspergillus flavus, ELISA, AOAC, TLC \n\n*Correspondence Author:  \n\nEmail: ga.gi.cha@hotmail.com \n\n \n\nIntroduction   \nMycotoxins are secondary metabolites produce by \n\ndifferent fungi under certain environmental \n\nconditions.   Mycotoxins cause acute kidney failure \n\n(ochratoxin), damage of central nervous system \n\n(tremogenic mycotoxin) and damage the upper \n\nrespiratory tract. Aflatoxins are the most potent and \n\nnaturally occurring mycotoxins produce by \n\nAspergillus flavus and A. parasiticus. Many factors \n\naffect the growth of fungi and contamination \n\naflatoxins on foods and feeds. Different factors \n\naffecting Aflatoxins contamination include the \n\nclimate of the region, the genotype of the crop \n\nplanted, soil type, minimum and maximum daily \n\ntemperatures, and daily net evaporation. \n\nContamination of toxins can occur at any time of \n\ngrowth of plant, pre and post harvesting periods and \n\nstorage conditions [1]. \n\n It was found that high doses (>6000mg) exposure of \n\naflatoxins may cause acute toxicity with lethal effect \n\nand prolonged exposure to small doses is \n\ncarcinogenic [2]. Mainly Aflatoxin B1 (AftB1) is the \n\npotent carcinogenic toxins to some animals and \n\nhumans [3], which is one of the leading cause of \n\ncancer deaths worldwide [4].", "start_char_idx": 47, "end_char_idx": 3421, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c96947df-d578-4758-bda5-99cfe40044a0": {"__data__": {"id_": "c96947df-d578-4758-bda5-99cfe40044a0", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9f273ac9-7086-4a58-9508-4e2d6ea06835", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "374a2b4180aab6aefbf39f2b4a0c6a1d8e26a9c4df09d15ebfdeca2969484773", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f303d78e-e789-4d48-bdde-c548406f2419", "node_type": "1", "metadata": {}, "hash": "678bb87253ac5cdc31c07eea369837e112afff53de9c45650ccb49cd6fb48b16", "class_name": "RelatedNodeInfo"}}, "text": "When animals are \n\nexposed to through the contaminated feeds, the toxin \n\nis converted into aflatoxinM1 and contaminate in their \n\nmilk. It is one of the sources of contamination \n\naflatoxins of dairy products [5]. The exposure of \n\naflatoxins causes many diseases such as \n\nhepatocellular carcinoma (HCC), impaired growth, \n\nimmune suppression etc. Exposure of aflatoxins and \n\nthe risks of toxic doses are more prevalent in the poor \n\nnations worldwide in both urban\u2013rural areas, \n\nhowever more strongly in the rural populations [6].  \n\nThese diseases are more common in most of the \n\ndeveloping countries. It was estimated that 550,000\u2013\n\n600,000 new HCC cases worldwide each year, of \n\nwhich about 25,200\u2013155,000 were due to aflatoxins \n\nexposure. Most of the people from developing \n\ncountries such as sub-Saharan Africa, Southeast Asia, \n\nand China including Nepal are suffering from HCC. \n\nTheir prevalence accounted largely due to aflatoxins \n\ncontamination in food [7]. It is essential to control \n\ncontamination of food and feeds for minimization of \n\noutbreak due to aflatoxins. The presence of AftB1 \n\nbiomarker reflects the formation of the reactive \n\nmetabolite and the level of DNA damage in the \n\nlivers. Enzyme Linked Immunosorbent Assay \n\n(ELISA) is one of the diagnostic test, depends upon \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:6-9                Shrestha and Mridha \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   7        Nepjol.info/index.php/njb \n\nprotein content, is used to determine aflatoxins \n\nqualitatively and quantitatively in the samples [8]. \n\nELISA and a monoclonal antibody against AFB1, \n\nAFB1 bound to albumin can be used to detect \n\naflatoxins in urine and blood samples. The presence \n\nof aflatoxins residues adducts, and metabolites are \n\nassayed directly in tissues, fluids and excreta [9] and \n\nanalyzed. The method is easy and inexpensive that \n\ndeveloped with the necessary reliability, accuracy, \n\nand sensitivity to bring immunoassay technology. \n\nMaterials and Methods \nAspergillus flavus was isolated from the atmosphere of \n\nKathmandu by gravity plate method. Lyophilized \n\nAflatoxins producing strains of A. flavus was obtained \n\nfrom United States Department of Agriculture and \n\nused as the reference to compare the Aflatoxin B1 \n\nproduction with that isolated from air. Aflatoxins \n\nstandard was purchased from Sigma Co. The \n\ndifferent strains of A. flavus producing aflatoxins \n\nwere screened on Aflatoxin Producing Medium \n\n(APM) as described by Donald et al. (1981) [10]. \n\nExtraction of Aflatoxin B1 was carried out as the \n\nmethod described by Abarca et al, (1988) and Chu \n\n(1987) [11, 12]. Detection of Aflatoxin B1 was done by \n\nThin Layer Chromatography (TLC) as the method \n\ndescribed by using pre-coated silica gel plates \n\n(Merck) [13]. The confirmation test was done by \n\nAssociation of Official Analytical Chemists (AOAC) \n\nmethod [14]. Quantization of AftB1 in different \n\nsamples were carried out by ELISA in various \n\nparameters such as incubation periods (7th, 9th, 11th, \n\n13th, and 15th, days), pH variations (4.5, 5.5 and 6.5), \n\ntemperatures (240, 280 and 320C), and concentrations \n\nof sucrose (1.5%, 2% and 2.5%) in Yeast Extract \n\nSucrose medium (YES) and different media like; \n\nSynthetic Low Salt medium (SLS), Coconut medium \n\n(CM),  natural media such as par-boiled rice, corn \n\nand groundnut described by Davis et al.1966 [15]. For \n\nthe study of production of Aflatoxin B1, three replicas \n\nwere maintained in each parameter.", "start_char_idx": 3422, "end_char_idx": 6964, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f303d78e-e789-4d48-bdde-c548406f2419": {"__data__": {"id_": "f303d78e-e789-4d48-bdde-c548406f2419", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c96947df-d578-4758-bda5-99cfe40044a0", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "434f41b18f3f6298cf8113901d00497d8f26f99a0b0998800e70a0900f85b866", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8fc7b39b-5568-45a6-88f9-b470979f5fd9", "node_type": "1", "metadata": {}, "hash": "ded98cb6c122d89c8ed2b4f7cf9c3b9353b79c1610cd6520a43fc0ee194cf16c", "class_name": "RelatedNodeInfo"}}, "text": "The \n\nexperimental results from this study were analyzed \n\nby SPSS 16.0 [10, 12]. \n\nResults  \nThe highest amounts of AftB1 (68.56 ng/ml) was \n\nrecorded in YES medium with 5.5 pH, 2% sucrose \n\nand  after 14 days of incubation at 28 \u00b11\u00b0C (Figure \n\n1A). The various temperatures have significant effect \n\non the production of AftB1 (Figure 2B). However, \n\nsucrose concentrations different and pH have no \n\nsignificant the effect, since p-values are more than \n\n0.050 (Figure 2A and Figure 2B).  \n\n \nFigure 1: Effect of A. Incubation periods B. Temperature on \n\nproduction of Aflatoxin B1 \n\n \nFigure 2: Effect of A. Sucrose concentration and B. pH in YES \n\nmedium on the production of Aflatoxin B1 \n\nSpearman's rho test showed that there is no \n\nsignificant linear relation between two variables- \n\nincubation period (time in days) and production of \n\nAftB1 (ng/mL) as the p-value is more than 5% level of \n\nsignificance (Table 1).  \n\nTable 1: Analysis of Aflatoxin B1 produced by A.flavus; \n\nTLC and ELISA method in various incubation periods. \n\nNote: ++ denotes for intensity of fluorescence \n\nKruskal-Wallis test showed that there is no \n\nsignificant effect of a particular medium on the \n\nproduction of AftB1 (the p-value of 0.998 is more than \n\n5% level of significance). Moreover, each of six \n\nmedium has equal effect on it. However, the highest \n\nNo. Incubation periods* \n\nAflatoxin B1 \n\nTLC ELISA ng/ml \n\n1 7 + 2.6 \n\n2 9 ++ 6.46 \n\n3 11 +++ 24.76 \n\n4 13 +++ 68.56 \n\n5 15 +++ 58.8 \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:6-9                Shrestha and Mridha \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   8        Nepjol.info/index.php/njb \n\nlevel (121.20ng/g) of alfatoxin B1 was obtained in \n\nnatural medium; groundnuts.  \n\n \n Note: YES = Yeast extract medium, SLS= Synthetic low salt \nmedium, CM= Coconut medium \n\nFigure 3: Effect of different media on the production of \n\nAflatoxin B1.in different incubation periods at 28 \u00b11\u00b0C. \n\nDiscussion  \nAflatoxins produced by food borne Aspergillus flavus \n\nand A. parasiticus cause several human diseases such \n\nas Hepatocellular carcinoma (HCC), or liver cancer. \n\nContamination of Aflatoxins can occur at any stage of \n\nfood production from pre-harvest to storage [16]. Its \n\ncontamination is determined by the most commonly \n\nused method; thin layer chromatography (TLC).   The \n\nthin layer chromatography method was used for \n\nscreening and quantification. Aflatoxins production is \n\nlow during early stages of growth that increases with \n\nthe decline of growth and the maximum production \n\nwas recorded after the stationary phase of growth, \n\nwhich soon decline in its production (Table 1 and \n\nFigure 1). Similarly, West et al. (1973) reported that \n\nthe aflatoxin level falls after reaching a maximum, \n\nwhich might be due to non-specific chemical \n\nmechanism for degradation of the toxin [17]. Smith \n\nand Moss (1985) also reported that very low \n\naflatoxins was produced during the phase of \n\nvigorous growth in a laboratory and when some \n\nnutritional factor runs out and limits growth toxins \n\nbiosynthesis occurs rapidly [18]. Factors that affect \n\naflatoxins contamination include the climate of the \n\nregion, the genotype of the crop planted, soil type, \n\nminimum and maximum daily temperatures, and \n\ndaily net evaporation. Wilson and Payne (1994) and \n\nFandohan et al.", "start_char_idx": 6965, "end_char_idx": 10332, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8fc7b39b-5568-45a6-88f9-b470979f5fd9": {"__data__": {"id_": "8fc7b39b-5568-45a6-88f9-b470979f5fd9", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f303d78e-e789-4d48-bdde-c548406f2419", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "022b718e1ab832caa7ebc2b3a3be87e660aaa7383fcf83e73fdaafa473333ec5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4d13ab7-7695-42d7-86ef-38b7d009d977", "node_type": "1", "metadata": {}, "hash": "11f8161e6264bdb4155085dcefcc8893fa45996b3c2dd2836cbf4e34d4c0e879", "class_name": "RelatedNodeInfo"}}, "text": "Wilson and Payne (1994) and \n\nFandohan et al. (2005) reported that many factors \n\naffect the growth of Aspergillus fungi and the level of \n\naflatoxins production in food [16, 19]. It is essential to \n\nreduce Aflatoxins contamination of food by \n\nproviding unsuitable condition to produce toxins. \n\nAftB1 production is also promoted by stress or \n\ndamage to the crop due to drought prior to harvest, \n\ninsect activity, poor timing of harvest, heavy rains at \n\nharvest and post-harvest, and inadequate drying of \n\nthe crop before storage [20]. Humidity, temperature, \n\nand aeration during drying and storage are also \n\nimportant factors for aflatoxins contamination.   \n\nThe most effective method of detection and \n\nquantification of aflatoxins contamination in foods \n\nand feeds is ELISA by which a very low amount of it \n\ncan be detected [12]. In this study the highest \n\namounts of AftB1 (68.56 ng/mL) was recorded in YES \n\nmedium with 5.5 pH, 2% sucrose and  after 14 days of \n\nincubation at 28 \u00b11\u00b0C. The study on the effect of \n\ndifferent parameters on Aflatoxin B1 production \n\nshowed that temperature has significant effect (P= \n\n0.050). However, different pH and sucrose \n\nconcentrations have no significant effect, since P= > \n\n0.050. This method can be employed for the detection \n\nand quantification of aflatoxins exposure of human \n\nand causes of hepatocellular carcinoma due to \n\nAspergillus flavus from Urine, blood samples. \n\nGroopman et al (1994) showed that aflatoxin \n\nmetabolites in urine reflect recent exposure (i.e. 2-3 \n\ndays) whereas the measurement of aflatoxins \n\nalbumin adducts in blood reflects exposure over a \n\nlonger period (i.e. 2-3 months) [21]. Qian et al (1994) \n\nstudies showed correlation of aflatoxins intakes to \n\nbiomarker levels and to disease [22].   \n\nConclusions \nThe spores of A. flavus under the favourable \n\nenvironmental conditions produce sufficient amount \n\nof AftB1 that effect on human health. AftB1 had been \n\ndetected and quantified by TLC and ELISA. Aflatoxin \n\nB1 could use as a marker for the diagnosis of various \n\ndiseases caused by Aspergillus species, especially A. \n\nflavus. More research is needed to determine \n\naflatoxins levels in biological specimens that are \n\nassociated with adverse health effects.  \n\nAcknowledgement \nWe are highly obliged to UNESCO for providing \n\nfellowship to execute the work in China and very \n\nmuch thankful to Prof. Dr. Zhang Yizeng, the Head \n\nof the Department of Molecular Biology, Sichuwan \n\nUniversity, Prof. Dr. Guangian Wang, Dept. of \n\nSanitary Technology, School of Public Health, West \n\nChina University of Medical Science, Chengdu, \n\nChina. We are grateful to Dr. Peterson SW, \n\nMicrobiologist, United States Department of \n\nAgriculture, Midwest Area, and National Center for \n\nAgriculture Utilization Research for providing \n\nlyophilized aflatoxins producing strains of A. flavus.  \n\nReferences \n1. Wilson DM, Payne GA: Factors Affecting \n\nAspergillus flavus Group Infection and Aflatoxin \nContamination of the Crops. The Toxicology of \n\nB \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1:6-9                Shrestha and Mridha \n\n  \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   9        Nepjol.info/index.php/njb \n\nAflatoxins: Human Health, Veterinary, and \nAgricultural Significance. DL Eaton and J D \nGroopman. San Diego, CA, Academic Press, Inc. \n1994: 309-325 \n\n2.", "start_char_idx": 10287, "end_char_idx": 13681, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4d13ab7-7695-42d7-86ef-38b7d009d977": {"__data__": {"id_": "d4d13ab7-7695-42d7-86ef-38b7d009d977", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8fc7b39b-5568-45a6-88f9-b470979f5fd9", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "b03bba747d6c49672003ff608751bf5b0733dabab3a892f2058b9becdd4539d8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c89f781a-6bbe-43ce-92af-9d2c8bdb9b88", "node_type": "1", "metadata": {}, "hash": "88277d6133361319399534cce767f9ad5cd33f68b76f7dba814aaf57ef406532", "class_name": "RelatedNodeInfo"}}, "text": "1994: 309-325 \n\n2. Groopman JD and Donahue KF: Aflatoxin, a \nhuman carcinogen: Determination in foods and \nbiological samples by monoclonal antibody \naffinity chromatography. J of Assoc. of Off. Anal. \nChem. 1988, 71: 861-867. \n\n3. IARC: Some naturally occurring substances: Food \nitems and constituents. IARC monographs on \nEvaluation of carcinogenic risk to humans 1993: 56 \n\n4. WHO: The Global Burden of Disease: 2004 \nUpdate. Geneva: World Health Organization; \n2008. [Accessed 27 April 2010].  \n\n5. Strosnider H, Azziz-Baumgartner E, Banziger M, \nBhat RV, Breiman R, Brune M: Workgroup report: \npublic health strategies for reducing aflatoxin \nexposure in developing countries. Environ Health \nPerspect, 2006, 114:1898\u20131903.  \n\n6. Plymoth A, Viviani S, Hainaut P: Control of \nhepatocellular carcinoma through Hepatitis B \nvaccination in areas of high endemicity: \nperspectives for global liver cancer \n\nprevention. Cancer Lett 2009, 286(1):15\u201321 \n7. Liu Y, Wu F: Global Burden of Aflatoxin-Induced \n\nHepatocellular Carcinoma: A Risk Assessment \nEnvironmental Health Perspectives, 2010, 118(6): \n818-824. Published online 2010 Feb 19 \n doi:  10.1289/ehp.0901388. \n\n8. Ozaslan M, Caliskan \u0130, Kilic IH, Karagoz ID: \nApplication of the ELISA and HPLC test for \ndetection of aflatoxin in Pistachio, Scientific \nResearch and Essays 2011, 6(14):2913-2917.  \n\n9. Bennett JW, Klich M: Mycotoxins. Clinic Microbio \n2003, 16(3):497\u2013516. \n\n10. Donald T, Wicklow OL, Shotwell LA, Gordon : Use \nof aflatoxin producing ability medium to \ndistinguish aflatoxin producing strains of \n\nA.flavus. Appl.Eviron. Micribiol. 1981, 41: 697-699. \n11. Abarca ML, Bragulat MR, Bruguera MT and \n\nCabanes FJ: Comparison of some screening \nmethods for aflatoxigenic moulds. \nMycopathologia, 1988 104: 75-79. \n\n12. Chu FS: Current immunochemical method of \naflatoxin in groundnuts and groundnut products. \nIn Aflatoxin contamination of groundnut. \nICRISAT. India: Proc Int Works. 1987 :6-9. \n\n13. Ren P, Ahearn DG, Crow SA: Comparative study \nof Aspergillus mycotoxin production on enriched \nmedia and construction material. J Ind Microbiol \nBiotechnol 1999, 23:209-213 \n\n14. Horwitz E: Official methods of analysis of the \nAssociation of Official Analytical Chemists, \nAOAC, Washington D.C. 1975: 462- 467. \n\n15. Davis ND, Diener UL, Eldridge:  Production of \naflatoxins B1 and G1 by Aspergillus flavus in \nsemisynthetic medium. App. Microbiol. 1966, \n14(3): 370-380. \n\n16. Wilson DM, Payne GA: Factors Affecting \nAspergillus flavus Group Infection and \nAflatoxin Contamination of the Crops. The \nToxicology of Aflatoxins: Human Health, \nVeterinary, and Agricultural Significance. DL \nEaton and J D Groopman. San Diego, CA, \nAcademic Press, Inc. 1994: 309-325. \n\n17.", "start_char_idx": 13663, "end_char_idx": 16404, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c89f781a-6bbe-43ce-92af-9d2c8bdb9b88": {"__data__": {"id_": "c89f781a-6bbe-43ce-92af-9d2c8bdb9b88", "embedding": null, "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-41", "node_type": "4", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "a44b4307ee8c205965b6246c6aa0bd50ae0132ad6c655b8e5345a8484ab72c19", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4d13ab7-7695-42d7-86ef-38b7d009d977", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "51dd8b8b6730921fc8be8b568655e34565fa42480f928b1208b09119a324616c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d16c6502-8864-435e-8f10-6a3da8053b52", "node_type": "1", "metadata": {}, "hash": "c1d6c9485f7054e3b0578afdd949db1a7d08819591ce3009fc477164429da19e", "class_name": "RelatedNodeInfo"}}, "text": "1994: 309-325. \n\n17. West S, Wyatt RD, Hamilton PB: Improved Yield \nof Aflatoxin by Incremental Increases of \nTemperature. Applied Microbiol 1973, 25:1018-\n1019. \n\n18. Smith JE, Moss MO Mycotoxins Formation, \nAnalysis and significance. John E. Smith & Sons \nChicherster,New York. Brisbane Tokonto. \nSingapore, 1985. \n\n19. Fandohan P, Zoumenou D, Hounhouiganb DJ, \nMarasasc WFO, Wingfieldd MJ, Helle  K: Fate of \naflatoxins and fumonisins during the processing \n\nof maize into food products in Benin. Int J Food \nMicrobiol 2005, 98(3): 249-59. \n\n20. Hell K, Cardwell KF: The influence of storage \npractices on aflatoxin contamination in maize in \nfour agroecological zones of Benin, West Africa. J \nStored Prod Res 2000. 36(4): 365-382. \n\n21. Groopman JD, Wogan GN, Roebuck BD, Kensler \nTW:  Molecular biomarkers for aflatoxins and \ntheir application to human cancer prevention. \nCancer Res 1994, 1(54) (Suppl 7):1907-1911 \n\n22. Qian GS, Ross RK, Yu MC, Yuan JM, Gao \nYT, Henderson BE, Wogan GN, Groopman JD:  A \nfollow-up study of urinary markers of aflatoxin \nexposure and liver cancer risk in Shanghai, \nPeople's Republic of China. Cancer Epidemiol \nBiomarkers Prev 1994, 1:3-10.", "start_char_idx": 16384, "end_char_idx": 17565, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d16c6502-8864-435e-8f10-6a3da8053b52": {"__data__": {"id_": "d16c6502-8864-435e-8f10-6a3da8053b52", "embedding": null, "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-42", "node_type": "4", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "88575e43941292e37b5da2c6cf4d99d64ee6910b278b88fffcbc4dce7acdbad4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c89f781a-6bbe-43ce-92af-9d2c8bdb9b88", "node_type": "1", "metadata": {"identifier": "njb-41", "author": "Shrestha, Geeta Rajbhandari; Mridha, Amin Udhin", "title": "Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus", "date": "2015-12-30", "file": "njb-41.pdf"}, "hash": "b848a685ddac7bbfde00019e090ab2e35771d25bc869c51153f7ed79b5eef643", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cea9cc2e-9712-42ab-8dbd-474988411ba0", "node_type": "1", "metadata": {}, "hash": "b094eed6068d804789abe346224c854d0ee37af4ac78ab850c3180a814ff0652", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 2-5    ISSN 2091-1130 (Print) / ISSN 2467-9319 (online) \n\n \nORIGINAL RESEARCH ARTICLE \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal    2        Nepjol.info/index.php/njb \n\nAccumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus \n\nStrain Isolated from Paddy Field of Kathmandu University \n\nPremises \nDeepak Upreti, Naresh Prasad Sapkota, Bibek Aryal, Sangita Shakya*  \n\nDepartment of Biotechnology, Kathmandu University, Dhulikhel, Nepal  \n\nAbstract \nIn this study, the effect of applying nutrient limitation on the production of Poly-hydro-oxy-butyric acid (PHB) from \n\nsoil bacteria was examined.  PHB is a biodegradable polymer which provides a reserve of carbon and energy. PHB was \n\nextracted by chloroform dispersion method. The amount of synthesized PHB was determined as crotonic acid by \n\nspectrophotometry. We found that Nitrogen limiting condition stimulated PHB accumulation. The highest level of PHB \n\naccumulation was observed in DNB-6 strain which accumulated 31 % of the dry mass at 20 % glucose concentration. \n\nThe probabilistic identification of bacteria by PIBwin software version 1.9.2 showed that the strain DNB-6 was close in \n\nnature to Bacillus cereus. \n\nKeywords: Polyhydrooxybutyrate, Cell dry weight, Nutrient limitation, biodegradable, bioplastic.  \n\n*Corresponding author \n\nEmail: sangita@ku.edu.np \n\nIntroduction \nPoly-hydro-oxy-butyric acid (PHB) is a member of a \n\npolymer belonging to a group polyhydroalkanoate \n\n(PHA). A wide variety of prokaryotic organisms have \n\nbeen shown to accumulate this polymer, including \n\nnumerous heterotrophic and autotrophic aerobic \n\nbacteria, photosynthetic anaerobic bacteria, gliding \n\nbacteria, actinomycetes species, cyanobacteria [1]. \n\nPHB is a biodegradable thermoplastic which \n\nprovides a reserve of carbon and energy and \n\naccumulates as intracellular granules when grown \n\nunder nutrient limiting conditions [2]. Three distinct \n\nenzymatic reactions involved in the PHB biosynthetic \n\npathway. The initial reaction involves condensation \n\nof two acetyl-COA molecules to form acetoacetyl-\n\nCoA, which is catalyzed by \u00df-ketothiolase encoded \n\nby phbA. This step is followed by the reduction of \n\nacetoacetyl-CoA by an NADPH- dependent \n\nacetoacetyl-COA dehydrogenase encoded by phbB. \n\nThen, the (R)-3- Hydroxybutyrl- COA monomers are \n\npolymerized into PHB by PHB synthase encoded by \n\nphbC (Figure 1) [3].  \n\nThe synthetic plastics are made from petrochemicals \n\nwhich are not renewable. They do not readily \n\nbiodegrade and often the collection and transport of \n\nthis waste is difficult and expensive. Due to the non- \n\nbiodegradable characteristics of petrochemicals-\n\nderived plastic materials much interest has been \n\ncreated in the development of biodegradable plastics. \n\nBiodegradation is chemical degradation of materials \n\nbrought about by the action of naturally occurring \n\nmicroorganisms such as bacteria and algae [4]. \n\nBiodegradable plastics produced from renewable \n\nsources are considered a potential substitute for \n\nconventional petrochemical plastics because of their \n\nbiodegradability and non-toxicity characteristics [1, 5-\n\n7]. PHB is one of the potential raw materials for \n\nproducing biodegradable plastic. The PHB \n\nproduction capacities of bacteria have been \n\ninvestigated for possible application in industry. \n\nHowever, the use of PHB in industrial applications \n\nhas been hampered mainly by their high production \n\ncost compared to petrochemical-based polymers \n\n[8,9].", "start_char_idx": 47, "end_char_idx": 3593, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cea9cc2e-9712-42ab-8dbd-474988411ba0": {"__data__": {"id_": "cea9cc2e-9712-42ab-8dbd-474988411ba0", "embedding": null, "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-42", "node_type": "4", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "88575e43941292e37b5da2c6cf4d99d64ee6910b278b88fffcbc4dce7acdbad4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d16c6502-8864-435e-8f10-6a3da8053b52", "node_type": "1", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "7c77448a6e495a7c0f4061387601bf8dd3866cc4ee8ed1d9e621a9f9f2396900", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aa9681c2-2cca-4a2b-9678-08621734b160", "node_type": "1", "metadata": {}, "hash": "a8aeb076b334ad2b1374764b34bcc286ceeedeff74f4aa0df2fa8cd4b067bbbe", "class_name": "RelatedNodeInfo"}}, "text": "The condition for bacterial PHB production can \n\nbe met in soil, due to its heterogeneous nature. It may \n\nbecome a limiting factor for bacterial growth \n\nespecially in some nitrogen poor (carbon-rich) sites. \n\nSoil Bacillus species have been shown to accumulate \n\nPHB during the sporulation of bacterial growth. PHB \n\nproduction by the isolate has been favoured by the \n\nglucose concentration [10]. \n\n \n\n\n\nNepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 2-5       Upreti et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   3        Nepjol.info/index.php/njb \n\n \n\nThis study makes an effort to evaluate the PHB-\n\nproducing efficiency of Bacillus species isolated from \n\nsoil, located in the paddy field of Kathmandu \n\nUniversity premises under nutrient limited \n\nconditions. The polymer has been extracted from a \n\nselected Bacillus species strain and characterized \n\npartially. \n\nMaterials and Methods  \nIsolation and purification of soil Bacteria  \n The soil sample for the isolation of bacteria was \n\ncollected during the months of March to May. Soil \n\nSamples were collected from five different areas of \n\npaddy field of Kathmandu University premises.  \n\nFifteen soil isolates were named as (DNB-1 \u2013 DNB-15) \n\nand further screened randomly to check their PHB-\n\nproducing efficiency. Each gram of the sample was \n\nsuspended in 9 ml of sterile distilled water and \n\nshaken vigorously for 2 min. The sample was heated \n\nat 80 \u00b0C for 10 minutes in water bath. After heating, \n\ndilution of 10-3, 10-4, 10-5, 10-6 g/ml were prepared \n\nfrom the soil suspension for plate counts and spread \n\non Nutrient Agar medium. After incubation at 30 \u00b0C \n\nfor 24- 48 hours, serially diluted plates were picked \n\nup and examined microscopically. As standard \n\npractice, plates having 30-300 colonies were chosen \n\nfor isolating the single bacterial colony.  \n\nThe spore morphology, gram staining, motility and \n\nseveral biochemical tests were carried out to \n\ncharacterize the bacteria. The isolate was identified \n\non the basis of comparison of these characters with \n\nthose described in Bergey\u2019s Manual of Determinative \n\nbacteriology [11]. \n\nScreening for PHB production  \nTo test the production of PHB, seed culture of \n\nisolated colonies were grown in 10 ml Nutrient broth \n\nand incubated for 24 hours at 30 \u00b0C. Later, the seed \n\nculture was transferred to 50 ml in 250 ml Erlenmeyer \n\nflasks with a 2% (v/v) inoculums and incubated at 30 \n\n\u00b0C with vigorous orbital shaking at 150 rpm. The \n\nculture was grown to a stationary phase and the \n\ncultures were examined by fluorescence microscopy \n\nafter staining with nile red. It has been proved that \n\nnile red is an excellent vital stain for the detection of \n\nintracellular lipids. The cells were flame-fixed on \n\nslide glass, then a drop of 0.1 \u00b5g/ml nile red solution \n\nwas added to the smear. After being heated over a \n\nflame for 1 second, the smear was covered with a \n\ncover-glass and examined under the fluorescence \n\nmicroscope. Nile red staining gives strong \n\nfluorescence [12]. \n\nPHB Analysis \nDry cell mass was treated with a dispersion \n\ncontaining chloroform and 80% Sodium hypochlorite \n\nin water at a 3:1 ratio. The mixture was then \n\nincubated at 30 \u00b0C for 1 hour and then centrifuged at \n\n10,000 g for 10 minutes. Three different layers were \n\nformed. The upper phase was a hypochlorite \n\nsolution, the middle phase contained non-PHB cell \n\nmaterial and undisrupted cells, and the bottom phase \n\nwas chloroform containing PHB. The upper phase \n\nwas removed first with a pipette. The chloroform \n\nlayer was also drawn using a pipette.", "start_char_idx": 3594, "end_char_idx": 7203, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aa9681c2-2cca-4a2b-9678-08621734b160": {"__data__": {"id_": "aa9681c2-2cca-4a2b-9678-08621734b160", "embedding": null, "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-42", "node_type": "4", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "88575e43941292e37b5da2c6cf4d99d64ee6910b278b88fffcbc4dce7acdbad4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cea9cc2e-9712-42ab-8dbd-474988411ba0", "node_type": "1", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "b5006e634cb21bfc6b16f81df398835ffe972ed6e214659657721f17526fad1b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4a905fec-05d9-4d71-ab53-5809ca916090", "node_type": "1", "metadata": {}, "hash": "ea68beda0b0c4a592f1c64f9ea916fb54dd2ca7423baa4e4f979786f6a7113dc", "class_name": "RelatedNodeInfo"}}, "text": "The chloroform \n\nlayer was also drawn using a pipette. The PHB was \n\nthen extracted from the chloroform layer by slowly \n\nintroducing the chloroform into ten volumes of ice \n\ncold methanol with continuous stirring. \n\nChemical Determination of PHB \nChloroform extract was dried at 40 \u00b0C and 10ml of \n\nconcentrated sulphuric acid was added. Then they \n\nwere heated at 100 \u00b0C in a water bath for 20 min. \n\nAfter cooling, the sample was transferred to a silica \n\ncuvette and the absorbance at wavelength of 235 nm \n\nis measured against a sulphuric acid as a blank. \n\nResults \nOf the fifteen samples evaluated for PHB production, \n\nDNB-6 isolate accumulated the maximum PHB \n\n(31.91%) of dry cell weight. The isolate was identified \n\nas a member of genus Bacillus when biochemical and \n\nmorphological test was matched to Bergey\u2019s Manual of \n\nDeterminative Bacteriology. Citrate utilization, growth \n\npattern, carbohydrate utilization and number of other \n\ntests as summarized in the (Table 1, Figure 2). When \n\nthe results of these tests were inserted in PIBwin \n\nsoftware version 1.9.2, the strain DNB-6 was \n\nidentified to close in nature to Bacillus cereus with ID \n\nscore of 0.76.  \n\n \n  \n\nA B  \n\nFigure 2 A, B: Gram staining and Endospore test. A: Gram \nstaining of DNB-6, B: Endospore of strain DNB-6. \n\nGrowth curve analysis  \nThe growth curve was plotted under nitrogen \n\ndeficient media (NDM), phosphate deficient media \n\n(PHDM), and potassium deficient media (PDM), \n\nsulfur deficient media (SDM) (Figure 3).", "start_char_idx": 7149, "end_char_idx": 8662, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4a905fec-05d9-4d71-ab53-5809ca916090": {"__data__": {"id_": "4a905fec-05d9-4d71-ab53-5809ca916090", "embedding": null, "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-42", "node_type": "4", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "88575e43941292e37b5da2c6cf4d99d64ee6910b278b88fffcbc4dce7acdbad4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aa9681c2-2cca-4a2b-9678-08621734b160", "node_type": "1", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "2984eb4580cf2bcb317fcdb52392401adc3681c9457f64c786488f448d8c6536", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "870899db-1a0a-408d-bc1c-ee89997cfd19", "node_type": "1", "metadata": {}, "hash": "6d6fb0b7ceaa2e2419d689bdf33d8ffe3fb9ab79092a5c4745495497cd8155a7", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 2-5       Upreti et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   4        Nepjol.info/index.php/njb \n\n \n\nTable 1: PBB production and characteristics of DNB-6 \n\n \n\nIt was observed that the DNB-6 showed good growth  \n\nat nitrogen deficient media (Figure 3).  The growth \n\ncurve was plotted up to 72 hours in 6 hours interval \n\nof time at 20% glucose concentration (Figure 3). \n\n \n\nFigure 3: Comparison of Growth curve of DNB-6 under \nnitrogen deficient media (NDM), Phosphate deficient \nmedia (PHDM), Potassium deficient media (PDM) and \nsulfur deficient media (SDM) at 20% Glucose concentration. \n\nPHB yield analysis  \n\nProduction of PHB was maximum at 48 hours \n\n(Figure 4) when DNB-6 strain was subjected to \n\nnitrogen limiting at 20% glucose concentration. \n\n \n\nFigure 4: Time profile of PHB accumulation under nitrogen \ndeficient media at 20 % Glucose concentration (1: 24 hr, 2: \n48 hr, 3: 72hr and 4: 96hr).  \n\nThe intensity of fluorescence was checked at different \n\ntime interval under nitrogen limitation by \n\nfluorescence microscope (Figure 5A, 5B, 5C, 5D). \n\nTime profile of growth and PHB production \n\naccumulation have indicated that maximum PHB \n\nproduction during starting of stationary phase and \n\ndegraded during late stationary phase. \n\n  \n\n  \n\nA B\n\nC D\n\nFigure 5: PHB intensity under fluorescent microscope. A: \nPHB intensity at 24 hours of strain DNB-6, B: PHB intensity \nat 48 hours of strain DNB-6, C: PHB intensity at 72 hours \nstrain DNB-6 D: PHB intensity at 96 hours of Strain DNB-6. \n\nDiscussion  \nGrowth curve analysis showed that PHB was a \n\ngrowth associated product and its accumulation is \n\nsignificantly increased when all cultures reached \n\nfrom exponential phase to till-stationary phase. \n\nDifferent intensity of PHB obtained at different time \n\nalso coincided with the PHB yield at different time. \n\nThe analysis of PHB yield was carried out in nitrogen \n\ndeficient media because the growth curve as well as \n\nPHB intensity observed under this condition was \n\nbetter than in other conditions. The bacteria were \n\nharvested at 24 hours, 48 hours, 72 hours and 96 \n\nhours. The yield percentage of PHB was maximum at \n\n48 hours at 20% glucose concentration. This reflected \n\nthat by this time the bacteria had already entered the \n\nstationary phase. It was observed that the bacteria \n\nstart accumulating PHB granules in its stationary \n\nphase (Anupam et al., 1999). This high yield of \n\npolymer could be because of using glucose as the \n\ncarbon source. Bacillus cereus when grown on \n\nstructurally unrelated carbon such as fructose, \n\nsucrose and gluconate has produced interesting other \n\npolymer beside PHB [13]. \n\nThere was variability in PHB accumulation at \n\ndifferent time profile. The highest PHB producing \n\nefficiency was found in isolate DNB-6. The maximum \n\nPHB yield was 31.91% of dry weight under nitrogen \n\ndeficient media at 20% glucose concentration. The \n\nrecovery process was carried by chloroform \n\ndispersion method. The PHB yield was parallel to the \n\nbacterial growth.  \n\nIn conclusion, soil bacterial isolate found were \n\ncapable of accumulating PHB as an energy material.", "start_char_idx": 8667, "end_char_idx": 11867, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "870899db-1a0a-408d-bc1c-ee89997cfd19": {"__data__": {"id_": "870899db-1a0a-408d-bc1c-ee89997cfd19", "embedding": null, "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-42", "node_type": "4", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "88575e43941292e37b5da2c6cf4d99d64ee6910b278b88fffcbc4dce7acdbad4", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4a905fec-05d9-4d71-ab53-5809ca916090", "node_type": "1", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "70b3c456f46e78acfc3b4c95c2d225eafb5a50b57d891db48f006d9eb7efcd3c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "72f3ff96-7bea-44a1-a37e-87fe55ff338f", "node_type": "1", "metadata": {}, "hash": "1272f52b93e1f1daa20ed89730c09a2fcb38b20623237e8a681ae34ba7277f07", "class_name": "RelatedNodeInfo"}}, "text": "Nepal Journal of Biotechnology. D e c .  2 0 1 5  Vol. 3, No. 1: 2-5       Upreti et al. \n\n \n\n \n\n\u00a9NJB, Biotechnology Society of Nepal   5        Nepjol.info/index.php/njb \n\n \n\nThe present study showed that the maximum PHB \n\nproduction was under nitrogen limitation condition \n\nwhich agrees well with other study that suggests \n\nPHB production was maximum under nutrient \n\nlimiting condition. \n\nAcknowledgements \nThis research was supported by Kathmandu \n\nUniversity, Department of Biotechnology. \n\nAuthor Contributions \n\nAssistant Prof. Sangita Shakya designed and \nsupervised the project. Deepak Upreti prepared the \nmanuscript. Deepak Upreti, Naresh Prasad Sapkota \nand Bibek Aryal share the equal amount of \ncontributions in this work.  \n\nReferences \n\n1. Lara LM, Huisman GW: Metabolic Engineering of \nPoly (3-Hydroxyalkanoates): From DNA to Plastic. \nMicrobiol Mol Biol Rev 1999, 63: 21-53. \n\n2. Choi J, Lee SY: Process analysis and economic \nevaluation for Poly (3-hydroxybutyrate) production \nby fermentation. Bioprocess Engineering 1997, 17: \n335-342. \n\n3. Stockdale H, Ribbons DW, Dawes EA: Occurrence of \nPoly-\u00df-Hydroxybutyrate in the Azotobacteriaceae. J \nBacteriol 1968, 95: 1798-1803. \n\n4. Kyrikou I, Briassoulis, D: Biodegradation of \nAgriculture Plastic Films: A Critical Review. J Polym \nEnviron 2007, 15: 125-150. \n\n5. Wang F, Lee YL: Poly (3-Hydroxybutyrate) \nproduction with High Productivity and High \nPolymer Content by a Fed-Batch Culture of \nAlcaligenes latus under Nitrogen Limitation. Appl \nEnviron Microbiol 1997, 63: 3703-3706. \n\n6. Singh M, Patel S, Kalia V: Bacillus Subtilis as \npotential producer for polyhydroxyalkanoates. \nMicrob Cell Fact 2009, 8: 38. \n\n7. Ojumu TV, Yu J, Solomon BO: Production of \nPolyhydroxyalkanoates, a bacterial biodegradable \npolymer .A J Biotechnol 2004, 3: 18-24. \n\n8. Byrom D: Polymer synthesis by microorganisms: \ntechnology and economics. Trends Biotechnol 1987, 5: \n246-250. \n\n9. Chio J, Lee Y: Factors affecting the economics of \nPolyhydroxyalkanotate production by bacterial \nfermentation. Appl Microbiol Biotechnol 1999, 51: 13-\n21. \n\n10. Anupam M, Banerjee R, Paul AK: Accumulation of \nPoly (3-hydroxybutyric Acid) by some soil \nstreptomyces. Curr Microbiol 1999, 39: 153-158. \n\n11. Holt JG: Bergey\u2019s Manual of Determinative \nBacteriology. Lippincott Williams and Wilkins \nBaltimore; 1994. \n\n12. Greenspan P, Mayer EP, Fowler SD. Nile red: \nSelective Fluorescent stain for intracellular lipid \ndroplets. J Cell Biol 1985, 100: 965-973. \n\n13. Stockdale H, Ribbons DW, Dawes EA: Occurrence of \nPoly-\u00df-Hydroxybutyrate in the Azotobacteriaceae. J \nBacteriol 1968, 95: 1798-1803", "start_char_idx": 11872, "end_char_idx": 14512, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "72f3ff96-7bea-44a1-a37e-87fe55ff338f": {"__data__": {"id_": "72f3ff96-7bea-44a1-a37e-87fe55ff338f", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "870899db-1a0a-408d-bc1c-ee89997cfd19", "node_type": "1", "metadata": {"identifier": "njb-42", "author": "Upreti, Deepak; Sapkota, Naresh Prasad; Aryal, Bibek; Shakya, Sangita", "title": "Accumulation of Poly-hydroxy-butyric Acid (PHB) by Bacillus Strain Isolated from Paddy Field of Kathmandu University Premises", "date": "2015-12-30", "file": "njb-42.pdf"}, "hash": "b961ce6bbdebeb6c0a29735507ac5f47cbeed2a53d82f30df36f163b457bf168", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "df9bd401-be34-44d3-ab1b-2f31b4103cd6", "node_type": "1", "metadata": {}, "hash": "4fe510353a95b6e5f2536adf8422c3dde29dadd0b253bb730d721f4781af436c", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - review article 3 Joshi Author Copy revised.docx\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 72 \n\n REVIEW ARTICLE \n\n \nAssociation\t\n \u00a0Mapping\t\n \u00a0for\t\n \u00a0Improvement\t\n \u00a0of\t\n \u00a0Quantitative\t\n \u00a0Traits\t\n \u00a0in\t\n \u00a0Plant\t\n \u00a0Breeding\t\n \u00a0\nPopulations\t\n \u00a0\n\nUmesh R. Rosyara1 and Bal K. Joshi2 \n \n\n1\t\n \u00a0South\t\n \u00a0Dakota\t\n \u00a0State\t\n \u00a0University,\t\n \u00a0Plant\t\n \u00a0Science\t\n \u00a0Department,\t\n \u00a0Brookings,\t\n \u00a0South\t\n \u00a0Dakota,\t\n \u00a0USA\t\n \u00a0\n2\t\n \u00a0Biotechnology\t\n \u00a0Unit,\t\n \u00a0NARC,\t\n \u00a0Khumaltar,\t\n \u00a0PO\t\n \u00a0Box\t\n \u00a01135\t\n \u00a0Kathmandu,\t\n \u00a0Nepal\t\n \u00a0\n*Correspondence\t\n \u00a0Author\t\n \u00a0recent\t\n \u00a0address:\t\n \u00a0joshibalak@yahoo.com\t\n \u00a0\n\n \n\nABSTRACT \n\nDNA-based molecular markers have been extensively utilized for mapping of genes and \nquantitative trait loci (QTL) of interest based on linkage analysis in mapping populations. This is in \ncontrast to human genetics that use of linkage disequilibrium (LD)-based mapping for fine mapping \nof QTLs using single nucleotide polymorphisms.  LD based association mapping (AM) has promise \nto be used in plants. Possible use of such approach may be for fine mapping of genes / QTLs, \nidentifying favorable alleles for marker aided selection and cross validation of results from linkage \nmapping for precise location of genes / QTLs of interest.  In the present review, we discuss \ndifferent mapping populations, approaches, prospects and limitations of using association mapping \nin plant breeding populations. This is expected to create awareness in plant breeders in use of AM \nin crop improvement activities.   \n\n \nKew words: Association mapping, plant breeding, DNA marker, quantitative trait loci \n\n \n\nINTRODUCTION \n\n \nThe development and use of molecular \n\nmarkers for the detection and exploitation of \n\nDNA polymorphism in plant and animal \n\nsystems is one of the most significant \n\ndevelopments in the field of molecular biology \n\nand biotechnology. Of mapping techniques, \n\nlinkage based mapping is popular in mapping \n\ngenes in self and cross pollinated crop species. \n\nThe objective of such genetic mapping is to \n\nidentify simply inherited markers in close \n\nproximity to genetic factors affecting  \n\n \n\n \n\n \n\nquantitative traits (quantitative trait loci, or \n\nQTL). This localization relies on processes \n\nthat create a statistical association between \n\nmarker and QTL alleles and processes that \n\nselectively reduce that association as a \n\nfunction of the marker distance from the QTL. \n\nWhen using crosses between inbred parents to \n\nmap QTL, we create in the F1 hybrid complete \n\nassociation between all marker and QTL \n\nalleles that derive from the same parent.", "start_char_idx": 50, "end_char_idx": 3019, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "df9bd401-be34-44d3-ab1b-2f31b4103cd6": {"__data__": {"id_": "df9bd401-be34-44d3-ab1b-2f31b4103cd6", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "72f3ff96-7bea-44a1-a37e-87fe55ff338f", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "8c7e11ca2112d82e7405e07b9ceab30bfd8d84df5dfae4358f5732faf3c447eb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "01d2e134-db6a-4297-ad03-2a5c35210c91", "node_type": "1", "metadata": {}, "hash": "f6ebdff099a44a579b4bac3342d6ec982e1d909690552083e30552c94dcb4bea", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 73 \n\nRecombination in the meioses that lead to \n\ndoubled haploid, F2, or recombinant inbred \n\nlines reduces the association between a given \n\nQTL and markers distant from it. \n\nUnfortunately, arriving at these generations of \n\nprogeny requires relatively few meioses such \n\nthat even markers that are far from the QTL \n\n(e.g. 10 cM) remain strongly associated with \n\nit. Such long-distance associations hamper \n\nprecise localization of the QTL. One approach \n\nfor fine mapping is to expand the genetic map, \n\nfor example through the use of advanced \n\nintercross lines, such as F6 or higher \n\ngenerational lines derived by continual \n\ngenerations of outcrossing the F2 [1]. In such \n\nlines, sufficient meioses have occurred to \n\nreduce disequilibrium between moderately \n\nlinked markers.  \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nWhen these advance generation lines are \n\ncreated by selfing, the reduction in \n\ndisequilibrium is not nearly as great as that \n\nunder random mating. The central problem \n\nwith any of the above approaches for fine \n\nmapping is the limited number of meioses that \n\nhave occurred and (in the case of advanced \n\nintercross lines) the cost of propagating lines \n\nto allow for a sufficient number of meioses. \n\nAn alternative approach is association \n\nmapping (AM), taking advantage of events \n\nthat created association in the relatively distant \n\npast. Assuming many generations, and \n\ntherefore meioses, have elapsed since these \n\nevents, recombination will have removed \n\nassociation between a QTL and any marker not \n\ntightly linked to it. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nF2 / BC \n\nPedigree \n\nAssociation Mapping  \n\n4\n0 \n\n10 \n\n2 \n\nRecombinant \nInbred Lines \n\nNear Isogenic \nLines \n\nPositional \ncloning \n\nIntermated \nrecombinant inbreds  \n\nR\nes\n\nea\nrc\n\nh \nti\n\nm\ne \n\n(Y\nea\n\nrs\n) \n\n1 \n\n5 \n\n1 1 x 10 4 1 x 10 7 \n\nResolution  (bp) \n\nA\nlle\n\nle\n n\n\num\nbe\n\nr \n \n\nFig.1: Schematic comparison of various methods for identifying nucleotide polymorphism trait association in terms of \nresolution, research time and allele number. BC, backcross.", "start_char_idx": 3028, "end_char_idx": 5537, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "01d2e134-db6a-4297-ad03-2a5c35210c91": {"__data__": {"id_": "01d2e134-db6a-4297-ad03-2a5c35210c91", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "df9bd401-be34-44d3-ab1b-2f31b4103cd6", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "10072953d0138aba40f514b1b5feb103babfe7400acf1f36f621cd5ac5865460", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f25faa59-061d-4246-bad6-444e31e11350", "node_type": "1", "metadata": {}, "hash": "cbb4b7f3c44d0d895036d672c0e491b19e0ca5b106ffc285c0cf389f4df944ae", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 74 \n\nAM, also known as association analysis (AA) \n\nor linkage disequilibrium mapping is a method \n\nthat relies on linkage disequilibrium to study \n\nthe relationship between phenotypic variation \n\nand genetic polymorphisms [2]. Thus Linkage \n\nmapping counts recombination between \n\nmarkers and the unknown genes whereas \n\nassociation mapping measure correlation \n\nbetween marker alleles and trait allele in a \n\npopulation (linkage disequilibrium).  \n\nAssociation mapping allows for much finer \n\nmapping than standard bi-parental cross \n\napproaches. Time requirement and resolution \n\nof association mapping is compared with other \n\ntypes of mapping approaches (Figure 1).  \n\n \nLinkage Disequilibrium  \n\n \nLinkage disequilibrium (LD) is the nonrandom \n\ncombination of alleles at two genetic loci, \n\nwhich in random mating populations is mostly \n\ngenerated by mutation and genetic drift, and \n\ndecays by recombination. The trend of LD \n\ndecay is shown in graphs with different \n\nrecombination fractions (Figure 2). Therefore, \n\nLD will be observed between two loci if they \n\nare in tight linkage or if the haplotype is recent \n\n(Hedrick, 2005). Mutations are rare events \n\nhence, it is expected that most mutations \n\nhappened many generations ago and should be \n\nin linkage equilibrium with other loci, unless \n\nthey are very closely linked. While significant \n\nLD in random mating populations is evidence \n\nof tight linkage, population perturbations like \n\nmigration, inbreeding, and selection can build \n\nup LD among loosely linked or even unlinked \n\nloci. Therefore, the characteristics of the \n\npopulation under study must be recognized \n\nwhen conducting AA or AM and interpreting \n\nits results.  \n\n \nStudies have shown that LD levels vary both \n\nwithin and between species (for detail, [2]. For \n\nexample, LD extends less than 1000 bp [3] for \n\nmaize landraces and roughly 2000 bp for \n\ndiverse maize inbred lines [4], but can be as \n\nhigh as 100 kb for commercial elite inbred \n\nlines [5]. LD decay can also vary considerably \n\nfrom locus to locus. For example, significant \n\nLD was observed up to 4 kb for the Y1 locus \n\n(encoding phytonene synthase), but was seen \n\nat only 1 kb for PSY2 (a putative phytonene \n\nsynthase) in the same maize population [6]. \n\nBeside the outbred maize, many LD studies \n\nhave also been carried out in other plant \n\nspecies [7-12]. \n\n \nA variety of mechanisms generate linkage \n\ndisequilibrium, and several of these can \n\noperate simultaneously. The two most \n\ncommon mechanisms include populations \n\nexpanding from a small number of founders \n\nand through admixture. The haplotypes present \n\nin the founders will be more frequent than \n\nexpected under equilibrium. Three special \n\ncases are noteworthy. First, genetic drift \n\naffects gametic phase disequilibrium (GPD) by \n\nthis mechanism in that a population", "start_char_idx": 5543, "end_char_idx": 8812, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f25faa59-061d-4246-bad6-444e31e11350": {"__data__": {"id_": "f25faa59-061d-4246-bad6-444e31e11350", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "01d2e134-db6a-4297-ad03-2a5c35210c91", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "fa59e8131c1bb75159d5efb9bf660b6bff42f4d06902e5e1e20dcef7824314f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e64bec56-ed45-48bf-a654-205241e49dbc", "node_type": "1", "metadata": {}, "hash": "58ac3b97e6235a3e49406e8c7bf23c6bd5539b1ccb9fc215e000447471edc5ea", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 75 \n\nexperiencing drift derives from fewer \n\nindividuals than its present size. Second, by \n\nconsidering an individual with a new mutation \n\nas a founder, we see that its descendants will \n\npredominantly receive the mutation and loci \n\nlinked to it in the same phase. Linked marker \n\nalleles will therefore be in GPD with the \n\nmutant allele. Finally, an extreme case arises \n\nin the F2 population derived from the cross of \n\ntwo inbred lines. Here, all individuals derive \n\nfrom a single F1 founder genotype and \n\nassociation between loci can be predicted \n\nbased on their mapping distance. Second, \n\ngametic phase disequilibrium arises in \n\nstructured populations when allelic frequencies \n\ndiffer at two loci across subpopulations, \n\nirrespective of the linkage status of the loci. \n\nAdmixed populations, formed by the union of \n\npreviously separate populations into a single \n\npanmictic one, can be considered a case of a \n\nstructured population where sub-structuring \n\nhas recently ceased. \n\n \n\n \nFig. 2. Decay of linkage disequilibrium with time for four \ndifferent recombination fractions (\u03b8).  \nFor unlinked loci, \u03b8 = 0.5 and LD decays rapidly \nwithin a small number of generations. For \nclosely linked loci, the decay in LD is extremely \nslow. D, Coefficient of linkage disequilibrium \n(Source: [13]). \n\nMETHODS FOR ASSOCIATION \n\nMAPPING \n\n \nMulti-parent Advanced Generation \n\nIntercross \n\n \nIn the advanced intercross [1], F2 individuals \n\nare intermated for several generations before \n\nmapping. The successive rounds of \n\nrecombination cause LD to decay and the \n\nprecision of QTL location to increase. This \n\napproach has now been extended to include \n\npopulations with multiple parents, to take into \n\naccount information from multiple linked \n\nmarkers [14, 15] and to prioritize candidate \n\npolymorphisms [16, 17]. The multiparent \n\nadvanced generation intercross (MAGIC) was \n\nfirst proposed and applied to mice [14] and is \n\ndescribed as heterogenous stock. Recently \n\nmore successes are described [18]. In both \n\ncrops and animals, an advantage of the method \n\nis that a population can be established \n\ncontaining lines that capture the majority of \n\nthe variation available in the gene pool. \n\nAlthough it might take several years before \n\nthese populations are suitable for fine \n\nmapping, they are cheap to set up and their \n\nvalue as mapping resources increases with \n\neach generation. In plants, MAGIC can be \n\nused to combine coarse mapping with low \n\nmarker densities on lines derived from an early \n\ngeneration, with fine mapping using lines \n\nderived from a more advanced generation of \n\ncrossing and a higher marker density. If such", "start_char_idx": 8817, "end_char_idx": 11902, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e64bec56-ed45-48bf-a654-205241e49dbc": {"__data__": {"id_": "e64bec56-ed45-48bf-a654-205241e49dbc", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f25faa59-061d-4246-bad6-444e31e11350", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "0976fc75e2c3232ee658d10bd0845a056f106ebaa4e36de1e9287ba7faa9bd83", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "32251b7d-021c-44ef-89fd-073429a1aa17", "node_type": "1", "metadata": {}, "hash": "eeff77a49b09e3fe6ad0230cf9fe8eac4b242bc03514b651257d52dbbce623a6", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 76 \n\npopulations were established now, they would \n\nbe well placed to exploit the advances in \n\ngenomics technology and reduction in \n\ngenotyping and sequencing costs predicted to \n\noccur in the next few years [16, 19, 20]. \n\n \nThe Transmission Disequilibrium Test  \n\n \nThe ability to map QTL in collections of \n\nbreeders\u2019 lines, old landraces or samples from \n\nnatural populations has great potential. In these \n\npopulations, LD often decays more rapidly \n\nthan in controlled crosses. Furthermore, \n\nphenotypic data often already exist, saving \n\ntime and money. The challenge is to \n\ndistinguish QTL\u2013marker associations arising \n\nfrom LD between closely linked markers from \n\nspurious background associations. The first \n\nand most robust method of achieving this was \n\nthe transmission disequilibrium test (TDT) \n\nintroduced by Richard Spielman and \n\ncolleagues in 1993 [21]. The TDT provides a \n\nway of detecting linkage in the presence of \n\ndisequilibrium [21]. Neither linkage alone nor \n\ndisequilibrium alone (i.e. between unlinked \n\nmarkers) will generate a positive result so the \n\nTDT is an extremely robust way of controlling \n\nfor false positives. At its simplest, multiple \n\nfamilies consisting of two parents and a single \n\nprogeny are collected, as shown in Figure 3. \n\nStarting from such trios, different models have \n\nbeen evolved since then and some new models \n\nallow nuclear families [22] to extended family \n\n[23] for quantitative trait analysis in addition \n\nto qualitative traits. The test of association for \n\nextended families allows use of available \n\ngenotypic and phenotypic data from family of \n\nany size and structure. Different possible types \n\nof families that can be analyzed are shown in \n\nFigure 4.  \n\n \n\nFigure 3. The transmission disequilibrium test \n\nIn the simplest case, progeny are selected for \n\nan extreme phenotype and transmissions to the \n\nprogeny from heterozygous parents counted. \n\nIn the case shown, The A allele is transmitted \n\nto affected offspring four times out of five \n\nThe single progeny in each family is usually \n\nselected for an extreme phenotype. In human \n\ngenetics this typically means they are affected \n\nby the disease under study. Parents and \n\nprogeny are genotyped, but only parents \n\nheterozygous at the marker locus are included \n\nin the analysis. From each parent, one allele \n\nmust be transmitted to the progeny and one is \n\nnot transmitted. Over all families, a count is \n\nmade of the number of transmissions and non-\n\ntransmissions. In the absence of linkage \n\nbetween QTL and marker, the expected ratio \n\nof transmission to nontransmission is 1:1. In \n\nthe presence of linkage it is distorted to an \n\nextent that depends on the strength of LD", "start_char_idx": 11907, "end_char_idx": 15048, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "32251b7d-021c-44ef-89fd-073429a1aa17": {"__data__": {"id_": "32251b7d-021c-44ef-89fd-073429a1aa17", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e64bec56-ed45-48bf-a654-205241e49dbc", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "00d0213a0ef6319d9be7199b8cadc0454d745ce34a38503a69b5b9a967f148b5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "51ed4d32-3476-4da9-92b9-1a4c0f2ed1c8", "node_type": "1", "metadata": {}, "hash": "3165f7dcf6b8e19163a6b805d4854d5891174e6e3f23791cda24b8fadcd6b1b7", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 77 \n\nbetween the marker and QTL. The distortion is \n\ntested in a chi-squared test. Power depends on \n\nthe strength of LD and on the effectiveness of \n\nselection of extreme progeny in driving \n\nsegregation away from expectation. This \n\nelegant test is extremely robust to the effects \n\nof population structure, but is susceptible to an \n\nincrease in false positive results generated by \n\ngenotype error and biased allele calling [24].  \n\nThis risk can be reduced by modeling \n\ngenotype errors and missing data in the \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nanalysis [25-27] or by comparing the \n\ntransmission ratio for extreme phenotypes \n\nwith that for control individuals or for the \n\nopposite extreme. The TDT has been \n\nextended to study haplotype transmissions, \n\nquantitative traits, the use of sib pairs rather \n\nthan parents and progeny, and information \n\nfrom extended pedigrees. TDT and other \n\nfamily-based association tests are reviewed \n\nelsewhere [28].  \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nFig. 4. Different type of families for association mapping elite inbred lines \n \n\n \n\nThree way cross Four way cross  \n\nGrand \nparents \n\nParents \n\nOffspring \n\nA. Extended pedigree  \n\nB. Nuclear family pedigree  \n \n\nC.  Trios", "start_char_idx": 15054, "end_char_idx": 16753, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "51ed4d32-3476-4da9-92b9-1a4c0f2ed1c8": {"__data__": {"id_": "51ed4d32-3476-4da9-92b9-1a4c0f2ed1c8", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "32251b7d-021c-44ef-89fd-073429a1aa17", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "d1e13aa59fa1494110dc40c6604a3921d10a4e8dbfa81f55b9fcf1ada69961b8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d3a6016e-a023-48d4-bcaa-12a46064811c", "node_type": "1", "metadata": {}, "hash": "ce5223b1fbef2893e53ff266a9d7d57afa47f179fb9ee6e996744106c02ba166", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 78 \n\nIn crops, parental and progeny lines are \n\nusually separated by several generations of \n\ngametogenesis rather than by one. In this case, \n\nthe TDT is still valid, but might no longer be \n\nso robust, the process of breeding might itself \n\ndistort segregation patterns. A family-based \n\nassociation test that is applicable to plant \n\nbreeding programs has recently been proposed \n\n[29]. The authors point out that for candidate \n\ngene studies, this method is more cost \n\neffective than the alternative methods \n\ndescribed below given that no additional \n\ncontrol markers are required. However, some \n\npower will be lost because only progeny \n\nderived from F1s known to have a \n\nheterozygous marker genotype are \n\ninformative.  \n\n \nGenomic Control \n\nPopulation structure arising from recent \n\nmigration and population admixture will \n\ngenerate LD between a trait and markers \n\ndistributed over the whole genome. This can \n\nbe detected by studying whether the \n\ndistribution of the test statistic for association, \n\nestimated empirically from a set of genome-\n\nwide distributed markers, differs from the \n\nexpected null distribution. This is the basis of \n\ngenomic control (GC) [30, 31]. To estimate the \n\nempirical distribution accurately would require \n\nmany markers. However, all that is required is \n\nto estimate the mean test statistic and compare \n\nit with its expected value (1.0 for a 1 degree of \n\nfreedom chi-squared test) for which only ~50 \n\nmarkers are needed [32]. If the average chi-\n\nsquared at a set of 50 control markers is much \n\ngreater than 1.0, population structure is \n\nindicated. For any candidate marker, the null-\n\nhypothesis is now no longer absence of \n\nassociation between it and the trait. Rather, it \n\nis that there is no association above the \n\nbackground level resulting from population \n\nstructure. To test for this, we simply divide the \n\nobserved chi-squared between the candidate \n\nand trait by the average chisquared at the \n\ncontrol markers and look up the p-value of the \n\nadjusted chi-squared in the usual manner. \n\n \nGC is valid for any single degree of freedom \n\ntest. Preferably, the control markers should \n\nloosely match the test marker in allele \n\nfrequency, but this is not crucial [31]. For \n\nquantitative traits, the difference between trait \n\nmeans for each marker class is usually tested \n\nin a t test. Provided the number of observations \n\nis reasonably large, t2 is distributed as a 1 \n\ndegree of freedom chi-squared and GC can \n\nstill be carried out. More recent work has \n\nsuggested that greater accuracy is achieved by \n\ntreating the test statistic as an F test with one \n\ndegree of freedom (df) in the numerator and \n\ndegrees of freedom in the denominator equal \n\nto the number of control loci [33]. More \n\nsophisticated versions of GC are available. \n\nWith large numbers of candidate \n\npolymorphisms to test, the majority are not \n\nexpected to be genuinely associated with the \n\ntrait. In this case, procedures and software are", "start_char_idx": 16760, "end_char_idx": 20172, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d3a6016e-a023-48d4-bcaa-12a46064811c": {"__data__": {"id_": "d3a6016e-a023-48d4-bcaa-12a46064811c", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "51ed4d32-3476-4da9-92b9-1a4c0f2ed1c8", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "1af5bd1b9d3586f2c61e65072e0e0e7c8c4df17ab8bb917e48b939d508eb2a04", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "da4a4422-4b7f-4284-9309-09e65e8eab80", "node_type": "1", "metadata": {}, "hash": "6bf10e8fc6c060619552af42c49b0d0e973cc1e2c1d1af6c5005c6d1bd35ed2f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 79 \n\navailable in which, in effect, the candidate \n\nmarkers act as their own controls. GC has also \n\nbeen extended to control for bias in accuracy \n\nof genotyping between DNA samples from \n\ndifferent origins [34] and to tests with >1 df \n\n[35]. GC also corrects for unknown kinship \n\namong collections of lines [30]. The presence \n\nof related lines can greatly increase the \n\nfrequency of false positives. For many crop \n\ndatasets this will be the greatest source of bias. \n\nThe correction of the false positive rate using \n\nGC comes at a cost: power is always \n\ndecreased. This loss of power can be great in \n\ncases of extreme population subdivision. \n\nFurthermore, because loci can vary in their \n\ndifferentiation between populations, the \n\nuniform adjustment of GC might be \n\ninsufficient for some candidate polymorphisms \n\nand overcorrect at others. \n\n \nStructured Association \n\n \nStructured association (SA) provides a \n\nsophisticated approach to detecting and \n\ncontrolling population structure [36-38]. \n\nAgain, additional markers are required, \n\nrandomly distributed across the genome. Just \n\nas for GC, recent migration and population \n\nadmixture are assumed to generate LD among \n\nunlinked and loosely linked markers that have \n\nyet to decay fully. However, we expect the \n\nparental populations themselves to be in \n\nlinkage equilibrium. By trial and error one \n\ncould allocate the individuals in our sample to \n\nparental populations such that disequilibrium \n\nwithin populations was minimized. One could \n\nthen include information on population \n\nmembership in the test of association. This is \n\nthe approach taken for SA. First individuals \n\nare allocated to populations, then this \n\ninformation is used to control for population \n\nmembership in the test of association [36-38]. \n\nTo allocate individuals to populations we need \n\nto know in advance how many populations \n\nthere are. If unknown, this can be estimated: \n\nthe allocation process is repeated for different \n\npossible numbers and the best fitting selected. \n\nNevertheless, deciding on population number \n\ncan be problematic. The computer program \n\nSTRUCTURE [37] uses computationally \n\nintensive methods to partition individuals into \n\npopulations. Many individuals or lines will not \n\nbelong uniquely to one, but will be the \n\ndescendents of crosses between two or more \n\nancestral populations. STRUCTURE also \n\nestimates the proportion of ancestry \n\nattributable to each population. Following \n\nallocation of individuals to populations, the \n\ntest for association is carried out in a model \n\nfitting exercise. Here, the principle is that \n\nvariation attributable to population \n\nmembership is accounted for first, using \n\nestimates of population membership from \n\nSTRUCTURE, and then the presence of any \n\nresidual association between the marker and \n\nphenotype is tested. For example, to test for \n\nassociation between a quantitative trait and a \n\nmicrosatellite, the trait is first regressed on the", "start_char_idx": 20177, "end_char_idx": 23578, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "da4a4422-4b7f-4284-9309-09e65e8eab80": {"__data__": {"id_": "da4a4422-4b7f-4284-9309-09e65e8eab80", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d3a6016e-a023-48d4-bcaa-12a46064811c", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "b2d16a2278c115f4590da15c5ae8ccaca94de4b0f77c2820e9e663e94b73963c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c047a166-1a94-4d4d-aa74-1252379d40b4", "node_type": "1", "metadata": {}, "hash": "b21b964cab8d2bc08949bf4c3d16b501af4607079a3c19deafc1d9d01ce36a52", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 80 \n\nestimated coefficients of population \n\nmembership and then on the marker \u2013 coded as \n\na factor as if in an analysis of variance [39]. \n\nSA is effective in detecting and adjusting for \n\nthe presence of population structure, but does \n\nnot deal with consanguinity within \n\npopulations. Recently, Ed Buckler\u2019s group \n\nintroduced a method in which population \n\nmembership is estimated using STRUCTURE \n\nand kinship among varieties is estimated \n\nempirically from a second set of control \n\nmarkers [40]. The analysis takes into account \n\nboth population structure and the correlation \n\nbetween individuals that results from their \n\nrelationships. This method is implemented in \n\nthe software TASSEL. \n\n \nASSOCIATION MAPPING IN PLANT \n\nBREEDING POPULATIONS \n\n \nScientific plant breeding is a recent activity \n\nthat normally involves a narrow genetic pool, \n\nsuch that breeding populations can be traced \n\nback to relatively few original parents, \n\nnormally landraces, within a relatively small \n\nnumber of generations (e.g. [41, 42]. Under \n\nthis scenario, mutations play a minor role and \n\nmost of the observed LD is expected to reflect \n\nthe haplotypes of the original parents. \n\nMoreover, because there were few \n\nopportunities for recombination between the \n\ntime of introduction of a parent and the \n\npresent, LD in some plant breeding \n\npopulations may not reliably indicate tight \n\nlinkage. Between unlinked loci, LD can be \n\ncaused by simultaneous selection of \n\ncombinations of alleles at different genes, \n\nincluding epistasis, and by population structure \n\n[43]. Both phenomena should be common in \n\nplant breeding populations. \n\n \nSelection should affect LD in parts of the \n\ngenome related to traits that are relevant for \n\nthe breeding program. This source of distortion \n\nshould be taken into consideration in the \n\ninterpretation of results of AA in a case-\n\nspecific manner. In contrast, population \n\nstructure is expected to affect the pattern of LD \n\nover the whole genome and must be controlled \n\na priori for correct association analysis [38]. \n\nMost of the literature on AA refers to human \n\npopulations or theoretical panmitic \n\npopulations. There is limited information and \n\ndiscussion about applications of this technique \n\nto plant breeding. As the information \n\ngenerated by QTL studies accumulates, a \n\nmethod is needed to convert efficiently that \n\ninformation into practical tools for plant \n\nselection. Association analysis can be an \n\neffective approach for closing the gap between \n\nQTL analysis and marker-assisted selection. \n\n \nThe objective of this review paper is to discuss \n\npotential applications of association mapping \n\nfor plant breeding populations.  Plant breeding \n\npopulations include basically three types - \n\ngermplasm bank collections, synthetic \n\npopulations, and elite lines.", "start_char_idx": 23583, "end_char_idx": 26836, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c047a166-1a94-4d4d-aa74-1252379d40b4": {"__data__": {"id_": "c047a166-1a94-4d4d-aa74-1252379d40b4", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "da4a4422-4b7f-4284-9309-09e65e8eab80", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "1bb1f8bc656a8264a94102d4e27dc6b4196bf45c51b0851fc1709da46c651eaa", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ccf4dbdb-1a33-46d4-9506-553d4903c638", "node_type": "1", "metadata": {}, "hash": "d6107cc5d45a64736494f224c7a789b939b5f7567f4b691a367a5159b5e7c07a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 81 \n\nChoice of Populations for AM in Plant \n\nBreeding Programs \n\n \nIn a plant breeding program, three main types \n\nof populations could be considered for \n\nimplementation of AM: germplasm bank \n\ncollections, elite breeding materials, and \n\nsynthetic populations. The application of AA \n\ndiffers among those populations in several \n\naspects (Table 1). For efficient integration of \n\nAM with other methods currently in use, \n\nmaterial that is routinely generated and \n\nevaluated should be used for both purposes. In \n\nthe case of germplasm banks, core collections \n\nare expected to represent most of the genetic \n\nvariability with a manageable number of \n\naccessions [44], and thus are suitable for \n\ngenetic studies. In the case of elite materials, \n\nthe sample could be composed by lines and \n\nchecks evaluated in regional trials. For \n\nsynthetic populations, the evaluation unit \n\nshould be also the association unit (or closely \n\nrelated to it), whether it is an individual or a \n\nfamily. \n\n \nGermplasm Bank Core Collections \n\n \nSamples representing the genetic diversity of a \n\nspecies are attractive for AM because of the \n\nwide allele diversity encompassed. Methods of \n\nselection of core collections often involve \n\ngenotyping unlinked markers to compute \n\ngenetic distances, thus providing information \n\nabout population structure. The process of \n\nselection of a minimum sample with maximum \n\nvariation has a normalizing effect that is \n\nexpected to reduce population structure and \n\nLD between unlinked loci, thus creating a \n\nsituation favorable for association analysis \n\n[45]. A difficulty likely to occur in this type of \n\nmaterial is related to genetic heterogeneity \n\nwithin samples. Landraces and natural \n\npopulations often consist of open-pollinated \n\nvarieties or mixtures of genotypes, and the \n\nDNA extraction, genotyping, and phenotyping \n\nschemes must account for this variability.  \n\n \n\nCore collections are useful materials for AM \n\nof qualitative traits, such as disease resistance \n\nor special quality characteristics (color, aroma, \n\netc). Studies focusing on domestication-related \n\ntraits such as seed dormancy, shattering, or \n\ninflorescence type also could require wide \n\nphenotypic variation, beyond the limits of \n\ncultivated germplasm [46]. Conversely, the \n\nbroad genetic variability of those collections \n\nnormally make them unsuitable for analysis of \n\nquantitative traits because part of the \n\naccessions would be unadapted to growing \n\nconditions and prevalent diseases, resulting in \n\npoor precision of trait measurement.  \n\nCommon ancestors of distantly related \n\nindividuals occurred many generations ago; \n\ntherefore, LD is expected to have decayed to \n\nshort genetic distances. For this reason, AM in \n\ncore collections will probably require \n\ncandidate genes or major QTL mapped within \n\nnarrow confidence intervals [47]. Compared \n\nwith linkage-based fine mapping and", "start_char_idx": 26842, "end_char_idx": 30181, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ccf4dbdb-1a33-46d4-9506-553d4903c638": {"__data__": {"id_": "ccf4dbdb-1a33-46d4-9506-553d4903c638", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c047a166-1a94-4d4d-aa74-1252379d40b4", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "dfe4a7c356b4312e5b130756e8310610390b2ab2151de779d8bd53f8f0af5952", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4ee9fea-a8b8-4eb9-a74f-1c604d01ccc3", "node_type": "1", "metadata": {}, "hash": "21a4b5289494132f6a0855ea3e5a4b2c861de620ef81f0a131031046afbb63d3", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 82 \n\nTable 1. Comparison of different types of populations for association analysis. Depends on the collection, conservation and \nsampling schemes \n \nAspects of \nassociation mapping \n\nGermplasm bank \n \n\nElite material \n \n\nSynthetic populations \n \n\nSamples Entries of a core \ncollection \n\nInbred lines and \ncultivars \n\nIndividual plants or \nprogenies \n\nSample turnover Static Gradually substituted Ephemeral \nSource of \nphenotypic data \n\nPhenotypic \nscreenings \n\nReplicated yield trials Evaluation for recurrent \nselection \n\nType of traits High heritability and \ndomestication traits \n\nLow heritability, \nyield \n\nDepends on the \nevaluation scheme \n\nLevel of LD Low High Intermediate \nPopulation \nstructuring \n\nMedium High Low \n\nAllele diversity \namong samples \n\nHigh Low Intermediate \n\nAllele diversity \nwithin samples \n\nVariable  1 allele 1 or 2 alleles  \n\nResolution of AM High Low Intermediate and \nincreasing \n\nPower of AM Low High Intermediate and \ndecreasing \n\nApplication of \nsignificant markers \n\nMarker-assisted \nbackcross \n\nMarker-assisted \nselection \n\nIncorporation in selection \nindex \n\n \nDepends on the collection, conservation and  sampling schemes. For diploid species. (Source: [45]. \n\n \n\npositional cloning [48] the AM approach \n\nwould offer the advantage of simultaneously \n\ndetecting the effect and screening the \n\ngermplasm for useful alleles. Significant \n\nmarkers would be useful for introgression of \n\nthe new variation into elite germplasm through \n\nmarker-assisted backcrossing [49], while \n\nmarkers used for population structure inference \n\ncould be used to speed up the recovery of the \n\nrecurrent parent genome. Theoretical \n\nprojections indicate that the use of two markers \n\nper chromosome for selection against the \n\ndonor genotype could shorten the transfer by \n\nabout two generations [50]  \n\n \n \n\nElite Lines and Cultivars \n\n \nMaximum relative efficiency of marker-\n\nassisted selection compared with phenotypic \n\nselection is expected when heritability is low \n\nand markers capture a significant portion of  \n\nthe variation for the trait [51].  \n\nElite lines are desirable materials for AM of \n\nlow heritability traits, including yield, yield \n\ncomponents, and tolerance to abiotic stresses \n\nbecause elite lines are genetically stable and \n\nare well adapted to normal growing conditions.  \n\n \nIn plant breeding programs, there is normally a \n\nlarge body of phenotypic data accumulated for", "start_char_idx": 30186, "end_char_idx": 33032, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4ee9fea-a8b8-4eb9-a74f-1c604d01ccc3": {"__data__": {"id_": "d4ee9fea-a8b8-4eb9-a74f-1c604d01ccc3", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ccf4dbdb-1a33-46d4-9506-553d4903c638", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "3badd3a3a403e9b9f97c1d64a0b5756f8f3c9be4c292ec4af70a178c15219149", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6fa161f0-4cff-4128-a694-e47852b489b6", "node_type": "1", "metadata": {}, "hash": "da58c5bdf80946fbad0ab56d057f5e69db42559589c7e69d410851b94940d9b6", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 83 \n\nelite lines and cultivars from replicated field \n\nexperiments over locations and years. Use of \n\nthose data for AM requires statistical models \n\naccounting for covariances introduced both by \n\nexperimental design (years, locations, \n\nreplicates) and polygenic effects. Moreover, \n\nthose data are often unbalanced because new \n\nlines are included in field trials each year, \n\nwhile other lines are discarded. Maximum \n\nlikelihood solutions of mixed-effects models \n\nyield minimum-variance unbiased estimates of \n\nallele effects from unbalanced data, taking into \n\naccount the correlation structure of the data \n\n[52]. Mixed-effects models were used to \n\nanalyze plant height, disease resistance, and \n\ngrain moisture in maize [53] and grain size and \n\nmilling quality in wheat [45].  \n\n \nPopulation structure can be prominent in elite \n\nmaterial because it is common for closely \n\nrelated lines to be admitted to advanced trials. \n\nIf pedigrees are known, the relationships \n\namong the lines can be determined [41] and \n\nused to control for polygenic effects [54]. In \n\nthis case, it is not essential to estimate \n\npopulation structure through unlinked markers, \n\nalthough there may still be interest in marker \n\ndata as a genetic fingerprint for variety \n\nprotection [55] and for purity control of seed \n\nproduction.  \n\n \nA typical elite plant breeding pool is derived \n\nfrom few founders in the recent past, and is \n\nsubmitted to intense selection. For those \n\nreasons, LD is expected to be high in this \n\nmaterial, and the first experimental results \n\nconfirm this expectation [3, 5]. Although AM \n\nin elite lines may not offer much improved \n\nresolution compared with QTL analysis in \n\nbiparental mapping populations, there are at \n\nleast two important advantages: a substantially \n\nhigher level of polymorphism and detection of \n\nfavorable alleles directly in the target \n\npopulation. Elite lines are natural candidates \n\nfor crossing to generate the next round of \n\nbreeding, and significant markers could be \n\nused for marker-assisted selection in the \n\nprogeny.  \n\n \nSynthetic Populations \n\n \nAlthough the potential of synthetic populations \n\nfor AM is largely unknown, they might be the \n\nplant breeding materials that best approximate \n\nthe assumption of random mating because \n\nsynthetics are normally designed and \n\nmaintained to minimize inbreeding. Population \n\nstructure is expected to be mild or absent, \n\nwhich is an important advantage of synthetics \n\nfor AM. If the experimental material represents \n\na single intermating population, the power of \n\nAM is maximized and the risk of false \n\nassociations is minimized [56]. Nevertheless, \n\npopulation structure can still occur because of \n\ndifferences in flowering time, plant height, and \n\nother traits that may lead to assortative mating. \n\n \nGenotypic information could be useful in all \n\nphases of population breeding. In the choice of", "start_char_idx": 33037, "end_char_idx": 36371, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6fa161f0-4cff-4128-a694-e47852b489b6": {"__data__": {"id_": "6fa161f0-4cff-4128-a694-e47852b489b6", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4ee9fea-a8b8-4eb9-a74f-1c604d01ccc3", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "8451e53515ebf4e3da4a6aa0e168222e71d52b0cf35205a9b59c025a306fbea7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f88765db-526a-4a59-b9e5-40a18e138513", "node_type": "1", "metadata": {}, "hash": "dbd67e67a1bb3eabf5aec34ed7c7073292b321bca000e5d85adf3b8c13d304f1", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 84 \n\nparents to form the population, knowledge of \n\nthe genetic distance among lines would be \n\nuseful to achieve a compromise between high \n\nmeans for agronomic traits and high allelic \n\nvariability. By genotyping samples of \n\nsubsequent cycles with unlinked markers, \n\nbreeders can monitor changes in allele \n\ndiversity, effective population size, and \n\npopulation structure [57, 58]. The allele \n\ndiversity of synthetic populations depends on \n\nthe number and divergence of parents and the \n\nintensity of selection applied. The level of LD \n\nin synthetic populations is expected to be high \n\nin the initial generations, such that a genome \n\nscan could detect large chromosome segments \n\nassociated with traits, and trace them back to \n\nparental haplotypes. In subsequent \n\ngenerations, the decay of LD by recombination \n\nwould favor increasingly refined mapping. \n\nHowever, synthetic populations are often \n\nsubmitted to recurrent selection, a breeding \n\nscheme consisting of successive cycles of \n\nevaluation, selection, and recombination [59]. \n\nIntense selection could build up LD by \n\nfavoring allelic combinations or by promoting \n\ngenetic drift [6]. For this reason, populations \n\nsubjected to mild or no selection would be \n\npreferred for AM. [60] developed a population \n\nfor association analysis from the Illinois \n\nhigh/low oil populations, with 10 generations \n\nof recombination without selection.  \n\n \n\nA short time frame is a fundamental \n\ncharacteristic of plant breeding populations for \n\nAM, compared with natural populations. \n\nTherefore, in plant breeding populations, the \n\nmost significant association does not \n\nnecessarily indicate the position of the gene \n\n[45]. In the long term, linkage becomes the \n\nmajor factor defining the association between \n\nQTL or gene and marker, and only closely \n\nlinked markers remain in high LD; however, \n\nthe time required to achieve this situation is \n\nlonger than most breeding programs have been \n\nin existence. For this reason, AM in plant \n\nbreeding programs should be considered a \n\nmethod of detection of markers for indirect \n\nselection, rather than a method for fine-\n\nmapping QTL [45]. To alleviate this problem, \n\nthe breeder should use methods like recurrent \n\nselection, which maximizes the heterozygosity \n\nand the opportunities for recombination.  \n\n \nThe resolving power of LD mapping depends \n\non how rapidly LD decays with genetic \n\ndistance. This varies between populations of \n\nlandraces, wild progenitors and modern \n\ncultivars as a result of the diverse history to \n\nwhich crop plants have been subjected since \n\ntheir domestication [61]. In some populations, \n\nLD will decay so rapidly that they are best \n\nsuited for fine mapping, whereas in others the \n\ndecay might be so slow that whole genome \n\nscans are practical. In crops where collections \n\nof contemporary, historical and wild material \n\nexist, selection of different sets of lines might \n\npermit both fine and coarse mapping [61]. \n\nHowever, in most crops, marker density is", "start_char_idx": 36376, "end_char_idx": 39819, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f88765db-526a-4a59-b9e5-40a18e138513": {"__data__": {"id_": "f88765db-526a-4a59-b9e5-40a18e138513", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6fa161f0-4cff-4128-a694-e47852b489b6", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "a6ec65a44f42b2e083efd265988bde168905997ad207f3a2d416fb2f7fd3b415", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0f1078bc-217d-4074-82f5-29963b0b665b", "node_type": "1", "metadata": {}, "hash": "5d31fe395944a4ce365f0b3a4299c7da744a634db63b8a95206c0145975f5233", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 85 \n\ncurrently too low for genome scans. Before \n\nattempting these, power calculations should \n\ndemonstrate that, given the rate of decay of LD \n\nin the population to be studied, the density of \n\nmarkers and their allele frequency distribution \n\nare adequate to detect linked QTL accounting \n\nfor specified proportions of the phenotypic \n\nvariation. Population size is also important. An \n\nLD mapping experiment will almost always \n\nhave lower power than a family based linkage \n\nmapping experiment of equivalent size: if 100 \n\nlines are just sufficient for a family based \n\nlinkage mapping study, they will be too few \n\nfor LD mapping. For these reasons this is \n\nbelieved that the best use of LD mapping is to \n\nrefine the location of QTL identified in family \n\nbased linkage mapping and candidate gene \n\nstudies. While linkage mapping methods offer \n\na high power to detect QTL in genome-wide \n\napproaches, association mapping methods \n\nhave the merit of a high resolution to detect \n\nQTL [4]. Linkage and association analysis are \n\nthought to be complementary to each other in \n\nterms of providing prior knowledge, cross-\n\nvalidation, and statistical power [62]. So if \n\nboth analyses are done this is expected to help \n\nin proper location of QTLs.  Longer term, \n\nprospects for high-throughput genotyping and \n\nsequencing might make whole-genome scans \n\nby LD mapping more feasible. The challenge \n\nis to identify and create the appropriate \n\npopulations so that computational, analytical \n\nand profiling advances can be rapidly \n\nharnessed by the crop science community. For \n\nplant breeding application, at current situation \n\nAM could be useful for validating location of \n\nQTL of interest and identifying favorable \n\nallele for marker aided selection. Once a \n\ngenetic marker has been demonstrated to be \n\nassociated with a phenotypic trait of interest, it \n\ncan be used as a selection target to obtain an \n\nindirect response in the trait. In recurrent \n\nselection, markers could be used to store \n\ninformation acquired from phenotypic \n\nevaluations, which can be used for selection in \n\nlater cycles. Likewise, in pedigree breeding, \n\nmarkers could carry information about yield \n\npotential from the phase of replicated field \n\ntrials to the phase of single-plant selection, \n\nwhen evaluation of yield cannot be made with \n\nreasonable precision.  \n\n \nCONCLUSION \n\n \nWith the availability of high density maps in a \n\nnumber of crop plants, the whole genome \n\nsequences in model plants like Arabidopsis \n\nand rice, and the sequences of gene rich \n\nregions in crops like sorghum, maize and \n\nwheat the association mapping tool have future \n\nfor increasing applications. Even though most \n\nof plant breeders\u2019 populations could not be \n\nused for fine mapping as such the association \n\nmapping could be helpful in identification of \n\nfavorable alleles for marker aided selection \n\nand cross validation of results of linkage based \n\nmapping.  \n\n \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 39824, "end_char_idx": 43284, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0f1078bc-217d-4074-82f5-29963b0b665b": {"__data__": {"id_": "0f1078bc-217d-4074-82f5-29963b0b665b", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f88765db-526a-4a59-b9e5-40a18e138513", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "a3f195553ba571d7b6540e0f6facdc8a4228b174e5d7f9451dd9d6fba413909f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3ea77933-0971-473d-bab5-f9108bb57624", "node_type": "1", "metadata": {}, "hash": "582b16767948ae6d95804a40cfa46c7b5a16db9460c81ba232e8d95b965c90ec", "class_name": "RelatedNodeInfo"}}, "text": "2012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 86 \n\nREFERENCES \n\n1. Darvasi A, Soller M: Advanced intercross \nlines, an experimental population for fine \ngenetic mapping. Genetics 1995, 141:1199-\n1207. \n\n2. Flint-Garcia SA, Thornsberry JM, Buckler \nES: Structure of linkage disequilibrium in \nplants. Annu. Rev. Plant Biol. 2003, 54:357-\n374. \n\n3. Tenaillon MI, Sawkins MC, Long AD, et al. \nPatterns of DNA sequence polymorphism \nalong chromosome 1 of maize (Zea mays \nssp. mays L.). Proc. Natl. Acad. Sci. USA \n2001, 98:9161-9166. \n\n4. Remington DL, Thornsberry JM, Matsuoka \nY, et al. Structure of linkage disequilibrium \nand phenotypic associations in the maize \ngenome. Proc. Natl. Acad. Sci. USA 2001, \n98:11479-11484. \n\n5. Ching A, Caldwell KS, Jung M, et al. SNP \nfrequency, haplotype structure and linkage \ndisequilibrium in elite maize inbred lines. \nBMC Genet. 2002, 3:19. \n\n6. Palaisa KA, Morgante M, Williams M, \nRafalski A: Contrasting effects of selection \non sequence diversity and linkage \ndisequilibrium at two phytoene synthase \nloci. Plant Cell 2003, 15:1795-1806. \n\n7. Ingvarsson PK: Nucleotide polymorphism \nand linkage disequilibrium within and \namong natural populations of European \naspen (Populus tremula L., Salicaceae). \nGenetics 2005, 169:945-953. \n\n8. Brown GR, Gill GP, Kuntz RJ, Langley CH, \nNeale DB: Nucleotide diversity and linkage \ndisequilibrium in loblolly pine. Proc. Natl. \nAcad. Sci. USA 2004, 101:15255-15260. \n\n9. Morrell PL, Toleno DM, Lundy KE, Clegg \nMT: Low levels of linkage disequilibrium in \nwild barley (Hordeum vulgare ssp.  \n\n \n\nspontaneum) despite high rates of self-\nfertilization. Proc. Natl. Acad. Sci. USA 2005, \n102:2442-2447. \n\n10. Garris AJ, McCouch SR, Kresovich S: \nPopulation structure and its effect on \nhaplotype diversity and linkage \ndisequilibrium surrounding the xa5 locus of \nrice (Oryza sativa L.). Genetics 2003, \n165:759-769. \n\n11. Hamblin MT, Mitchell SE, White GM, et \nal. Comparative population genetics of the \npanicoid grasses: sequence polymorphism, \nlinkage disequilibrium and selection in a \ndiverse sample of sorghum bicolor. Genetics \n2004, 167:471-483. \n\n12. Nordborg M, Borevitz JO, Bergelson J, et \nal. The extent of linkage disequilibrium in \nArabidopsis thaliana. Nat. Genet.", "start_char_idx": 43258, "end_char_idx": 45843, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3ea77933-0971-473d-bab5-f9108bb57624": {"__data__": {"id_": "3ea77933-0971-473d-bab5-f9108bb57624", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0f1078bc-217d-4074-82f5-29963b0b665b", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "ffa7e22d8804d6695a51938aa38f8407991c714a32c86ba40fcf5cfaa48b7f6d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0b955f6c-4ba8-4545-8558-3c6460e04c15", "node_type": "1", "metadata": {}, "hash": "e05372646ab39510bf51b4c8399d3aa8b9d00dad2bcf71d9cd5de4ae8eebb331", "class_name": "RelatedNodeInfo"}}, "text": "Nat. Genet. 2002, \n30:190-193. \n\n13. Mackay I, Powell W: Methods for linkage \ndisequilibrium mapping in crops. TRENDS \nin Plant Science 2007, \n12:doi:10.1016/j.tplants.2006.12.001. \n\n14. Mott R, Talbot CJ, Turri MG, Collins AC, \nFlint J: A method for fine mapping \nquantitative trait loci in outbred animal \nstocks. Proc. Natl. Acad. Sci. USA 2000, \n97:12649-12654. \n\n15. Mott R, Flint J: Simultaneous detection \nand fine mapping of quantitative trait loci \nin mice using heterogeneous stocks. Genetics \n2002, 160:1609-1618. \n\n16. Yalcin B, Flint J, Mott R: Using \nprogenitor strain information to identify \nquantitative trait nucleotides in outbred \nmice. Genetics 2005, 171:673-681. \n\n17. Valdar W, Solberg LC, Gauguier D, et al. \nGenome-wide genetic association of \ncomplex traits in heterogeneous stock mice. \nNat. Genet. 2006, 38:879-887. \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 87 \n\n18. Valdar W, Flint J, Mott R: Simulating the \ncollaborative cross: power of quantitative \ntrait loci detection and mapping resolution \nin large sets of recombinant inbred strains \nof mice. Genetics 2006, 172:1783-1797. \n\n19. Syvanen AC: Toward genome-wide SNP \ngenotyping. Nat. Genet. 2005, 57:S5-S10. \n\n20. Macdonald SJ, Pastinen T, Genissel A, \nCornforth TW, Long AD: A low-cost open-\nsource SNP genotyping platform for \nassociation mapping applications. Genome \nBiol. 2005, 6:R105. \n\n21. Spielman R, McGinnis S, Ewens WJ: \nTransmission test for linkage \ndisequilibrium: the insulin gene region and \ninsulin-dependent diabetes mellitus \n(IDDM). Am. J. Hum. Genet. 1993, 52:506-\n516. \n\n22. Abecasi s GR, Cookson WO, Cardon LR: \nPedigree tests of transmission \ndisequilibrium. Eur. J. Hum. Genet. 2000, \n8:545-551. \n\n23. Abecasis GR, Cardon LR, Cookson WOC: \nA general test of association for quantitative \ntraits in nuclear families. Am. J. Hum. Genet. \n2000, 66:279-292. \n\n24. Mitchell AA, Chakravarti A: Undetected \ngenotyping errors cause apparent \novertransmission of common alleles in the \ntransmission/disequilibrium test. Am. J. \nHum. Genet. 2003, 72:598-610. \n\n25. Allen AS, Rathouz PJ, Satten GA: \nInformative missingness in genetic \nassociation studies: Case-parent designs. \nAm. J. Hum. Genet.", "start_char_idx": 45832, "end_char_idx": 48416, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0b955f6c-4ba8-4545-8558-3c6460e04c15": {"__data__": {"id_": "0b955f6c-4ba8-4545-8558-3c6460e04c15", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3ea77933-0971-473d-bab5-f9108bb57624", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "888b9807c6be1dd2a6646a873ad7d14fb18a4059499cc7f8e2117b69346c1729", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c112b22e-5af3-4ff1-8af9-205087cf56b4", "node_type": "1", "metadata": {}, "hash": "0b2537948e174c2e7864229c0e683efe85279715aed92deb2fa4809bbebeaecf", "class_name": "RelatedNodeInfo"}}, "text": "Am. J. Hum. Genet. 2003, 72:671-680. \n\n26. Gordon DS, Heath C, Liu X, Ott J: A \ntransmission/disequilibrium test that allows \nfor genotyping errors in the analysis of \nsingle-nucleotide polymorphism data. Am. J. \nHum. Genet. 2001, 69:371-380. \n\n27. Gordon D, Haynes C, Johnnidis C, et al. A \ntransmission disequilibrium test for general \npedigrees that is robust to the presence of \nrandom genotyping errors and any number \nof untyped parents. Eur. J. Hum. Genet. \n2004, 12:752-761. \n\n28. Laird NM, Lange C: Family-based \ndesigns in the age of large-scale gene-\nassociation studies. Nat. Rev. Genet. 2006, \n7:385-394. \n\n29. Stich B, Melchinger AE, Piepho H, et al. A \nnew test for family-based association \nmapping with inbred lines from plant \nbreeding programs. Theor. Appl. Genet. \n2006, 113:1121-1130. \n\n30. Devlin B, Roeder K: Genomic control for \nassociation studies. Biometrics 1999, 55:997-\n1004. \n\n31. Reich DE, Goldstein DB: Detecting \nassociation in a casecontrol study while \ncorrecting for population stratification. \nGenet. Epidemiol. 2001, 20:4-16. \n\n32. Bacanu SA, Devlin B, Roeder K: \nAssociation studies for quantitative traits in \nstructured populations. Genet. Epidemiol. \n2002, 22:78-93. \n\n33. Devlin B, Bacanu SA, Roeder K: Genomic \ncontrol in the extreme. Nat. Genet. 2004, \n36:1129-1130. \n\n34. Clayton DG, Walker NM, Smyth DJ, et al. \nPopulation structure, differential bias and \ngenomic control in a large-scale, case-\ncontrol association study. Nat. Genet. 2005, \n37:1243-1246. \n\n35. Zheng G, Freidlin B, Gastwirth JL: Robust \ngenomic control for association studies. Am. \nJ. Hum. Genet. 2006, 78:350-356. \n\n36. Falush D, Stephens M, Pritchard JK: \nInference of population structure using \nmultilocus genotype data: linked loci and \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 88 \n\ncorrelated allele frequencies. Genetics 2003, \n164:1567-1587. \n\n37. Pritchard JK, Stephens M, Donnelly P: \nInference of population structure using \nmultilocus genotype data. Genetics 2000, \n155:945-959. \n\n38. Pritchard JK, Stephens M, Rosenberg NA, \nDonnelly P: Association mapping in \nstructured populations. Am. J. Hum. Genet. \n2000, 67:170-181. \n\n39.", "start_char_idx": 48398, "end_char_idx": 50943, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c112b22e-5af3-4ff1-8af9-205087cf56b4": {"__data__": {"id_": "c112b22e-5af3-4ff1-8af9-205087cf56b4", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0b955f6c-4ba8-4545-8558-3c6460e04c15", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "e7d001903b671b9aadb058da4d7d611b33e1211e0d314adfc2bf23a9d8a8b918", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fca90f3f-2817-4d20-a07e-cf1085e8057f", "node_type": "1", "metadata": {}, "hash": "3184e123620c2bf06faf7923c779c96986569da7ffa8a5d7e4d552a4e4f61102", "class_name": "RelatedNodeInfo"}}, "text": "Genet. \n2000, 67:170-181. \n\n39. Aranzana MJ, Kim S, Zhao K, et al. \nGenome-wide association mapping in \nArabidopsis identifies previously known \nflowering time and pathogen resistance \ngenes. PLoS Genet. 2005, 1:60. \n\n40. Yu J, Pressoir G, Briggs WH, et al. A \nunified mixed-model method for association \nmapping accounting for multiple levels of \nrelatedness. Nat. Genet. 2006, 38:203-208. \n\n41. Bered F, Barbosa-Neto JF, Carvalho FIF: \nGenetic variability in common wheat \ngermplasm based on coefficients of \nparentage. Genet. Mol. Biol. 2002, 25:211-\n215. \n\n42. Lu H, Redus MA, Coburn JR, et al. \nPopulation structure and breeding patterns \nof 145 US rice cultivars based on SSR \nmarker analysis. Crop Sci. 2005, 45:66-76. \n\n43. Hartl D, Clark A: Principles of population \ngenetics. Sunderland, MA: Sinauer; 1997. \n\n44. Zhang XR, Zhao YZ, Cheng Y, et al. \nEstablishment of sesame germplasm core \ncollection in China. Genet. Resour. Crop \nEvol. 2000, 47:273-279. \n\n45. Breseghello F, Sorrells ME: Association \nmapping of kernel size and milling quality \nin wheat (Triticum aestivum L.) cultivars. \nGenetics 2005, (doi:10.15. \n\n46. Clark RM, Linton E, Messing J, Doebley \nJF: Pattern of diversity in the genomic \n\nregion near the maize domestication gene \ntb1. Proc. Natl. Acad. Sci. USA 2004, \n101:700-707. \n\n47. Thornsberry JM, Goodman MM, Doebley \nJ, et al. Dwarf polymorphisms associate with \nvariation in flowering time. Nat. Genet. \n2001, 28:286-289. \n\n48. Yan L, Loukoianov A, Tranquilli G, et al. \nPositional cloning of the wheat vernalization \ngene VRN1. Proc. Natl. Acad. Sci. USA 2003, \n100:6263-6268. \n\n49. Frisch M, Melchinger AE: Selection \ntheory for marker-assisted backcrossing. \nGenetics 2005, 170:909-917. \n\n50. Hospital F, Chevalet C, Mulsant P: Using \nmarkers in gene introgression breeding \nprograms. Genetics 1992, 132:1199-1210. \n\n51. Lande R, Thompson R: Efficiency of \nmarker-assisted selection in the \nimprovement of quantitative traits. Genetics \n1990, 124:743-756. \n\n52. Pinheiro JC, Bates DM: Mixed-Effects \nModels in S and S-PLUS. New York: Springer; \n2000. \n\n53. Parisseaux B, Bernardo R: In-silico \nmapping of quantitative trait loci in maize. \nTheor. Appl. Genet. 2004, 109:508-514. \n\n54. Zhang YM, Mao Y, Xie C, et al. Mapping \nquantitative trait loci using naturally \noccurring genetic variance among \ncommercial inbred lines of maize (Zea mays \nL.). Genetics 2005, 169:2267-2275. \n\n55. R\u00f6der MS, Wendehake K, Korzun V, et al. \nConstruction and analysis of a \nmicrosatellite-based database of European \nwheat varieties. Theor. Appl. Genet. 2002, \n106:67-73. \n\n56. Cardon LR, Palmer LJ: Population \nstratification and spurious allelic \nassociation. Lancet 2003, 361:598-604.", "start_char_idx": 50912, "end_char_idx": 53622, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fca90f3f-2817-4d20-a07e-cf1085e8057f": {"__data__": {"id_": "fca90f3f-2817-4d20-a07e-cf1085e8057f", "embedding": null, "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-43", "node_type": "4", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "4063ead20f227bcaed9d9b5f306c590bfee0b18a6c7b09c1fef516991224fb9a", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c112b22e-5af3-4ff1-8af9-205087cf56b4", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "3e44d0be4e01eb1a4cc2f0904d6d9ce92932e33abb1578df19fa89639b6615a6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1f72d553-8666-4ea1-9fd0-fdcb2ad44229", "node_type": "1", "metadata": {}, "hash": "296b0d34588e40405a79df3e9a1d951b450eb7f0979a6bac9fc468fbf6c75084", "class_name": "RelatedNodeInfo"}}, "text": "Lancet 2003, 361:598-604. \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a072\t\n \u00a0\u2013\t\n \u00a089\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 89 \n\n57. Courtois B, Filloux D, Ahmadi N, et al. \nUsing molecular markers in rice population \nimprovement through recurrent selection. \nIn Population improvement: A way of \nexploiting the rice genetic resources of Latin \nAmerica. edited by Guimar\u00e3es EP Rome: \nFAO; 2005:52-74. \n\n58. Ramis CA, Badan CDC, Guimar\u00e3es EP, \nD\u00edaz A, Gamboa CE: Molecular markers as \ntools for rice population improvement. In \nPopulation improvement: A way of exploiting \nthe rice genetic resources of Latin America. \nedited by Guimar\u00e3es EP Rome: FAO; \n2005:75-94. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n59. Fehr WR: Principles of cultivar \ndevelopment: Theory and technique. Ames, \nIA: Macmillian; 1987. \n\n60. Laurie CC, Chasalow SD, LeDeaux JR, et \nal. The genetic architecture of response to \nlong-term artificial selection for oil \n\nconcentration in the maize kernel. Genetics \n2004, 168:2141-2155. \n\n61. Caldwell KS, Russell J, Langridge P, \nPowell W: Extreme population-dependent \nlinkage disequilibrium detected in an \ninbreeding plant species, Hordeum vulgare. \nGenetics 2006, 172:557-567. \n\n62. Wilson LM, Whitt SR, Ibanez AM, et al. \nDissection of maize kernel composition and \nstarch production by candidate gene \nassociation. Plant Cell 2004, 16:2719-2733.", "start_char_idx": 53597, "end_char_idx": 55298, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1f72d553-8666-4ea1-9fd0-fdcb2ad44229": {"__data__": {"id_": "1f72d553-8666-4ea1-9fd0-fdcb2ad44229", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fca90f3f-2817-4d20-a07e-cf1085e8057f", "node_type": "1", "metadata": {"identifier": "njb-43", "author": "Rosyara, Umesh R; Joshi, Bal K", "title": "Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations", "date": "2022-07-11", "file": "njb-43.pdf"}, "hash": "3ddf5bcd4de6656d11b4c266d1420d816abf07b85dc7021d58e4396cf94d4f1c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f7571db9-1ae1-452a-b7dd-c932b1d40e49", "node_type": "1", "metadata": {}, "hash": "1582560b8d7920e1de0cf20f3d599b35ef0b5c23ad2ccba398db04657244f036", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Paper 4 Deswal Author Copy.doc\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 62 \n\n REVIEW ARTICLE \n\n \nModels\t\n \u00a0of\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0antigen\t\n \u00a0receptor\t\n \u00a0activation:\t\n \u00a0the\t\n \u00a0puzzle\t\n \u00a0still\t\n \u00a0remained\t\n \u00a0\n\nSumit Deswala,b \n\naDepartment of Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics and Faculty of Biology III, University of \nFreiburg, St\u00fcbeweg 51, 79108 Freiburg, Germany \nbSpemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany \n \nCorresponding address: Max Planck Institute of Immunobiology and Epigenetics, St\u00fcbeweg 51, 79108 Freiburg, Germany. E-mail: \ndeswal@immunbio.mpg.de, Tel.: +49-761-5108-314, FAX: +49-761-5108-423 \n\n \n\nAbstract \n\nT cell antigen receptor (TCR) is a protein-complex expressed on all T cells of the immune system \nand is responsible for the activation of T cells when infectious agent is presented by an antigen \npresenting cell (APC) in the form of peptides bound to the major histocompatibility complex \n(pMHC). Despite numerous studies it is not clear what biochemical changes upon binding of \nantigen ligand to the extracellular domains of TCR leads to activation of intracellular signaling (a \nprocess known as TCR triggering). This review summarizes possible biochemical mechanisms for \nTCR triggering and discusses their comparative limitations and advantages in explaining various \nexperimental observations. \n\nKeywords: T cell antigen receptor, activation, model \n\nIntroduction \n\nT cell antigen receptor (TCR or TCR-CD3) is \n\na protein complex expressed exclusively in T \n\ncells and is composed of the variable TCR \u03b1 \n\nand \u03b2 chains and the constant CD3\u03b3, CD3\u03b4, \n\nCD3\u03b5 and CD3\u03b6 chains (Figure 1). CD3\u03b3 and \n\nCD3\u03b4 chain are glycoproteins each of which \n\nform a heterodimer with non-glycosylated \n\nCD3\u03b5 chain. Along with the CD3\u03b6-\u03b6 \n\nhomodimer, these chains associate with the \n\nTCR\u03b1\u03b2 heterodimer to generate the full TCR-\n\nCD3 complex [1]. The TCR-CD3 is \n\nresponsible for activation of a T cell upon \n\nantigen encounter which then help in the \n\nactivation of B cells by releasing helper \n\ncytokines (helper T cells) or kill the target cell \n\ndirectly by the secretion of cytotoxic effector \n\nmolecules such as granzymes, perforin and \n\ngranulysin. \n\nAntigenic ligand binding to the TCR leads to \n\nthe phosphorylation of immunoreceptor", "start_char_idx": 50, "end_char_idx": 2785, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f7571db9-1ae1-452a-b7dd-c932b1d40e49": {"__data__": {"id_": "f7571db9-1ae1-452a-b7dd-c932b1d40e49", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1f72d553-8666-4ea1-9fd0-fdcb2ad44229", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "6bc8609ccfc5e1189bbb35dfd216dcefb135da03b4fdc716e55aae392c5bad98", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2a5c17b8-54ad-4753-bbbc-e407813033e8", "node_type": "1", "metadata": {}, "hash": "43319fe77af796194768b88061b177970154bf8df090becf0a0ca518f3420315", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 63 \n\ntyrosine-based activation motifs (ITAMs) of \n\nthe CD3 complex by the Src family protein \n\ntyrosine kinase Lck, resulting in the \n\nmembrane targeting and activation of another \n\nkinase, ZAP-70. Once activated, ZAP-70 \n\nphosphorylates several substrates, including \n\nthe transmembrane adaptor protein, linker for \n\nactivation of T cells (LAT). Phospho-LAT \n\nserves as an important point of divergence for \n\nsignals initiated from the TCR by recruiting \n\nseveral effector molecules to the plasma \n\nmembrane thus initiating multiple pathways \n\nessential for full T cell activation [2]. \n\n! \"\n\n# $%\n\n&'&\n\n#\n\n++\n+ --\n\nITAM\n\n--\n\nV\n\nC\n\nV\n\nC\n\n--\n\n \n\nFig.1: The TCR-CD3 complex. V and C represent variable and \nconstant domains respectively for the TCR \u03b1 and \u03b2. The \nhypervariable region that binds to the pMHC complex is shown \nin red color. \n\n \n\nAlthough the downstream signaling \n\nmechanisms of T cell activation have been \n\nexplored to quite detailed extent, the basic \n\nmechanism of how ligand binding to \n\nextracellular domains of TCR\u03b1\u03b2 leads to \n\ndownstream signaling remained unclear.  \n\nSeveral models of TCR triggering have been \n\nproposed based on the experimental findings, \n\nbut none of the models so far could explain \n\nevery aspect of TCR triggering. Broadly, these \n\nmodels can be grouped in three major \n\nprocesses which involve aggregation, \n\nconformational change or segregation (Figure \n\n2). This review summarizes the proposed \n\nmodels of TCR triggering and discusses their \n\ncomparative abilities for explaining various \n\nexperimental observations (Table 1). \n\n1. Antigen induced clustering  \n\nSeveral forms of clustering have been \n\nproposed as the triggering mechanism for the \n\nTCR. These include homodimer, heterodimer \n\nand the pseudodimer models as discussed \n\nbelow.", "start_char_idx": 2790, "end_char_idx": 5004, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2a5c17b8-54ad-4753-bbbc-e407813033e8": {"__data__": {"id_": "2a5c17b8-54ad-4753-bbbc-e407813033e8", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f7571db9-1ae1-452a-b7dd-c932b1d40e49", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "b0a869e62be684a3fdef045bad335810d52ca053aaff99c3b27a1a3c93c21e3b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9bb3b841-ba7a-4b24-a9b8-9ea879381dec", "node_type": "1", "metadata": {}, "hash": "59ff5be8a2746c796ac55ec247db2ee8f4d3dd242025a2bca6f5b1fafcbe92b9", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 64 \n\nb c\n\nd e\n\nf\n\nLck\n\nTCR\n\npMHC\n\nLck LckLck\n\nbinding\n\nTCR\n\nLck\n\nCD45\n\npMHC\n\nLck Lck Lck LckLck\n\nbinding\n\nlipid raft\n\nLck\n\nTCR\n\npMHC\n\nCo-receptor\n\nLck\n\nTCR\n\npMHC\n\nLck\n\nTCR\n\npMHC\n\nLckLck\n\nCD45\n\nTCR\n\npMHC\n\nbinding\n\n!\n\nLck\nLck\n\nTCR\n\npMHC\n\nLck\n\nPullbinding\n\nTCR\n\npMHC\n\nbinding\n\na\n\ng h\n\nClustering models\n\nConformational change models\n\nSegregation models\n\n \n\nFig.2: Various models of TCR triggering. a) Homodimer model, b) Heterodimer model, c) Pseudo-dimer model, d) Membrane binding model, \ne) Kinetic deformation model, f) Permissive geometry model, g) Kinetic segregation, h) Lipid raft mediated segregation model. Full \ndescription of each model is given in the main text.", "start_char_idx": 5021, "end_char_idx": 6117, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9bb3b841-ba7a-4b24-a9b8-9ea879381dec": {"__data__": {"id_": "9bb3b841-ba7a-4b24-a9b8-9ea879381dec", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2a5c17b8-54ad-4753-bbbc-e407813033e8", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "183979cf761c90ed8660b11d1b58e9695a0d7d2d5030a706413ed517ef9f30e7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "260e8e4c-8884-4aa1-812f-53b647ae85e2", "node_type": "1", "metadata": {}, "hash": "32e440b21663923b48b527c8cc2bd180fd4e3af20c327d69e7b25b1b89245a0c", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 65 \n\nTable 1: Abilities of different models to explain various experimental observations. \n\nObservation\t\n \u00a0\n\t\n \u00a0\n\nTriggering\t\n \u00a0model\t\n \u00a0\n\t\n \u00a0\n\n\t\n \u00a0 Homodimer\t\n \u00a0 Heterodimer\t\n \u00a0 Pseudo-\ndimer\t\n \u00a0\n\nMembrane \nbinding\t\n \u00a0\n\nKinetic \ndeformation\t\n \u00a0\n\nPermissive \ngeometry\t\n \u00a0\n\nKinetic \nsegregation\t\n \u00a0\n\nLipid \nraft\t\n \u00a0\n\nPre-\u00ad\u2010clustered\t\n \u00a0\nTCR\t\n \u00a0\n\nNo\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0\n\nCoreceptor\t\n \u00a0\nindependent\t\n \u00a0\ntriggering\t\n \u00a0\n\nYes\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0\n\nTriggering\t\n \u00a0by\t\n \u00a0\nsoluble\t\n \u00a0\n\nmonomer\t\n \u00a0pMHC\t\n \u00a0\n\nNo\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0\n\nTriggering\t\n \u00a0by\t\n \u00a0\nanti-\u00ad\u2010CD3\t\n \u00a0\nantibodies\t\n \u00a0\n\nYes\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0\n\nActivation\t\n \u00a0by\t\n \u00a0\npervanadate\t\n \u00a0\n\nNo\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0\n\nInhibition\t\n \u00a0by\t\n \u00a0\ntruncated\t\n \u00a0CD45\t\n \u00a0\n\nNo\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0\n\t\n \u00a0\n\nInhibition\t\n \u00a0by\t\n \u00a0\nelongated\t\n \u00a0pMHC\t\n \u00a0\n\nNo\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0\n\t\n \u00a0\n\nTriggering\t\n \u00a0in\t\n \u00a0\nabsence\t\n \u00a0of\t\n \u00a0self\t\n \u00a0\n\npMHC\t\n \u00a0\n\nYes\t\n \u00a0 Yes\t\n \u00a0 No\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0 Yes\t\n \u00a0\n\n \n\na. Homodimer model \n\nClustering of TCR\u2013CD3 complexes following \n\nTCR engagement could lead to enhanced \n\nphosphorylation, for example by increasing \n\nthe proximity of associated Lck molecules, \n\nresulting in the activation of the second \n\nreceptor in the cluster by trans-\n\nautophosphorylation (or this enhanced \n\nproximity of kinases might lead to their trans-\n\nphosphorylation which enhance their activity \n\nto phosphorylate the receptor). This model is  \n\n \n\nsupported by the observations that artificial \n\nligands such as pMHC tetramer and the anti-\n\nTCR\u03b1\u03b2 and anti-CD3 antibodies that crosslink \n\nthe TCR lead to its activation whereas Fab \n\nfragment of these antibodies or the monomeric \n\npMHC in solution fail to activate the TCR [3]. \n\nAlso the observations that MHC class I [4, 5] \n\nand MHC class II [6] exist in pre-formed \n\nclusters, support the homodimer model. \n\nHowever, since several studies have now \n\nshown the existence of pre-clustered TCRs on \n\nthe T cell surface [7, 8], it is difficult to \n\nimagine that clustering alone could trigger the", "start_char_idx": 6123, "end_char_idx": 8789, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "260e8e4c-8884-4aa1-812f-53b647ae85e2": {"__data__": {"id_": "260e8e4c-8884-4aa1-812f-53b647ae85e2", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9bb3b841-ba7a-4b24-a9b8-9ea879381dec", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "d56a7d95eb0b1ae2b067c4ccab42d55e90e17c297144a99094efbc8408bb9e1c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "486a2e61-9ce2-45d6-b5a9-3ebee4c09d1f", "node_type": "1", "metadata": {}, "hash": "60ba59729bb15f6f96cd8c4cb1505ad61eecc20c7eab1c00605e3704e4a71b56", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 66 \n\nTCR. In fact in a study by Schamel and \n\ncolleagues [9], both clustering and \n\nconformational change were shown to be \n\nrequired for TCR triggering (permissive \n\ngeometry model, as discussed later). \n\nb.  Heterodimer model \n\nIn the heterodimer model [10], coreceptor \n\nCD8 or CD4 binding to the same pMHC \n\ncomplex as the TCR brings the coreceptor-\n\nassociated Lck kinase into proximity with \n\nTCR\u2013CD3 ITAMs and their phosphorylation. \n\nFurther it has been proposed that to fully \n\nactivate the TCR, a pMHC ligand need to \n\ninteract with a TCR\u2013CD8 pair with a \n\nthreshold time to induce stable zippering \n\nbetween the membrane-proximal domain of \n\nCD8 and the connecting peptide motif in the \n\nTCR\u03b1 [11]. This allows stable association of \n\nLck with the CD3 complex and results in \n\ncomplete phosphorylation of the CD3 ITAMs. \n\nA low-affinity pMHC ligand that interacts \n\nwith a TCR\u2013CD8 pair with less than threshold \n\ntime induces incomplete zippering and \n\ntherefore allows only transient Lck association \n\nand partial CD3 phosphorylation. However, \n\nthe observations that T cells can develop and \n\nfunction normally in the mice lacking both \n\nCD4 and CD8 suggest that coreceptors are not \n\nabsolutely required for TCR triggering and T \n\ncell activation [12, 13]. Also the fact that anti-\n\nCD3 antibodies and their F(ab\u2019)2 fragments \n\nthat do not engage the co-receptors can still \n\nactivate the TCR [14, 15], indicates that co-\n\nreceptors are not required for the TCR \n\ntriggering itself, though they might be \n\nimportant for full activation of a T cell. \n\nc. Pseudo-dimer model \n\nOnly a few MHCs carry high affinity agonist \n\npeptides for the TCRs, whereas a great \n\nmajority of MHCs carry endogenous self-\n\npeptides. On the basis of the crystallographic \n\nstudies which showed that the CD4 tail \n\nassociating with Lck was far from their own \n\nTCR/CD3 complex, the pseudodimer model \n\npostulates that two TCRs are brought together \n\nby binding low-affinity self or high-affinity \n\nagonist pMHC ligands and that the CD4 \n\nassociated with a TCR engaging the agonist \n\npMHC complex can assists in phosphorylation \n\nof neighboring TCR (engaged to low affinity \n\nself peptide) by the associated kinase Lck \n\n[16]. Thus according to this model, a \n\n\u2018pseudodimer\u2019 consists of one foreign and one \n\nself-antigen engaged receptor linked via CD4 \n\nmolecule is the primary unit of TCR signal \n\ninitiation module. \n\n2. Conformational change model \n\nSeveral studies have proposed conformational", "start_char_idx": 8794, "end_char_idx": 11708, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "486a2e61-9ce2-45d6-b5a9-3ebee4c09d1f": {"__data__": {"id_": "486a2e61-9ce2-45d6-b5a9-3ebee4c09d1f", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "260e8e4c-8884-4aa1-812f-53b647ae85e2", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "f23781c3a001df09b7bc1e61f82722142547c2ace8af8a827f992fa096b35e14", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1150ab7b-cd33-42d4-8d04-a2ed8ee9cdaa", "node_type": "1", "metadata": {}, "hash": "497056ec8489c7e9d5cc77b540ebdd32220c8f76e797702b1639b348a457f79e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 67 \n\nchange as a requirement for TCR triggering \n\n[17, 18]. However, how the ligand binding to \n\nTCR\u03b1\u03b2 ectodomains leads to a \n\nconformational change in the cytoplasmic tails \n\nof CD3 subunits is not clear. The situation is \n\ncomplicated by the fact that the crystal \n\nstructure of the fully assembled TCR-CD3 \n\ncomplex is not known, although individual \n\nsubunits have been crystallized partially [19-\n\n22]. Some of the important models for the \n\nmechanism of conformational change in TCR-\n\nCD3 are discussed below.  \n\na. Membrane binding model \n\nAccording to this model, cytoplasmic tails \n\nof CD3\u03b5 [23, 24] and CD3\u03b6 [25] associate \n\nwith the lipids present in the plasma \n\nmembrane of a T cell. Thereby, the ITAMs \n\nare buried in the plasma membrane, \n\nmaking them inaccessible for \n\nphosphorylation by the kinases. Ligand \n\nbinding leads to release of these CD3 \n\nchains from the plasma membrane, making \n\nthem accessible to kinase and hence the \n\nphosphorylation. \n\nb. Kinetic deformation model \n\nSeveral line of evidence have proposed that \n\nthe mechanical effects (such as pulling or \n\nshearing) of pMHC binding to the TCR leads \n\nto a piston-like displacement of the TCR\u2013CD3 \n\ncomplex [26, 27]. This induces a change in the \n\nconformation of the CD3 cytoplasmic \n\ndomains, allowing ITAM phosphorylation. \n\nc. Permissive geometry model \n\nBased on the experimental evidence for the \n\nexistence of TCR-CD3s in a pre-clustered \n\nform [7] and the fact that pMHC monomers in \n\nsolution fail to trigger TCR[28, 29] while \n\npMHC dimer or higher oligomer can induce \n\nTCR triggering, the permissive geometry \n\nmodel was proposed[30]. According to this \n\nmodel, before ligand binding, the mono and \n\nmultivalent TCR-CD3s exist in an \n\nautoinhibited state where ITAMs are \n\ninaccessible for phosphorylation by the kinase. \n\nSimultaneous dimeric (or higher order) ligand \n\nbinding to the two TCR-CD3 complexes leads \n\nto a scissor-like movement in the two TCR-\n\nCD3 complexes leading to exposure of their \n\ncytoplasmic tails, thus making the ITAMs \n\naccessible for phosphorylation.   \n\n3. Segregation Model \n\nIn a resting T cell, phosphorylation of the \n\nTCR-CD3 is kept in check by the active \n\nphosphotases. This is supported by the fact \n\nthat pervanadate, a phosphatase inhibitor,", "start_char_idx": 11713, "end_char_idx": 14417, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1150ab7b-cd33-42d4-8d04-a2ed8ee9cdaa": {"__data__": {"id_": "1150ab7b-cd33-42d4-8d04-a2ed8ee9cdaa", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "486a2e61-9ce2-45d6-b5a9-3ebee4c09d1f", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "c6336355ebf9eb01d911bb5c2a4455e496f9fdd2dbde56a43461f66ba3b9a7d5", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e6b973b1-880d-4775-9a1c-c45c79f6cf6c", "node_type": "1", "metadata": {}, "hash": "017d1e4e02dd6e46f6a2b0f798bfd23d15ea70dd49f952917b35e3d8db20a916", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 68 \n\ntreatment leads to strong phosphorylation of \n\nthe TCR-CD3 ITAMs and also initiates the \n\ndownstream signaling [31]. Thus any process \n\nthat favors the kinase-phosphtase balance \n\ntowards kinase, can lead to TCR-CD3 \n\nphosphorylation and the signaling. \n\nRedistribution of the TCR-CD3, kinases and \n\nphosphatases in which phosphatase is \n\nsegregated from the TCR can interfere with \n\nthe dephosphorylation. There are two \n\nmechanisms proposed for such ligand \n\ndependent re-distribution, which are discussed \n\nbelow.  \n\na. Kinetic segregation \n\nKinetic segregation model was proposed \n\nbased on the topological view of the cell \n\nsurface molecules at the T cell\u2013APC interface \n\n[32, 33]. The tight intercellular contact causes \n\nthe segregation of the molecules by sizes of \n\ntheir ectodomain resulting in physical \n\nseparation of TCR from the large inhibitory \n\ntyrosine phosphatase CD45, leading to stable \n\nphosphorylation of TCR\u2013CD3 ITAMs by Lck. \n\nThough sounding good in the context of a T-\n\ncell \u2013 APC contact, the model fails to explain \n\nthe activation of T cells by soluble anti-\n\nTCR\u03b1\u03b2 and anti-CD3 antibodies and the \n\npMHC tetramer [14, 34]. \n\nb. Lipid raft mediated segregation model \n\nLipid rafts are the detergent (such as Triton X-\n\n100, Brij-series, NP-40 or CHAPS)-resistant \n\nmembrane microdomains which are enriched \n\nin glycosphingolipids, cholesterol and lipid-\n\nmodified proteins such as the GPI-anchored \n\nproteins. Also these microdomains are \n\nenriched in double-acylated (myristoylated, \n\npalmitoylated) Src-family kinases and the \n\nimportant signaling molecules such as the \n\ncoreceptors CD4 and CD8 and the adaptor \n\nprotein LAT. Lipid raft mediated segregation \n\nmodel postulates that pMHC engagement \n\nresults in partitioning of the TCR\u2013CD3 \n\ncomplex into lipid rafts enriched in Lck and \n\ndeficient in CD45 [35, 36]. Partial support for \n\nthis model comes from the observations that \n\npalmitoylation-deficient (and raft excluded) \n\nmutants of Lck and LAT are functionally \n\ndefective. In addition, artificial targeting of \n\nother cytoplasmic molecules, such as SHP-1 \n\n[37], CD45 [38] or PLC\u03b3 [39], to membrane \n\nrafts has marked functional effects on TCR-\n\ninduced signaling. A connecting peptide in the \n\nTCR\u03b1 chain and the CD3\u03b4 chain were \n\nidentified as the critical sites essential for \n\neffective raft association. Mutations in these \n\ncomponents interfere with TCR signaling [40]. \n\nThe mechanism of directing association of the \n\nengaged TCR complex with lipid rafts is not \n\nclear but might be based on co-engagement of \n\nthe raft resident CD4/CD8 coreceptors. \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 14422, "end_char_idx": 17539, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e6b973b1-880d-4775-9a1c-c45c79f6cf6c": {"__data__": {"id_": "e6b973b1-880d-4775-9a1c-c45c79f6cf6c", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1150ab7b-cd33-42d4-8d04-a2ed8ee9cdaa", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "4b42651355a48e2d301f57718702cbfc60e1e68d94410b921caba2d66bb510fd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "538a6b01-89e8-429d-a515-8d8347bd8bc0", "node_type": "1", "metadata": {}, "hash": "e5d7663f3662f30e84ce02aea505353ba829ea5dcac6ffc50e5ba4592f2fda4e", "class_name": "RelatedNodeInfo"}}, "text": "2,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 69 \n\nConclusion \n\nAnalysis of various proposed mechanisms \n\nreveals that probably a combination of these \n\nmechanisms is responsible for TCR triggering. \n\nExistence of pre-clustered TCR molecules \n\nrules out the homodimerization as a \n\nrequirement for TCR triggering. It is possible \n\nthat CD3 ITAMs are inaccessible for \n\nphosphorylation by the kinase in the resting \n\nstate and some sort of conformational change \n\nis necessary for exposing the ITAMs, but \n\nadditional studies are required to find out what \n\nmechanism is responsible for such \n\nconformational change. Segregation models \n\nargue the balance of kinase mediated \n\nphosphorylation and phosphatase mediated \n\ndephosphorylation shifts in favour of kinase \n\nupon ligand binding. Though such segregation \n\nmight contribute for TCR triggering in the \n\ncontext of a T cell-antigen presenting cell \n\ninteraction, it seems not the sole mechanism of \n\nTCR triggering as the soluble anti-TCR or \n\nanti-CD3 antibodies which do not induce \n\nsegregation can still cause TCR triggering.  \n\nFurther studies on the structure of fully \n\nassembled TCR-CD3 complex will be \n\nrequired for a better understanding of the TCR \n\ntriggering. \n\nReferences \n\n1. Call\t\n \u00a0ME,\t\n \u00a0Pyrdol\t\n \u00a0J,\t\n \u00a0Wiedmann\t\n \u00a0M,\t\n \u00a0Wucherpfennig\t\n \u00a0\nKW:\t\n \u00a0The\t\n \u00a0organizing\t\n \u00a0principle\t\n \u00a0 in\t\n \u00a0the\t\n \u00a0formation\t\n \u00a0\nof\t\n \u00a0the\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0receptor-\u00ad\u2010CD3\t\n \u00a0complex.\t\n \u00a0Cell\t\n \u00a02002,\t\n \u00a0\n111:967-\u00ad\u2010979.\t\n \u00a0\n\t\n \u00a0\n\n2. Smith-\u00ad\u2010Garvin\t\n \u00a0JE,\t\n \u00a0Koretzky\t\n \u00a0GA,\t\n \u00a0Jordan\t\n \u00a0MS:\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0\nactivation.\t\n \u00a0 Annu\t\n \u00a0 Rev\t\n \u00a0 Immunol\t\n \u00a0 2009,\t\n \u00a0 27:591-\u00ad\u2010\n619.\t\n \u00a0\n\t\n \u00a0\n\n3. Cochran\t\n \u00a0 JR,\t\n \u00a0 Aivazian\t\n \u00a0 D,\t\n \u00a0 Cameron\t\n \u00a0 TO,\t\n \u00a0 Stern\t\n \u00a0 LJ:\t\n \u00a0\nReceptor\t\n \u00a0 clustering\t\n \u00a0 and\t\n \u00a0 transmembrane\t\n \u00a0\nsignaling\t\n \u00a0 in\t\n \u00a0 T\t\n \u00a0 cells.\t\n \u00a0 Trends\t\n \u00a0 Biochem\t\n \u00a0 Sci\t\n \u00a0 2001,\t\n \u00a0\n26(5):304-\u00ad\u2010310.\t\n \u00a0\n\t\n \u00a0\n\n4. Matko\t\n \u00a0J,\t\n \u00a0Bushkin\t\n \u00a0Y,\t\n \u00a0Wei\t\n \u00a0T,\t\n \u00a0Edidin\t\n \u00a0M:\t\n \u00a0Clustering\t\n \u00a0\nof\t\n \u00a0 class\t\n \u00a0 I\t\n \u00a0 HLA\t\n \u00a0 molecules\t\n \u00a0 on\t\n \u00a0 the\t\n \u00a0 surfaces\t\n \u00a0 of\t\n \u00a0\nactivated\t\n \u00a0 and\t\n \u00a0 transformed\t\n \u00a0 human\t\n \u00a0 cells.\t\n \u00a0 J\t\n \u00a0\nImmunol\t\n \u00a01994,\t\n \u00a0152(7):3353-\u00ad\u20103360.\t\n \u00a0\n\t\n \u00a0\n\n5. Fooksman\t\n \u00a0 DR,\t\n \u00a0 Gronvall\t\n \u00a0 GK,\t\n \u00a0 Tang\t\n \u00a0 Q,\t\n \u00a0 Edidin\t\n \u00a0 M:\t\n \u00a0\nClustering\t\n \u00a0class\t\n \u00a0I\t\n \u00a0MHC\t\n \u00a0modulates\t\n \u00a0sensitivity\t\n \u00a0of\t\n \u00a0\nT\t\n \u00a0cell\t\n \u00a0recognition.", "start_char_idx": 17530, "end_char_idx": 20164, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "538a6b01-89e8-429d-a515-8d8347bd8bc0": {"__data__": {"id_": "538a6b01-89e8-429d-a515-8d8347bd8bc0", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e6b973b1-880d-4775-9a1c-c45c79f6cf6c", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "b4362c6254f8a5b3ce8c2f0d0a6f03f0f7846d131884523bf5ac109c59b57ac7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fceecb19-0863-4f99-9d2e-904cabe16614", "node_type": "1", "metadata": {}, "hash": "1045d315da9f69007bcf3aa91473d277779d4e97d6384069d1b13c1cfa4aea16", "class_name": "RelatedNodeInfo"}}, "text": "Journal\t\n \u00a0of\t\n \u00a0immunology\t\n \u00a02006,\t\n \u00a0\n176(11):6673-\u00ad\u20106680.\t\n \u00a0\n\t\n \u00a0\n\n6. Unternaehrer\t\n \u00a0 JJ,\t\n \u00a0 Chow\t\n \u00a0 A,\t\n \u00a0 Pypaert\t\n \u00a0 M,\t\n \u00a0 Inaba\t\n \u00a0 K,\t\n \u00a0\nMellman\t\n \u00a0 I:\t\n \u00a0 The\t\n \u00a0 tetraspanin\t\n \u00a0 CD9\t\n \u00a0 mediates\t\n \u00a0\nlateral\t\n \u00a0association\t\n \u00a0of\t\n \u00a0MHC\t\n \u00a0class\t\n \u00a0II\t\n \u00a0molecules\t\n \u00a0on\t\n \u00a0\nthe\t\n \u00a0 dendritic\t\n \u00a0 cell\t\n \u00a0 surface.\t\n \u00a0 Proceedings\t\n \u00a0 of\t\n \u00a0 the\t\n \u00a0\nNational\t\n \u00a0 Academy\t\n \u00a0 of\t\n \u00a0 Sciences\t\n \u00a0 of\t\n \u00a0 the\t\n \u00a0 United\t\n \u00a0\nStates\t\n \u00a0of\t\n \u00a0America\t\n \u00a02007,\t\n \u00a0104(1):234-\u00ad\u2010239.\t\n \u00a0\n\t\n \u00a0\n\n7. Schamel\t\n \u00a0 WW,\t\n \u00a0 Arechaga\t\n \u00a0 I,\t\n \u00a0 Risueno\t\n \u00a0 RM,\t\n \u00a0 van\t\n \u00a0\nSanten\t\n \u00a0 HM,\t\n \u00a0 Cabezas\t\n \u00a0 P,\t\n \u00a0 Risco\t\n \u00a0 C,\t\n \u00a0 Valpuesta\t\n \u00a0 JM,\t\n \u00a0\nAlarcon\t\n \u00a0 B:\t\n \u00a0 Coexistence\t\n \u00a0 of\t\n \u00a0 multivalent\t\n \u00a0 and\t\n \u00a0\nmonovalent\t\n \u00a0TCRs\t\n \u00a0explains\t\n \u00a0high\t\n \u00a0sensitivity\t\n \u00a0and\t\n \u00a0\nwide\t\n \u00a0 range\t\n \u00a0 of\t\n \u00a0 response.\t\n \u00a0 J\t\n \u00a0 Exp\t\n \u00a0 Med\t\n \u00a0 2005,\t\n \u00a0\n202:493-\u00ad\u2010503.\t\n \u00a0\n\t\n \u00a0\n\n8. Lillemeier\t\n \u00a0 BF,\t\n \u00a0 Mortelmaier\t\n \u00a0 MA,\t\n \u00a0 Forstner\t\n \u00a0 MB,\t\n \u00a0\nHuppa\t\n \u00a0JB,\t\n \u00a0Groves\t\n \u00a0JT,\t\n \u00a0Davis\t\n \u00a0MM:\t\n \u00a0TCR\t\n \u00a0and\t\n \u00a0Lat\t\n \u00a0are\t\n \u00a0\nexpressed\t\n \u00a0on\t\n \u00a0separate\t\n \u00a0protein\t\n \u00a0islands\t\n \u00a0on\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0\nmembranes\t\n \u00a0and\t\n \u00a0concatenate\t\n \u00a0during\t\n \u00a0activation.\t\n \u00a0\nNat\t\n \u00a0Immunol\t\n \u00a02010,\t\n \u00a011(1):90-\u00ad\u201096.\t\n \u00a0\n\t\n \u00a0\n\n9. Minguet\t\n \u00a0 S,\t\n \u00a0 Swamy\t\n \u00a0 M,\t\n \u00a0 Alarcon\t\n \u00a0 B,\t\n \u00a0 Luescher\t\n \u00a0 IF,\t\n \u00a0\nSchamel\t\n \u00a0 WW:\t\n \u00a0 Full\t\n \u00a0 activation\t\n \u00a0 of\t\n \u00a0 the\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0\nreceptor\t\n \u00a0 requires\t\n \u00a0 both\t\n \u00a0 clustering\t\n \u00a0 and\t\n \u00a0\n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 70 \n\nconformational\t\n \u00a0 changes\t\n \u00a0 at\t\n \u00a0 CD3.\t\n \u00a0 Immunity\t\n \u00a0\n2007,\t\n \u00a026(1):43-\u00ad\u201054.\t\n \u00a0\n\t\n \u00a0\n\n10. Trautmann\t\n \u00a0 A,\t\n \u00a0 Randriamampita\t\n \u00a0 C:\t\n \u00a0 Initiation\t\n \u00a0 of\t\n \u00a0\nTCR\t\n \u00a0signalling\t\n \u00a0revisited.\t\n \u00a0Trends\t\n \u00a0Immunol\t\n \u00a02003,\t\n \u00a0\n24(8):425-\u00ad\u2010428.\t\n \u00a0\n\t\n \u00a0\n\n11.", "start_char_idx": 20168, "end_char_idx": 22285, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fceecb19-0863-4f99-9d2e-904cabe16614": {"__data__": {"id_": "fceecb19-0863-4f99-9d2e-904cabe16614", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "538a6b01-89e8-429d-a515-8d8347bd8bc0", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "8b9f02a3a43cc41da5d79f76470c65fd3386dbb6ec2b776f558e3e87452eac54", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5360ef63-a3a5-4826-a2bf-7b36def43bbd", "node_type": "1", "metadata": {}, "hash": "e809e031ea06adc9e84eb58417b6af93a0f4ae1ca06cc982b911e8b59971fa30", "class_name": "RelatedNodeInfo"}}, "text": "11. Palmer\t\n \u00a0 E,\t\n \u00a0 Naeher\t\n \u00a0 D:\t\n \u00a0 Affinity\t\n \u00a0 threshold\t\n \u00a0 for\t\n \u00a0\nthymic\t\n \u00a0 selection\t\n \u00a0 through\t\n \u00a0 a\t\n \u00a0 T-\u00ad\u2010cell\t\n \u00a0 receptor-\u00ad\u2010co-\u00ad\u2010\nreceptor\t\n \u00a0 zipper.\t\n \u00a0 Nat\t\n \u00a0 Rev\t\n \u00a0 Immunol\t\n \u00a0 2009,\t\n \u00a0\n9(3):207-\u00ad\u2010213.\t\n \u00a0\n\t\n \u00a0\n\n12. Locksley\t\n \u00a0 RM,\t\n \u00a0 Reiner\t\n \u00a0 SL,\t\n \u00a0 Hatam\t\n \u00a0 F,\t\n \u00a0 Littman\t\n \u00a0 DR,\t\n \u00a0\nKilleen\t\n \u00a0N:\t\n \u00a0Helper\t\n \u00a0T\t\n \u00a0cells\t\n \u00a0without\t\n \u00a0CD4:\t\n \u00a0control\t\n \u00a0of\t\n \u00a0\nleishmaniasis\t\n \u00a0 in\t\n \u00a0 CD4-\u00ad\u2010deficient\t\n \u00a0 mice.\t\n \u00a0 Science\t\n \u00a0\n1993,\t\n \u00a0261(5127):1448-\u00ad\u20101451.\t\n \u00a0\n\t\n \u00a0\n\n13. Schilham\t\n \u00a0 MW,\t\n \u00a0 Fung-\u00ad\u2010Leung\t\n \u00a0 WP,\t\n \u00a0 Rahemtulla\t\n \u00a0 A,\t\n \u00a0\nKuendig\t\n \u00a0 T,\t\n \u00a0 Zhang\t\n \u00a0 L,\t\n \u00a0 Potter\t\n \u00a0 J,\t\n \u00a0 Miller\t\n \u00a0 RG,\t\n \u00a0\nHengartner\t\n \u00a0H,\t\n \u00a0Mak\t\n \u00a0TW:\t\n \u00a0Alloreactive\t\n \u00a0cytotoxic\t\n \u00a0T\t\n \u00a0\ncells\t\n \u00a0can\t\n \u00a0develop\t\n \u00a0and\t\n \u00a0function\t\n \u00a0in\t\n \u00a0mice\t\n \u00a0lacking\t\n \u00a0\nboth\t\n \u00a0 CD4\t\n \u00a0 and\t\n \u00a0 CD8.\t\n \u00a0 European\t\n \u00a0 journal\t\n \u00a0 of\t\n \u00a0\nimmunology\t\n \u00a01993,\t\n \u00a023(6):1299-\u00ad\u20101304.\t\n \u00a0\n\t\n \u00a0\n\n14. Chang\t\n \u00a0 TW,\t\n \u00a0 Kung\t\n \u00a0 PC,\t\n \u00a0 Gingras\t\n \u00a0 SP,\t\n \u00a0 Goldstein\t\n \u00a0 G:\t\n \u00a0\nDoes\t\n \u00a0OKT3\t\n \u00a0monoclonal\t\n \u00a0antibody\t\n \u00a0react\t\n \u00a0with\t\n \u00a0an\t\n \u00a0\nantigen-\u00ad\u2010recognition\t\n \u00a0 structure\t\n \u00a0 on\t\n \u00a0 human\t\n \u00a0 T\t\n \u00a0\ncells?\t\n \u00a0Proc\t\n \u00a0Natl\t\n \u00a0Acad\t\n \u00a0Sci\t\n \u00a0U\t\n \u00a0S\t\n \u00a0A\t\n \u00a01981,\t\n \u00a078(3):1805-\u00ad\u2010\n1808.\t\n \u00a0\n\t\n \u00a0\n\n15. Kaye\t\n \u00a0J,\t\n \u00a0Janeway\t\n \u00a0CA,\t\n \u00a0Jr.:\t\n \u00a0The\t\n \u00a0Fab\t\n \u00a0fragment\t\n \u00a0of\t\n \u00a0a\t\n \u00a0\ndirectly\t\n \u00a0 activating\t\n \u00a0 monoclonal\t\n \u00a0 antibody\t\n \u00a0 that\t\n \u00a0\nprecipitates\t\n \u00a0 a\t\n \u00a0 disulfide-\u00ad\u2010linked\t\n \u00a0 heterodimer\t\n \u00a0\nfrom\t\n \u00a0a\t\n \u00a0helper\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0clone\t\n \u00a0blocks\t\n \u00a0activation\t\n \u00a0by\t\n \u00a0\neither\t\n \u00a0allogeneic\t\n \u00a0Ia\t\n \u00a0or\t\n \u00a0antigen\t\n \u00a0and\t\n \u00a0self-\u00ad\u2010Ia.\t\n \u00a0J\t\n \u00a0Exp\t\n \u00a0\nMed\t\n \u00a01984,\t\n \u00a0159(5):1397-\u00ad\u20101412.\t\n \u00a0\n\t\n \u00a0\n\n16. Krogsgaard\t\n \u00a0M,\t\n \u00a0Li\t\n \u00a0QJ,\t\n \u00a0Sumen\t\n \u00a0C,\t\n \u00a0Huppa\t\n \u00a0JB,\t\n \u00a0Huse\t\n \u00a0\nM,\t\n \u00a0 Davis\t\n \u00a0 MM:\t\n \u00a0 Agonist/endogenous\t\n \u00a0 peptide-\u00ad\u2010\nMHC\t\n \u00a0 heterodimers\t\n \u00a0 drive\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 activation\t\n \u00a0 and\t\n \u00a0\nsensitivity.\t\n \u00a0Nature\t\n \u00a02005,\t\n \u00a0434(7030):238-\u00ad\u2010243.\t\n \u00a0\n\n17. Levin\t\n \u00a0 SE,\t\n \u00a0 Weiss\t\n \u00a0 A:\t\n \u00a0 Twisting\t\n \u00a0 tails\t\n \u00a0 exposed:\t\n \u00a0 the\t\n \u00a0\nevidence\t\n \u00a0for\t\n \u00a0TCR\t\n \u00a0conformational\t\n \u00a0change.", "start_char_idx": 22282, "end_char_idx": 24311, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5360ef63-a3a5-4826-a2bf-7b36def43bbd": {"__data__": {"id_": "5360ef63-a3a5-4826-a2bf-7b36def43bbd", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fceecb19-0863-4f99-9d2e-904cabe16614", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "015112c0a15d8759333c06041552a282ce6fff263cf7b7cdce15621351843b9b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "19af00ea-5f4d-4dc4-b3f6-007947e14ea9", "node_type": "1", "metadata": {}, "hash": "c34ce5326a3e8513e13e071238713ebecaaa403dcf683035381e288496549f36", "class_name": "RelatedNodeInfo"}}, "text": "J\t\n \u00a0Exp\t\n \u00a0\nMed\t\n \u00a02005,\t\n \u00a0201(4):489-\u00ad\u2010492.\t\n \u00a0\n\t\n \u00a0\n\n18. Gil\t\n \u00a0 D,\t\n \u00a0 Schamel\t\n \u00a0 WW,\t\n \u00a0 Montoya\t\n \u00a0 M,\t\n \u00a0 Sanchez-\u00ad\u2010\nMadrid\t\n \u00a0F,\t\n \u00a0Alarcon\t\n \u00a0B:\t\n \u00a0Recruitment\t\n \u00a0of\t\n \u00a0Nck\t\n \u00a0by\t\n \u00a0CD3\t\n \u00a0\nepsilon\t\n \u00a0 reveals\t\n \u00a0 a\t\n \u00a0 ligand-\u00ad\u2010induced\t\n \u00a0\n\nconformational\t\n \u00a0 change\t\n \u00a0 essential\t\n \u00a0 for\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0\nreceptor\t\n \u00a0signaling\t\n \u00a0and\t\n \u00a0synapse\t\n \u00a0formation.\t\n \u00a0Cell\t\n \u00a0\n2002,\t\n \u00a0109(7):901-\u00ad\u2010912.\t\n \u00a0\n\t\n \u00a0\n\n19. Sun\t\n \u00a0 ZY,\t\n \u00a0 Kim\t\n \u00a0 ST,\t\n \u00a0 Kim\t\n \u00a0 IC,\t\n \u00a0 Fahmy\t\n \u00a0 A,\t\n \u00a0 Reinherz\t\n \u00a0 EL,\t\n \u00a0\nWagner\t\n \u00a0 G:\t\n \u00a0 Solution\t\n \u00a0 structure\t\n \u00a0 of\t\n \u00a0 the\t\n \u00a0\nCD3epsilondelta\t\n \u00a0 ectodomain\t\n \u00a0 and\t\n \u00a0 comparison\t\n \u00a0\nwith\t\n \u00a0CD3epsilongamma\t\n \u00a0as\t\n \u00a0a\t\n \u00a0basis\t\n \u00a0for\t\n \u00a0modeling\t\n \u00a0\nT\t\n \u00a0 cell\t\n \u00a0 receptor\t\n \u00a0 topology\t\n \u00a0 and\t\n \u00a0 signaling.\t\n \u00a0 Proc\t\n \u00a0\nNatl\t\n \u00a0Acad\t\n \u00a0Sci\t\n \u00a0U\t\n \u00a0S\t\n \u00a0A\t\n \u00a02004,\t\n \u00a0101(48):16867-\u00ad\u201016872.\t\n \u00a0\n\t\n \u00a0\n\n20. Arnett\t\n \u00a0 KL,\t\n \u00a0 Harrison\t\n \u00a0 SC,\t\n \u00a0 Wiley\t\n \u00a0 DC:\t\n \u00a0 Crystal\t\n \u00a0\nstructure\t\n \u00a0of\t\n \u00a0a\t\n \u00a0human\t\n \u00a0CD3-\u00ad\u2010epsilon/delta\t\n \u00a0dimer\t\n \u00a0\nin\t\n \u00a0complex\t\n \u00a0with\t\n \u00a0a\t\n \u00a0UCHT1\t\n \u00a0single-\u00ad\u2010chain\t\n \u00a0antibody\t\n \u00a0\nfragment.\t\n \u00a0 Proc\t\n \u00a0 Natl\t\n \u00a0 Acad\t\n \u00a0 Sci\t\n \u00a0 U\t\n \u00a0 S\t\n \u00a0 A\t\n \u00a0 2004,\t\n \u00a0\n101(46):16268-\u00ad\u201016273.\t\n \u00a0\n\t\n \u00a0\n\n21. Sun\t\n \u00a0 ZYS,\t\n \u00a0 Seok\t\n \u00a0 Kim\t\n \u00a0 K,\t\n \u00a0 Wagner\t\n \u00a0 G,\t\n \u00a0 Reinherz\t\n \u00a0 EL:\t\n \u00a0\nMechanisms\t\n \u00a0 contributing\t\n \u00a0 to\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 receptor\t\n \u00a0\nsignaling\t\n \u00a0 and\t\n \u00a0 assembly\t\n \u00a0 revealed\t\n \u00a0 by\t\n \u00a0 the\t\n \u00a0\nsolution\t\n \u00a0structure\t\n \u00a0of\t\n \u00a0an\t\n \u00a0ectodomain\t\n \u00a0fragment\t\n \u00a0\nof\t\n \u00a0the\t\n \u00a0CD3eg\t\n \u00a0heterodimer.\t\n \u00a0Cell\t\n \u00a02001,\t\n \u00a0105:913-\u00ad\u2010\n923.\t\n \u00a0\n\t\n \u00a0\n\n22. Kjer-\u00ad\u2010Nielsen\t\n \u00a0L,\t\n \u00a0Dunstone\t\n \u00a0MA,\t\n \u00a0Kostenko\t\n \u00a0L,\t\n \u00a0Ely\t\n \u00a0LK,\t\n \u00a0\nBeddoe\t\n \u00a0 T,\t\n \u00a0 Mifsud\t\n \u00a0 NA,\t\n \u00a0 Purcell\t\n \u00a0 AW,\t\n \u00a0 Brooks\t\n \u00a0 AG,\t\n \u00a0\nMcCluskey\t\n \u00a0J,\t\n \u00a0Rossjohn\t\n \u00a0J:\t\n \u00a0Crystal\t\n \u00a0structure\t\n \u00a0of\t\n \u00a0the\t\n \u00a0\nhuman\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 receptor\t\n \u00a0 CD3\t\n \u00a0 epsilon\t\n \u00a0 gamma\t\n \u00a0\nheterodimer\t\n \u00a0 complexed\t\n \u00a0 to\t\n \u00a0 the\t\n \u00a0 therapeutic\t\n \u00a0\nmAb\t\n \u00a0 OKT3.\t\n \u00a0 Proc\t\n \u00a0 Natl\t\n \u00a0 Acad\t\n \u00a0 Sci\t\n \u00a0 U\t\n \u00a0 S\t\n \u00a0 A\t\n \u00a0 2004,\t\n \u00a0\n101(20):7675-\u00ad\u20107680.\t\n \u00a0\n\t\n \u00a0\n\n23.", "start_char_idx": 24315, "end_char_idx": 26320, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "19af00ea-5f4d-4dc4-b3f6-007947e14ea9": {"__data__": {"id_": "19af00ea-5f4d-4dc4-b3f6-007947e14ea9", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5360ef63-a3a5-4826-a2bf-7b36def43bbd", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a3f855bfd4029110d41a8acdbd9cdb4e83f713144eee9f883df79d3ec6cc9f87", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "965f6c80-3ccf-4f17-b871-dbb7ae16f7f9", "node_type": "1", "metadata": {}, "hash": "14223e5a191aba7ae704aaadc294463419438c77997d502a318ee078f24a4558", "class_name": "RelatedNodeInfo"}}, "text": "23. Xu\t\n \u00a0C,\t\n \u00a0Gagnon\t\n \u00a0E,\t\n \u00a0Call\t\n \u00a0ME,\t\n \u00a0Schnell\t\n \u00a0JR,\t\n \u00a0Schwieters\t\n \u00a0\nCD,\t\n \u00a0 Carman\t\n \u00a0 CV,\t\n \u00a0 Chou\t\n \u00a0 JJ,\t\n \u00a0 Wucherpfennig\t\n \u00a0 KW:\t\n \u00a0\nRegulation\t\n \u00a0 of\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 receptor\t\n \u00a0 activation\t\n \u00a0 by\t\n \u00a0\ndynamic\t\n \u00a0membrane\t\n \u00a0binding\t\n \u00a0of\t\n \u00a0the\t\n \u00a0CD3epsilon\t\n \u00a0\ncytoplasmic\t\n \u00a0 tyrosine-\u00ad\u2010based\t\n \u00a0 motif.\t\n \u00a0 Cell\t\n \u00a0 2008,\t\n \u00a0\n135(4):702-\u00ad\u2010713.\t\n \u00a0\n\t\n \u00a0\n\n24. Deford-\u00ad\u2010Watts\t\n \u00a0 LM,\t\n \u00a0 Tassin\t\n \u00a0 TC,\t\n \u00a0 Becker\t\n \u00a0 AM,\t\n \u00a0\nMedeiros\t\n \u00a0 JJ,\t\n \u00a0 Albanesi\t\n \u00a0 JP,\t\n \u00a0 Love\t\n \u00a0 PE,\t\n \u00a0 Wulfing\t\n \u00a0 C,\t\n \u00a0\nvan\t\n \u00a0Oers\t\n \u00a0NS:\t\n \u00a0The\t\n \u00a0cytoplasmic\t\n \u00a0tail\t\n \u00a0of\t\n \u00a0the\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0\nreceptor\t\n \u00a0 CD3\t\n \u00a0 epsilon\t\n \u00a0 subunit\t\n \u00a0 contains\t\n \u00a0 a\t\n \u00a0\nphospholipid-\u00ad\u2010binding\t\n \u00a0 motif\t\n \u00a0 that\t\n \u00a0 regulates\t\n \u00a0 T\t\n \u00a0\ncell\t\n \u00a0 functions.\t\n \u00a0 J\t\n \u00a0 Immunol\t\n \u00a0 2009,\t\n \u00a0 183(2):1055-\u00ad\u2010\n1064.\t\n \u00a0\n\t\n \u00a0\n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a062\t\n \u00a0\u2013\t\n \u00a071\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 71 \n\n25. Aivazian\t\n \u00a0 D,\t\n \u00a0 Stern\t\n \u00a0 LJ:\t\n \u00a0 Phosphorylation\t\n \u00a0 of\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0\nreceptor\t\n \u00a0zeta\t\n \u00a0is\t\n \u00a0regulated\t\n \u00a0by\t\n \u00a0a\t\n \u00a0lipid\t\n \u00a0dependent\t\n \u00a0\nfolding\t\n \u00a0 transition.\t\n \u00a0 Nat\t\n \u00a0 Struct\t\n \u00a0 Biol\t\n \u00a0 2000,\t\n \u00a0\n7(11):1023-\u00ad\u20101026.\t\n \u00a0\n\t\n \u00a0\n\n26. Ma\t\n \u00a0Z,\t\n \u00a0Sharp\t\n \u00a0KA,\t\n \u00a0Janmey\t\n \u00a0PA,\t\n \u00a0Finkel\t\n \u00a0TH:\t\n \u00a0Surface-\u00ad\u2010\nanchored\t\n \u00a0 monomeric\t\n \u00a0 agonist\t\n \u00a0 pMHCs\t\n \u00a0 alone\t\n \u00a0\ntrigger\t\n \u00a0 TCR\t\n \u00a0 with\t\n \u00a0 high\t\n \u00a0 sensitivity.\t\n \u00a0 PLoS\t\n \u00a0 Biol\t\n \u00a0\n2008,\t\n \u00a06(2):e43.\t\n \u00a0\n\n27. Ma\t\n \u00a0 Z,\t\n \u00a0 Janmey\t\n \u00a0 PA,\t\n \u00a0 Finkel\t\n \u00a0 TH:\t\n \u00a0 The\t\n \u00a0 receptor\t\n \u00a0\ndeformation\t\n \u00a0 model\t\n \u00a0 of\t\n \u00a0 TCR\t\n \u00a0 triggering.\t\n \u00a0 Faseb\t\n \u00a0 J\t\n \u00a0\n2008,\t\n \u00a022(4):1002-\u00ad\u20101008.\t\n \u00a0\n\t\n \u00a0\n\n28.", "start_char_idx": 26317, "end_char_idx": 28242, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "965f6c80-3ccf-4f17-b871-dbb7ae16f7f9": {"__data__": {"id_": "965f6c80-3ccf-4f17-b871-dbb7ae16f7f9", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "19af00ea-5f4d-4dc4-b3f6-007947e14ea9", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "6681de6f7b6479885ca545a15c6f6acfd114a87ed3a604bf489f4eb1fe95284f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2b57e889-9385-46e0-971d-d0e1cbf69fe9", "node_type": "1", "metadata": {}, "hash": "afefeec05485214af5bdb6498248da0a353e9393b932d93c4b059ed5ed4ed5b5", "class_name": "RelatedNodeInfo"}}, "text": "28. Stone\t\n \u00a0JD,\t\n \u00a0Stern\t\n \u00a0LJ:\t\n \u00a0CD8\t\n \u00a0T\t\n \u00a0Cells,\t\n \u00a0Like\t\n \u00a0CD4\t\n \u00a0T\t\n \u00a0Cells,\t\n \u00a0\nAre\t\n \u00a0 Triggered\t\n \u00a0 by\t\n \u00a0 Multivalent\t\n \u00a0 Engagement\t\n \u00a0 of\t\n \u00a0\nTCRs\t\n \u00a0 by\t\n \u00a0 MHC-\u00ad\u2010Peptide\t\n \u00a0 Ligands\t\n \u00a0 but\t\n \u00a0 Not\t\n \u00a0 by\t\n \u00a0\nMonovalent\t\n \u00a0 Engagement.\t\n \u00a0 J\t\n \u00a0 Immunol\t\n \u00a0 2006,\t\n \u00a0\n176(3):1498-\u00ad\u20101505.\t\n \u00a0\n\t\n \u00a0\n\n29. Cochran\t\n \u00a0 JR,\t\n \u00a0 Cameron\t\n \u00a0 TO,\t\n \u00a0 Stern\t\n \u00a0 LJ:\t\n \u00a0 The\t\n \u00a0\nrelationship\t\n \u00a0of\t\n \u00a0MHC-\u00ad\u2010peptide\t\n \u00a0binding\t\n \u00a0and\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0\nactivation\t\n \u00a0 probed\t\n \u00a0 using\t\n \u00a0 chemically\t\n \u00a0 defined\t\n \u00a0\nMHC\t\n \u00a0 class\t\n \u00a0 II\t\n \u00a0 oligomers.\t\n \u00a0 Immunity\t\n \u00a0 2000,\t\n \u00a0\n12(3):241-\u00ad\u2010250.\t\n \u00a0\n\t\n \u00a0\n\n30. Minguet\t\n \u00a0 S,\t\n \u00a0 Schamel\t\n \u00a0 WWA:\t\n \u00a0 A\t\n \u00a0 permissive\t\n \u00a0\ngeometry\t\n \u00a0model\t\n \u00a0for\t\n \u00a0TCR-\u00ad\u2010CD3\t\n \u00a0activation.\t\n \u00a0Trends\t\n \u00a0\nBiochem\t\n \u00a0Sci\t\n \u00a02008,\t\n \u00a033(2):51-\u00ad\u201057.\t\n \u00a0\n\t\n \u00a0\n\n31. Secrist\t\n \u00a0 JP,\t\n \u00a0 Burns\t\n \u00a0 LA,\t\n \u00a0 Karnitz\t\n \u00a0 L,\t\n \u00a0 Koretzky\t\n \u00a0 GA,\t\n \u00a0\nAbraham\t\n \u00a0RT:\t\n \u00a0Stimulatory\t\n \u00a0effects\t\n \u00a0of\t\n \u00a0the\t\n \u00a0protein\t\n \u00a0\ntyrosine\t\n \u00a0 phosphatase\t\n \u00a0 inhibitor,\t\n \u00a0 pervanadate,\t\n \u00a0\non\t\n \u00a0 T-\u00ad\u2010cell\t\n \u00a0 activation\t\n \u00a0 events.\t\n \u00a0 J\t\n \u00a0 Biol\t\n \u00a0 Chem\t\n \u00a0 1993,\t\n \u00a0\n268(8):5886-\u00ad\u20105893.\t\n \u00a0\n\t\n \u00a0\n\n32. Choudhuri\t\n \u00a0K,\t\n \u00a0Wiseman\t\n \u00a0D,\t\n \u00a0Brown\t\n \u00a0MH,\t\n \u00a0Gould\t\n \u00a0K,\t\n \u00a0\nvan\t\n \u00a0der\t\n \u00a0Merwe\t\n \u00a0PA:\t\n \u00a0T-\u00ad\u2010cell\t\n \u00a0receptor\t\n \u00a0triggering\t\n \u00a0is\t\n \u00a0\ncritically\t\n \u00a0 dependent\t\n \u00a0 on\t\n \u00a0 the\t\n \u00a0 dimensions\t\n \u00a0 of\t\n \u00a0 its\t\n \u00a0\npeptide-\u00ad\u2010MHC\t\n \u00a0 ligand.\t\n \u00a0 Nature\t\n \u00a0 2005,\t\n \u00a0\n436(7050):578-\u00ad\u2010582.\t\n \u00a0\n\t\n \u00a0\n\n33. Davis\t\n \u00a0 SJ,\t\n \u00a0 van\t\n \u00a0 der\t\n \u00a0 Merwe\t\n \u00a0 PA:\t\n \u00a0 The\t\n \u00a0 kinetic-\u00ad\u2010\nsegregation\t\n \u00a0model:\t\n \u00a0TCR\t\n \u00a0triggering\t\n \u00a0and\t\n \u00a0beyond.\t\n \u00a0\nNat\t\n \u00a0Immunol\t\n \u00a02006,\t\n \u00a07(8):803-\u00ad\u2010809.\t\n \u00a0\n\t\n \u00a0\n\n34. Boniface\t\n \u00a0JJ,\t\n \u00a0Rabinowitz\t\n \u00a0JD,\t\n \u00a0Wulfing\t\n \u00a0C,\t\n \u00a0Hampl\t\n \u00a0J,\t\n \u00a0\nReich\t\n \u00a0 Z,\t\n \u00a0 Altman\t\n \u00a0 JD,\t\n \u00a0 Kantor\t\n \u00a0 RM,\t\n \u00a0 Beeson\t\n \u00a0 C,\t\n \u00a0\nMcConnell\t\n \u00a0 HM,\t\n \u00a0 Davis\t\n \u00a0 MM:\t\n \u00a0 Initiation\t\n \u00a0 of\t\n \u00a0 signal\t\n \u00a0\n\ntransduction\t\n \u00a0 through\t\n \u00a0 the\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 receptor\t\n \u00a0\nrequires\t\n \u00a0 the\t\n \u00a0 multivalent\t\n \u00a0 engagement\t\n \u00a0 of\t\n \u00a0\npeptide/MHC\t\n \u00a0 ligands\t\n \u00a0 [corrected].\t\n \u00a0 Immunity\t\n \u00a0\n1998,\t\n \u00a09(4):459-\u00ad\u2010466.\t\n \u00a0\n\t\n \u00a0\n\n35. Horejsi\t\n \u00a0 V:\t\n \u00a0 Lipid\t\n \u00a0 rafts\t\n \u00a0 and\t\n \u00a0 their\t\n \u00a0 roles\t\n \u00a0 in\t\n \u00a0 T-\u00ad\u2010cell\t\n \u00a0\nactivation.", "start_char_idx": 28239, "end_char_idx": 30393, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2b57e889-9385-46e0-971d-d0e1cbf69fe9": {"__data__": {"id_": "2b57e889-9385-46e0-971d-d0e1cbf69fe9", "embedding": null, "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-44", "node_type": "4", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "a175e1f11b4211b8115d128d767f9384054e62ffbd040f1dd74584d1d78b84e0", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "965f6c80-3ccf-4f17-b871-dbb7ae16f7f9", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "02f6b2f081b97dafb40a09aa106c0c7a9159dd1433cce7d21e61b0de42510317", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2fe32167-c5bb-4597-9272-b4135bda7a4e", "node_type": "1", "metadata": {}, "hash": "2ce9b58b290f89a669efedaddb930b855fe7d9acb0e5ba5b48795969007c64a2", "class_name": "RelatedNodeInfo"}}, "text": "Microbes\t\n \u00a0Infect\t\n \u00a02005,\t\n \u00a07(2):310-\u00ad\u2010316.\t\n \u00a0\n\t\n \u00a0\n\n36. Horejsi\t\n \u00a0 V:\t\n \u00a0 The\t\n \u00a0 roles\t\n \u00a0 of\t\n \u00a0 membrane\t\n \u00a0\nmicrodomains\t\n \u00a0 (rafts)\t\n \u00a0 in\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 activation.\t\n \u00a0\nImmunol\t\n \u00a0Rev\t\n \u00a02003,\t\n \u00a0191:148-\u00ad\u2010164.\t\n \u00a0\n\t\n \u00a0\n\n37. Su\t\n \u00a0MW,\t\n \u00a0Yu\t\n \u00a0CL,\t\n \u00a0Burakoff\t\n \u00a0SJ,\t\n \u00a0Jin\t\n \u00a0YJ:\t\n \u00a0Targeting\t\n \u00a0Src\t\n \u00a0\nhomology\t\n \u00a0 2\t\n \u00a0 domain-\u00ad\u2010containing\t\n \u00a0 tyrosine\t\n \u00a0\nphosphatase\t\n \u00a0 (SHP-\u00ad\u20101)\t\n \u00a0 into\t\n \u00a0 lipid\t\n \u00a0 rafts\t\n \u00a0 inhibits\t\n \u00a0\nCD3-\u00ad\u2010induced\t\n \u00a0 T\t\n \u00a0 cell\t\n \u00a0 activation.\t\n \u00a0 Journal\t\n \u00a0 of\t\n \u00a0\nimmunology\t\n \u00a02001,\t\n \u00a0166(6):3975-\u00ad\u20103982.\t\n \u00a0\n\t\n \u00a0\n\n38. He\t\n \u00a0X,\t\n \u00a0Woodford-\u00ad\u2010Thomas\t\n \u00a0TA,\t\n \u00a0Johnson\t\n \u00a0KG,\t\n \u00a0Shah\t\n \u00a0\nDD,\t\n \u00a0 Thomas\t\n \u00a0 ML:\t\n \u00a0 Targeting\t\n \u00a0 of\t\n \u00a0 CD45\t\n \u00a0 protein\t\n \u00a0\ntyrosine\t\n \u00a0 phosphatase\t\n \u00a0 activity\t\n \u00a0 to\t\n \u00a0 lipid\t\n \u00a0\nmicrodomains\t\n \u00a0on\t\n \u00a0the\t\n \u00a0T\t\n \u00a0cell\t\n \u00a0surface\t\n \u00a0inhibits\t\n \u00a0TCR\t\n \u00a0\nsignaling.\t\n \u00a0 European\t\n \u00a0 journal\t\n \u00a0 of\t\n \u00a0 immunology\t\n \u00a0\n2002,\t\n \u00a032(9):2578-\u00ad\u20102587.\t\n \u00a0\n\t\n \u00a0\n\n39. Veri\t\n \u00a0 MC,\t\n \u00a0 DeBell\t\n \u00a0 KE,\t\n \u00a0 Seminario\t\n \u00a0 MC,\t\n \u00a0\nDiBaldassarre\t\n \u00a0 A,\t\n \u00a0 Reischl\t\n \u00a0 I,\t\n \u00a0 Rawat\t\n \u00a0 R,\t\n \u00a0 Graham\t\n \u00a0 L,\t\n \u00a0\nNoviello\t\n \u00a0 C,\t\n \u00a0 Rellahan\t\n \u00a0 BL,\t\n \u00a0 Miscia\t\n \u00a0 S\t\n \u00a0 et\t\n \u00a0 al:\t\n \u00a0\nMembrane\t\n \u00a0 raft-\u00ad\u2010dependent\t\n \u00a0 regulation\t\n \u00a0 of\t\n \u00a0\nphospholipase\t\n \u00a0 Cgamma-\u00ad\u20101\t\n \u00a0 activation\t\n \u00a0 in\t\n \u00a0 T\t\n \u00a0\nlymphocytes.\t\n \u00a0 Molecular\t\n \u00a0 and\t\n \u00a0 cellular\t\n \u00a0 biology\t\n \u00a0\n2001,\t\n \u00a021(20):6939-\u00ad\u20106950.\t\n \u00a0\n\t\n \u00a0\n\n40. Werlen\t\n \u00a0 G,\t\n \u00a0 Hausmann\t\n \u00a0 B,\t\n \u00a0 Palmer\t\n \u00a0 E:\t\n \u00a0 A\t\n \u00a0 motif\t\n \u00a0 in\t\n \u00a0\nthe\t\n \u00a0alphabeta\t\n \u00a0T-\u00ad\u2010cell\t\n \u00a0receptor\t\n \u00a0controls\t\n \u00a0positive\t\n \u00a0\nselection\t\n \u00a0 by\t\n \u00a0 modulating\t\n \u00a0 ERK\t\n \u00a0 activity.\t\n \u00a0 Nature\t\n \u00a0\n2000,\t\n \u00a0406:422-\u00ad\u2010426.", "start_char_idx": 30397, "end_char_idx": 32017, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2fe32167-c5bb-4597-9272-b4135bda7a4e": {"__data__": {"id_": "2fe32167-c5bb-4597-9272-b4135bda7a4e", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2b57e889-9385-46e0-971d-d0e1cbf69fe9", "node_type": "1", "metadata": {"identifier": "njb-44", "author": "Deswal, Sumit", "title": "Models of T cell antigen receptor activation: the puzzle still remained", "date": "2022-07-11", "file": "njb-44.pdf"}, "hash": "e58219f2cdb36ef6452b016111202d60193dd7ae2ca1e4a24f82f37aaf97d197", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b780d769-eb85-47c0-b67e-ee9d3d7a16d7", "node_type": "1", "metadata": {}, "hash": "f94744ed8fefa19542b98e7e3a98a1cf2fc32e2e8f37ccf657ee93b04dbf8f76", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Review article 1_Depali.doc\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 53\t\n \u00a0\n\n\t\n \u00a0 REVIEW\t\n \u00a0ARTICLE\t\n \u00a0\n\n \nAn\t\n \u00a0Overview\t\n \u00a0of\t\n \u00a0Computational\t\n \u00a0Approaches\t\n \u00a0in\t\n \u00a0Structure\t\n \u00a0Based\t\n \u00a0Drug\t\n \u00a0Design\t\n \u00a0\n\nDipali Singh*, Anushree Tripathi, Gautam Kumar \n \n1Department of Bioinformatics, Indian Institute of Information Technology-Allahabad, India. \n*Corresponding Address: Indian Institute of Information Technology-Allahabad, 211012, India.  \nEmail: ibi2010002@iiita.ac.in, dipali.d4@gmail.com \n \nShort title: Computational Approaches in SBDD \n\n \nAbstract \n\nDrug design is a costly and difficult process. Drug must fulfill several criteria of being active, non-\ntoxic and bioavailable. The conventional way of synthesizing drugs is a monotonous process. But \ncomputer aided drug design is a proficient way to overcome the tedious process of conventional \nmethod. Drugs can be designed computationally by structure or target based drug designing (SBDD). \nThis review summarizes the methods of structure based drug design, usage of related softwares and a \ncase study that explores to find a suitable drug (lead) molecule for the mutated state of H-Ras protein \nin order to prevent complex formation with Raf protein. \n\n \nKeywords: computer aided drug design, structure based drug design, Ras-protein \n\n \n\nIntroduction \n\nTraditionally, new drugs were generated from \n\nplants and other natural products through \n\naccidental observations and discoveries. Leads \n\nfor new drug were generated from screening of \n\norganic compounds. Increasing information on \n\nthe three dimensional structure of the biological \n\ntarget has paved path for structure based drug \n\ndesign. The rapid progress in the field of \n\ngenomic, proteomic, and structural biology has  \n\nincreased the opportunities for future drug lead \n\ndiscovery. The antihypertensive drug, captopril, \n\nan angiotensin-converting enzyme (ACE) \n\ninhibitor was the first success story in structure- \n\nbased drug design [1]. \n\n \nKubinyi  has reviewed success stories of \n\nstructure based drug design in the search for \n\nnew, potent and selective HIV protease \n\ninhibitors, thrombin inhibitors, neuraminidase \n\ninhibitors and integrin receptor antagonists [1]. \n\nAnderson in his review paper mentioned that \n\ntwo of the first drugs to reach the market using \n\nSBDD were Amprenavir and Nelfinavir \n\ndeveloped against HIV protease [2]. Structure-", "start_char_idx": 50, "end_char_idx": 2847, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b780d769-eb85-47c0-b67e-ee9d3d7a16d7": {"__data__": {"id_": "b780d769-eb85-47c0-b67e-ee9d3d7a16d7", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2fe32167-c5bb-4597-9272-b4135bda7a4e", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "2a186505f3078ece925848192750ab2a302b380f3d5dc7e416650444f49db5ec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "15c91ea6-42bd-4fa6-b203-6d1014072704", "node_type": "1", "metadata": {}, "hash": "34113fb081fb9c49f01c02dd25efc614db937b9491ee752b33a0a383c0e8de39", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 54\t\n \u00a0\n\nbased drug design can successfully contribute \n\nto the discovery process at different stages. It \n\ncan be used at a very early stage at which no \n\nleads are available [3]. \n\n \n1. Overview of the Process \n\n \nProteins 3D structures are generally used by \n\nSBDD to assist for the development and design \n\nof new lead (drug compounds). The overall \n\nprocess of SBDD (figure 1) would be divided \n\nmainly into two parts: \n\n \na. Docking Ligands \n\n \nProteins are flexible molecules and they adjust \n\ntheir shape to place bound ligands through \n\nrotation of bonds. SBDD allows to dock \n\nligand/drug molecules into protein active sites \n\nand to visualize the movement that occurs in \n\namino acid side chains. \n \n\nb. Lead  Optimization \n \n\nLead optimization is a technique of refining 3D \n\nstructures of drug molecules and it promotes \n\nthe binding of drug to protein active sites. In \n\nthis technique, researches gradually modify the \n\nstructure of the drug compound by docking \n\nevery specific structure of a drug compound in \n\nactive site of protein, and calculating their \n\nextent of interactions. \n\n!\n\n!\"#$%&'()*#$+,$-'.&/'&.#0123#4$5.&6$5#3)6*!\n\n!\n\n\t\n \u00a0\nFig.1: The Outline of Structure-Based Drug Design \n\n \n\n2. Design Process \n\na. Choice of drug target \n\nThe target should be closely linked to cause of \n\nhuman disease and binds to a small molecule, \n\ngenerally a protein, in order to carry out a \n\nfunction. Drug target are usually protein having \n\na well-defined binding pocket. SBDD against \n\nRNA targets with well-defined secondary \n\nstructure has also been effective [2]. \n\nAfter the identification of target, structure can \n\nbe determined following any of the methods: \n\n1.   X-ray crystallography \n\n2.  Nuclear magnetic resonance Spectroscopy \n\n(NMR) \n\n3.   Computational methods (Modelling) \n\n4.   Atomic Force Field Microscopy (AFM) \n \n\n3. Drug Design Methods \n\nOnce identification of structure and target site \n\nis completed, there are number of ways to \n\ndevelop lead based on the structure of the target", "start_char_idx": 2852, "end_char_idx": 5297, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "15c91ea6-42bd-4fa6-b203-6d1014072704": {"__data__": {"id_": "15c91ea6-42bd-4fa6-b203-6d1014072704", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b780d769-eb85-47c0-b67e-ee9d3d7a16d7", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "8f4ee49042de1183d9d410fa984759c65cbd2080ee66699e556a27a376cb2bf7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "50c9dab9-be1d-4f77-bf8c-26aaa1c89c49", "node_type": "1", "metadata": {}, "hash": "23cb5bd2d940b95955b3544ca28cc58b26279d7f52c2a6b8715087018a26947f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 55\t\n \u00a0\n\nwhich can be categorized as computer aided \n\nversus experimental. In experimental method, \n\nhigh-throughput screening is performed with \n\ncombinatorial chemistry and thousands of \n\nmolecules are tested for biochemical effects. \n\nComputer-aided methods can be classified into \n\n3 categories. \n\na.   Database searching and docking methods \n\nb.   De novo drug design methods \n\nc.   Ligand binding scoring functions \n\n \na. Database searching and docking methods \n\n \nWidely  used computational docking methods \n\nare DOCK, CONCORD, AUTODOCK, FLO98 \n\nand FLEXX. DOCK systematically attempts to \n\nfit each compound from a database to the target \n\nstructure\u2019s binding site in such a way that in the \n\ndatabase, three or more atoms of the molecule \n\noverlap with a set of predefined site points in \n\nthe target binding site [7]. The default method \n\nfor site point generation involves creating an \n\ninverse surface of the binding site. This is \n\nspecified by the set of overlapping spheres that \n\nfill the binding site and touch the molecular \n\nsurface at two points. The sphere centers (for \n\nall spheres with radii within a specified range) \n\nare used as site points. CONCORD is based on \n\nthe combination of geometry rules and \n\noptimization methods. It selects lowest energy \n\nconformer of the molecule then scores on grid \n\nusing different energy functions. On the basis \n\nof precalculated values for protein, each match \n\nis scored on a grid throughout the binding site \n\nof target molecule [7]. \n\n \nb. De novo drug design methods \n\n \nStructure based drug designing methods rely \n\nexclusively on ligand optimization approach \n\nbased on the study of protein active site \n\nproperties. There are three important categories \n\nof computational methods for the de novo \n\ndesign of structure based ligands: fragment \n\npositioning methods, molecule growth \n\nmethods, and fragment methods coupled to \n\ndatabase searches [6]. \n\n \nFragment positioning methods \n\n \nBasically these methods are based on the \n\nselection of structures of individual functional \n\ngroups or fragments from predefined library \n\nwhich fill the active site of enzyme [5]. Two \n\nwell-known programs which predict \n\nenergetically favorable binding site positions \n\nfor chemical fragments are GRID and MCSS   \n\n(Multiple Copy Simultaneous Search). GRID \n\ncalculates protein interaction energies for \n\nfunctional groups on a grid surrounding the \n\ntarget structure. It includes non-bonded \n\ninteraction like hydrogen bonding, electrostatic \n\nand Van der Waals. It is mainly useful for \n\nmodifying existing lead compounds. Limitation \n\nof GRID is that the sphere probe must be", "start_char_idx": 5302, "end_char_idx": 8366, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "50c9dab9-be1d-4f77-bf8c-26aaa1c89c49": {"__data__": {"id_": "50c9dab9-be1d-4f77-bf8c-26aaa1c89c49", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "15c91ea6-42bd-4fa6-b203-6d1014072704", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "39c94d964fe11350ecc325234dbcda2d461cf37dd33a21f80555d6f29a2baa75", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0d94234b-de9d-45a8-a07a-b2d3aef3a097", "node_type": "1", "metadata": {}, "hash": "f0002f5c44e2bbbc339f2d98e72d1016a62cda04e1ecc6a346b7268a411a3a2a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 56\t\n \u00a0\n\ncapable of making hydrogen bonds and must \n\nnot be in a linear arrangement [6]. In MCSS \n\nmethod, the probes are fully flexible and \n\nindividual atoms are represented by CHARMM \n\npotential energy function. The de novo drug \n\ndesigning approach involves three steps [6]. \n\nInitially it uses fragment positioning method. \n\nSecondly, clustering and connecting the \n\noptimally placed molecular fragments to form \n\nchemically sensible candidate ligands. Finally, \n\ndepicts the binding of proposed compounds \n\nwith another and to existing drugs. \n\n \nMolecule growth methods \n\n \nA fragment is fitted in the binding site of the \n\ntarget structure while ligand molecule is \n\nsuccessively built by bonding a further \n\nfragment to it. There are various molecule \n\ngrowth methods are available, including SMoG \n\n(Small Molecule Growth), GrowMol, \n\nGroupBuild and GenStar. \n\nSMoG uses simple model for ligand-protein \n\ninteractions as well as a knowledge-based \n\npotential. A large number of structures are \n\nstatistically analyzed by an efficient Monte \n\nCarlo molecular growth algorithm that \n\ngenerates molecules through the adjoining of \n\nfunctional groups directly in the binding region \n\n[7]. \n\nGrowMol generates ligand structures from a \n\nlibrary of atom as well as small functional \n\ngroup types and is scored based on its chemical \n\ncomplementarities with nearby atoms to the \n\nbinding site of the target. GroupBuild is similar \n\nto GrowMol, it uses a predefined library of \n\nchemical fragments and scores candidate \n\nfragment positions depending on force field to \n\nget candidate small molecule ligands fragment \n\nby fragement. GenStar generates chemically \n\nreasonable structure which fills active site of \n\nenzyme. The proposed molecules provide good \n\nsteric contact with the enzyme and also exist in \n\nlow energy conformation. These structures \n\nconsist of sp3 hybridized carbons which are \n\ngrown sequentially, but which can also branch \n\nor form rings. Atoms  are grown from \n\npredocked inhibitor core. For each new atom \n\ngenerated by the program, several hundred \n\ncandidate positions representing a range of \n\nreasonable bond lengths, bond angles, and \n\ntorsion angles are considered. Then, each \n\ncandidate is scored, with a simple enzyme \n\ncontact model. From the highest scoring cases, \n\npositions are chosen at random.  Duplicate \n\nstructures may be removed applying variety of \n\ncriteria. Energy of compounds may be \n\nminimized and displayed using standard \n\nmodeling programs. \n\n \nFragment methods coupled to database \nsearches \n \nIt is an integrated approach for fragment", "start_char_idx": 8371, "end_char_idx": 11389, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0d94234b-de9d-45a8-a07a-b2d3aef3a097": {"__data__": {"id_": "0d94234b-de9d-45a8-a07a-b2d3aef3a097", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "50c9dab9-be1d-4f77-bf8c-26aaa1c89c49", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "53dfac9a80243bb1e4c564f74cbff5a25a147f6d52ecd68bb9b871a2a36b8b9d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ff236d7f-8b3a-4435-b7f0-01eceb627d9f", "node_type": "1", "metadata": {}, "hash": "4a7c441199e7abd553cc0e0249d97a8418c2fea17693c38f7929fdd8b33d0e3a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 57\t\n \u00a0\n\npositioning methods and database searching \n\ntechniques   either to extract those existing \n\nmolecules from a database that can be docked \n\nwith the preferred fragments in their most \n\nfavorable positions into the binding site or for \n\nde novo design. HOOK generates a database of \n\nmolecular skeletons without involving \n\nfunctional groups on the database molecules \n\nand then fit molecular skeletons into the target \n\nbinding site such that two MCSS functional \n\ngroup minima can be hooked by using docking. \n\nThen, it undergoes geometrical   superposition \n\nof two designated hooks in the skeletal \n\nmolecules and finally using inverted Lennard-\n\nJones type contact potential, the fit of the \n\nskeleton in the binding site in two functional   \n\ngroup minima, is scored. After validating \n\nscores, secondary searches are carried out to \n\nattach additional MCSS minima to the skeleton, \n\nif fit is acceptable [6]. CAVEAT is \n\ncomparatively faster method due to \n\nconsideration of interaction between the \n\nskeletal molecule and the binding site in post \n\nprocessing step. It is similar to HOOK in that it \n\ninvolves searching of a database of three-\n\ndimensional structures of small cyclic \n\nmolecules to connect optimally placed \n\nfragments in the binding site. In the database, \n\nspecific bonds of each molecule are represented \n\nas vectors, and the molecule is specified as a \n\nset of pairwise combinations of bond vectors. It \n\nfinds matches between pairs of bond vectors \n\nfrom the fragments of the query molecules and \n\nthe database molecules [6]. \n\n \nc. Ligand binding scoring functions \n\n \nThe ligands binding scoring functions are \n\nmajor determinant of the accuracy of scoring \n\nfunctions that ranks the lead compounds. \n\nFactors which contribute to ligand binding \n\ninclude hydrophobic effect, dispersion \n\ninteractions, hydrogen bonding, other \n\nelectrostatic interactions and solvation effects. \n\nWith increasing complexity, the various \n\napproaches for estimating binding affinities \n\ninclude scoring functions based on statistical \n\nanalysis of known structure of protein ligand \n\ncomplexes, physicochemical properties, force \n\nfield calculations, force field calculation with \n\nadded solvation corrections and free energy \n\nperturbation (FEP) calculations. SMoG pseudo \n\nenergy function is a scoring function based on \n\nstatistical analysis of high resolution X ray \n\nstructure. Currently knowledge based, \n\nregression based and first principle based \n\nmethods have been developed to rank lead \n\ncompounds [8]. \n\n \n \n\n4. Case Study \n\n \nIn the Ras subfamily, mainly K-RAS, H-RAS \n\nand N-RAS codes for those proteins which are", "start_char_idx": 11394, "end_char_idx": 14469, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ff236d7f-8b3a-4435-b7f0-01eceb627d9f": {"__data__": {"id_": "ff236d7f-8b3a-4435-b7f0-01eceb627d9f", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0d94234b-de9d-45a8-a07a-b2d3aef3a097", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e6c0d799d49e8a7329ff9258a23e9a918a782181387787faee4c93f35091249f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "50ede648-f0ac-4a59-b1b4-f9f7f3d3deb4", "node_type": "1", "metadata": {}, "hash": "e288fe1adb9732e658bdb580d2c39f8ab4f9b6fa24a0456ea5bbfc0f31b23d63", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 58\t\n \u00a0\n\nmade up of 189 amino acids with molecular \n\nweight 21kDA protein [9]. Guanosine \n\nnucleotide binding protein or G-proteins works \n\nin form of signaling switches with two states \n\nthat are active and inactive. Usually it is bound \n\nto the nucleotide GDP in the inactive state. On \n\nthe other hand, it is bound to GTP in the active \n\nstate. Guanine nucleotide exchange factors \n\n(GEFs) and GTPase activating proteins (GAPs) \n\nare mainly used in exchanging the bound \n\nnucleotide. Ras is associated with an intrinsic \n\nGTPase activity in which it can hydrolyze \n\nbound GTP into GDP. But, due to its less  \n\nefficiency, RasGAP is needed which is formed \n\nby binding of Ras and GAP and stabilizes the \n\nRas catalytic residues by releasing inorganic \n\nphosphate and ultimately leads to Ras molecule \n\nin GDP bound state for Ras inactivation. It has \n\nbeen found that mutations in the Ras family of \n\nproto-oncogenes are very commonly observed \n\nin 20% to 30% of all human tumors [10].The   \n\ninappropriate activation of the gene affects \n\nmalignant transformation, proliferation and \n\nsignal transduction [11], due to which, the \n\nmutated Ras P21  has a structure that disables \n\nits ability to bind with GTPase activating \n\nprotein (GAP) and creates an \n\nautophosphorylation site, keeping the Ras P21 \n\nin the GTP-bound activate state and \n\ncontributing to a malignant cell phenotype [12, \n\n13]. \n\nIn this context, target-based drug discovery is \n\nconsidered to be highly potential. The mutated \n\nH-Ras is perceived to be an important target to \n\ntreat colorectal and pancreatic cancer. A \n\nsuitable drug (lead) molecule can be searched \n\nfor the mutated state of H-Ras protein in order \n\nto prevent complex formation with Raf protein.  \n \n\na. Materials and Methods \n \n\nThe protein structures of H-RAS P21 mutant \n\n(PDB ID - 521P) and of Ras-binding domain \n\n(PDB ID-1WXM) were taken from Protein \n\nData Bank \n\nhttp://www.rcsb.org/pdb/home/home.do. \n\nThere were two methods used to predict \n\npotential binding site. \n\nIn the first approach, screening of ligand \n\nmolecules was carried out through BLAST \n\nsearch engine by submitting the mutated HRas \n\n(PDB ID: 521P) protein sequence to DrugBank \n\ndatabase: \n\nhttp://redpoll.pharmacy.ualberta.ca/drugbank/dr\n\nugBlast.htm. The DrugBank search showed \n\ntrifluoroethanol, S-oxymathionine and \n\nisopropanol as active ligands. \n\nIn a second approach, ChemBank ligand entries \n\nwere downloaded  from Ligand in \n\nSDF format and entries of ligand was used for \n\nvirtual screening and docking into effectors \n\nregion of mutated H-Ras by using Discovery \n\nStudio/LigandFit program to identify active", "start_char_idx": 14474, "end_char_idx": 17542, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "50ede648-f0ac-4a59-b1b4-f9f7f3d3deb4": {"__data__": {"id_": "50ede648-f0ac-4a59-b1b4-f9f7f3d3deb4", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ff236d7f-8b3a-4435-b7f0-01eceb627d9f", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "38aa9ae828da3885466cd884910685b856c76442f943f73f28c1b37a591c06a7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0394053c-88a6-4f03-b12b-2284bee6f0d2", "node_type": "1", "metadata": {}, "hash": "78b18655febaf03b247d2630a36dfa54aa68beef050ca2376e9290e8e03a7c06", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 59\t\n \u00a0\n\npotential drugs. The Ligand Fit docking \n\nalgorithm produced 10 different hits of ligand, \n\nsuch as YS035, nizatidine, leuhistin, 3-\n\naminopropanesulphonic acid, guanidine, \n\nacetamide, methoxamine, urea, aluminum \n\nfluoride and hydroxyurea from two different \n\nbinding site cavities that were encompassed in \n\neffectors region of mutated H-Ras. \n\n \n5. Results and Discussion \n\n \nLeaving all the other molecules, the 3-\n\naminopropanesulphonic acid was docked with \n\nenergy of -0.009 kcal/mol and hydroxyurea \n\nwith -3.014 kcal/mol. These two ligand \n\nmolecules were also found to obey the \n\nLipinski\u2019s rule of five. This Rule evaluates \n\ndrug ability, or finds a chemical compound \n\nwith some particular pharmacological \n\nproperties that can make it an orally active drug \n\nin humans. \n \n\nThis result correlates well with  earlier \n\nexperimental results [14, 15] and it depicts that \n\nthe identified binding conformations of these \n\ninhibitors are reliable and produce anti-tumor \n\neffects in a variety of solid tumor [16] and \n\nleukemia. 3-aminopropanesulfonic acid is a \n\nsynthetic   gammaaminobutyric acid (GABA) \n\nanalog. Hydroxyurea is an antineoplastic agent \n\nthat produces anti-tumor effects in animals and \n\nman in a various forms of solid tumor and \n\nwould be an effective drug to inhibit function \n\nof mutant H-Ras P21 protein, which will be \n\nable to arrest cell growth and cancer cell \n\nproliferation. From this study and previously \n\nreported experimental data in literature, we \n\nobserve that hydroxyurea and 3-\n\naminopropanesulphonic acid would be an \n\neffective drug to inhibit function of mutant H-\n\nRas P21 protein, which will in turn arrest the \n\nprocess of cell growth and proliferation of the \n\ncancer cell [17]. It was earlier reported that the \n\noral administration of hydroxyurea to 20 \n\npatients with chronic myelogenous leukemia, \n\nresulted in the decrease count of white blood \n\ncell [18]. \n\n \nConclusion \n\n \nThe major goal of structure-based drug design \n\nis to develop an efficient process that involves \n\na high resolution crystal structure of validated \n\nbiological target molecules and reliably \n\ngenerates an easily synthesized, high affinity \n\nsmall molecule with desirable pharmacological \n\nproperties. New advancement in the field of \n\nstructural genomics, proteomics and \n\nbioinformatics will enhance variety of \n\napproaches for structure based drug design. \n\n \nAcknowledgement \n\n \nWe are thankful to Dr. Pritish Varadwaj for the \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 17547, "end_char_idx": 20535, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0394053c-88a6-4f03-b12b-2284bee6f0d2": {"__data__": {"id_": "0394053c-88a6-4f03-b12b-2284bee6f0d2", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "50ede648-f0ac-4a59-b1b4-f9f7f3d3deb4", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "4208b8a9f602fc53ddc6c91bae2e7fe35ce254dbd526c331a9c78c2c8be2de71", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5951b466-d760-4665-bf91-3d982ef173c0", "node_type": "1", "metadata": {}, "hash": "dbb30ccb7ec07e495a0e5ca6b0f77b65866dfe680374161d8881c177a4909766", "class_name": "RelatedNodeInfo"}}, "text": "2011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 60\t\n \u00a0\n\ninformative lecture on Computer Aided Drug \n\nDesign course. We are also thankful to all the \n\nfaculties of Department of Bioinformatics, \n\nIndian   Institute of Information Technology, \n\nAllahabad, India for introducing and \n\nenlightening us to the bioinformatics field. \n \n\nReferences \n \n\n1. Kubinyi H: Structure-based design of \nenzyme inhibitors and receptor \nligands. Current Opinion in Drug \nDiscovery and Development 1998, \n1(1):4-15. \n \n\n2. Amy CA: The Process of Structure-\nBased Drug Design. Chemistry & \nBiology 2003, 10: 787-797. \n \n\n3. Shravanti K et al: A Review on \nStructure Based Drug Design of \nProtein Tyrosine Phosphatase 1B \nInhibitors for Target for obesity and \nType 2 Diabetes Mellitus. Journal of \nPharmacy Research 2010, 3(12): 2939-\n2940 \n \n\n4. Sistla R, Ghadiyaram C, Srinivasan NC \nand Subramanya HS: A Structure based \nstrategy for New Drug Discovery. \nInnovations in Pharmaceutical \ntechnology 2006,August 30, 20:18-23 \n \n\n5. Sergio HR, Mark AM. GroupBuild: A \nFragment-Based Method for De Novo \nDrug Design. J.Med. Chem 1993, 36: \n1700-1710 \n \n\n6. Robert SD, Eugene IS. SMoG: de Novo \nDesign Method Based on Simple, Fast, \nand Accurate Free Energy Estimates. \n1. Methodology and Supporting \n\nEvidence.  J. Am. Chem. Soc 1996, 118: \n11733-1174 \n \n\n7. Diane JM: Computational approaches \nto structure-based ligand design.    \nPharmacology & Therapeutics 1999, 84: \n179\u2013191. \n \n\n8. Holger G, Gerhard K: Statistical \npotentials and scoring functions \napplied to protein\u2013ligand binding, \nCurrent Opinion in Structural \nBiology. Elsevier 2001, 11(2):231-235. \n \n\n9. Valencia A, Chardin P, Wittinghofer A, \nSander C: The Ras protein family: \nEvolutionary tree and role of \nconserved amino acids. Biochemistry \n1991, 30: 4637-4648. \n \n\n10. Bos J: Ras oncogenes in human \ncancer: a review. Cancer Res 1989, 49 \n(17): 4682-4689.  \n \n\n11. Lodish H, Berk A, Zipursky SL, \nMatsudaira P, Baltimore D, Darnell J: \nChapter 25, Cancer. Molecular cell \nbiology (4th ed.). San Francisco: W.H. \nFreeman 2000. ISBN 0-7167-3706-X. \n \n\n12. Bos J: The ras gene family and human \ncarcinogenesis. Mutat Res. 1988, \n195(3): 255-71 \n\n \n13. Henson ES, Gibson SB: Surviving   cell   \n\ndeath   through   epidermal   growth   \nfactor   (EGF) signal transduction \npathways: Implications for cancer \ntherapy.", "start_char_idx": 20509, "end_char_idx": 23192, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5951b466-d760-4665-bf91-3d982ef173c0": {"__data__": {"id_": "5951b466-d760-4665-bf91-3d982ef173c0", "embedding": null, "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-45", "node_type": "4", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "e7487bf0c04fa24231557c077203723052106e8e6d1d1d78aa9025070b900b01", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0394053c-88a6-4f03-b12b-2284bee6f0d2", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "bd4cd98b934311f2a3911631f3bb8342d31be86c10d6c4554f6d9ab555470b1e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a3b60690-c41e-4656-bb3e-886b41f18e07", "node_type": "1", "metadata": {}, "hash": "8e6d46fdc0d7e29d3e31650544c6cc74f383a0ed8ddafcd07f96c56593924a26", "class_name": "RelatedNodeInfo"}}, "text": "Cell Signal 2006, 18: 2089-\n2097. \n \n\n14. Young CW, Schochetman G, Karnofsky \nDA: Hydroxyureainduced Inhibition \nof Deoxyribonucleotide Synthesis: \nStudies in Intact Cells. Cancer Res \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a053\u2013\t\n \u00a061\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0 61\t\n \u00a0\n\n1967, 27: 526-534. \n \n\n15. Akerblom L, Ehrenberg A, Graslund A, \nLankinen, Reichard P, Thelander L: \nOverproduction of the Free Radical of \nRibonucleotide Reductase in \nHydroxyurea-Resistant Mouse \nFibroblast 3T6 Cells.  Proc Natl ACAD \nSci USA 1981, 78: 2159-2163. \n \n\n16. Schwartz HS, Garofalo M, Sternberg SS, \nPhilips FS: Hydroxyurea: Inhibition of \nDeoxyribonucleic Acid Synthesis in \nRegenerating Liver of Rats. Cancer \nRes 1965, 25:1867-1870. \n \n\n17. Krackoff IH, Savel H, Murphy ML: \nPhase II studies of hydroxyurea (NSC-\n32065) in adults: clinical evaluation of \nCancer. Cancer Chemother Rep1964, \n40: 53-5. \n \n\n18. Kennedy BJ: Hydroxyurea therapy in \nchronic myelogenous leukemia. Cancer \n1972. 29: 1052-1055.", "start_char_idx": 23193, "end_char_idx": 24534, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a3b60690-c41e-4656-bb3e-886b41f18e07": {"__data__": {"id_": "a3b60690-c41e-4656-bb3e-886b41f18e07", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5951b466-d760-4665-bf91-3d982ef173c0", "node_type": "1", "metadata": {"identifier": "njb-45", "author": "Singh, Dipali; Tripathi, Anushree; Kumar, Gautam", "title": "An Overview of Computational Approaches in Structure Based Drug Design", "date": "2022-07-11", "file": "njb-45.pdf"}, "hash": "c1396cb8b2cf9dae6352f73a8f6276ffd785cb9e7c5f27c175ad21fb27d44106", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "63f69bbf-5068-4681-a553-db886841fcc2", "node_type": "1", "metadata": {}, "hash": "d2fcb49d93460cc0a4376f1faa0a63f7d79e5924206c892ac529a26e53234b44", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Paper 5 Patel.docx\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 37 \n\n ORIGINAL RESEARCH ARTICLE \n\n \n \n\nIsolation\t\n \u00a0and\t\n \u00a0Characterization\t\n \u00a0of\t\n \u00a0Bacterial\t\n \u00a0Endophytes\t\n \u00a0From\t\n \u00a0Lycopersicon\t\n \u00a0esculentum\t\n \u00a0\nPlant\t\n \u00a0and\t\n \u00a0Their\t\n \u00a0Plant\t\n \u00a0Growth\t\n \u00a0Promoting\t\n \u00a0Characteristics\t\n \u00a0\n\t\n \u00a0\n\nHardik A. Patel1, Rajesh K. Patel1*, Sunil M. Khristi1, Kruti Parikh1, Geetha Rajendran2 \n\n \n1 Ashok & Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh \n\nVidyanagar, Gujarat-388121, INDIA \n2 Sultan Qaboos University, Muscat, Department of Biology, Sultanate of Oman \n \nCorresponding author: rkpatel46@yahoo.com \n\n \nAbstract \n\n \nThe study was designed to isolate and characterize bacterial endophytes from root and stem of \nLycopersicon esculentum plant which was collected form different region of Gujarat. Total 18 isolates \nof endophytic bacteria were selected in which, all the endophytic bacteria produced one or the other \ndifferent characteristics involved in plant growth promotion. They either produced phytohormones like \nindole acetic acid, siderophore, protease, pectinase, organic acid showed antifungal activity, chromium \ntolerance and solubilized phosphate. Four of the strains among the 18 showed maximum positive \nresults of plant growth promoting regulators (PGPR) test and among them best probable isolate was \nselected and thus its 16SrDNA was amplified and sequenced. Only HR7 endophyte of tomato turned \nout to be Pseudomonas aeruginosa. It\u2019s a gram negative coccobacili, sporeforming motile bacilli and \nshow maximum PGPR activity. The results of our present studies indicated that above strains might be \nendophytic and therefore, were associated with the plant growth.   \n\n   \nKeywords: Lycopersicon esculentum, endophytic bacteria, PGPR, IAA, 16SrDNA \n\n \n\nIntroduction   \n\nThere are many endophytic and epiphytic \n\nbacteria are directly or indirectly involved in \n\nplant growth and development. Endophytic \n\nbacteria live in plant tissues without causing \n\nsubstantive harm to the host or gaining any \n\nbenefit other than a noncompetitive  \n\nenvironment inside the host. It has recently \n\nbeen demonstrated that bacterial endophytes \n\nmay also have beneficial effects on host plants, \n\nsuch as growth promotion and biological \n\ncontrol of pathogens [10, 25, 28].", "start_char_idx": 49, "end_char_idx": 2798, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "63f69bbf-5068-4681-a553-db886841fcc2": {"__data__": {"id_": "63f69bbf-5068-4681-a553-db886841fcc2", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a3b60690-c41e-4656-bb3e-886b41f18e07", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "ccba781fee39f2debb895f0e6568a64a3f03c998d97775602293d986b315ae68", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2ead232f-e83b-4ce2-8df1-e7a0b28653a4", "node_type": "1", "metadata": {}, "hash": "c0eb87d17f8a8ea84d7e1eff076d625a20eafb7b6d7f0122b64d3950ed83abe7", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 38 \n\nSome studies have indicated that the plant \n\ngrowth-promoting potential of endophytes is \n\nhigher than that of rhizosphere microbes [23,31], \n\nbut the role of bacterial endophytes in plant \n\ngrowth are not yet fully understood. Most of \n\nthese microorganisms are not pathogenic to the \n\nhost plant. Moreover, the association between \n\nthe plant and its endophytes is very often \n\nmutualistic. In 1926, endophytic growth was \n\nrecognized as a particular stage in the life of \n\nbacteria, described as an advanced stage if \n\ninfection and as having a close relationship with \n\nmutualistic symbiosis [22]. Since then, \n\nendophytes have been defined as \n\nmicroorganisms that could be isolated form \n\nsurface-sterilized plant organ [15]. Although the \n\npresence of bacterial endophytes in plants is \n\nvariable and, occasionally transient [32], they \n\nare also often capable of eliciting drastic \n\nphysiological changes that modulate the growth \n\nand development in the plant [8].  \n\n \nThe utilization of endophytic and epiphytic \n\nbacteria in agriculture production depends on \n\nour knowledge of the bacteria-plant interaction \n\nand our ability to maintain, manipulate and \n\nmodify beneficial bacteria population under field \n\ncondition [14]. Many PGPRs are known to \n\npromote plant growth by a variety of \n\nmechanisms: fixation of atmospheric nitrogen \n\nthat is transferred to the plant, production of \n\nsiderophores that chelate iron and make it \n\navailable to the plant root, solubilization of \n\nminerals such as phosphorus, and synthesis of \n\nphytohormones [12]. PGPR have been reported \n\nto directly enhance plant growth by the \n\nproduction of plant growth regulators, and \n\nimprovements in plant nutrient uptake [12,16] or \n\nindirectly by the production of metabolites like \n\nantibiotics, siderophores etc that decrease the \n\ngrowth of phytopathogens [12].  PGPRs can be \n\nof two different types when associated with host \n\ntissue that is endophytes or epiphytes; otherwise \n\nthey can be even rhizospheric bacteria that are \n\npresent in the root adhering soil. \n\n \nThe aim of the present study was to isolate and \n\ncharacterize the endophytic bacteria associated \n\nto root and stem part of Lycopersicon \n\nesculentum, to evaluate different characteristics \n\ninvolved in plant growth promotion. Result \n\nrevealed that four of the strains showed \n\nmaximum positive results of PGPR test and its \n\n16SrDNA was amplified and sequenced. \n\n \nMaterials and methods \n\n \nIsolation of endophytic bacteria form \n\nLycopersicon esculentum \n \nEndophytes strains were isolated from root and \n\nstem of Lycopersicon esculentum plant (Table \n\n1). Roots and stem part of plant were thoroughly", "start_char_idx": 2808, "end_char_idx": 5909, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2ead232f-e83b-4ce2-8df1-e7a0b28653a4": {"__data__": {"id_": "2ead232f-e83b-4ce2-8df1-e7a0b28653a4", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "63f69bbf-5068-4681-a553-db886841fcc2", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "95678c19580792600bf8d412c347d31b4d52e185d05855ca6b0c1fbdd0b14c9c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1e6cec9a-1768-4e21-be67-bfbbbd67191a", "node_type": "1", "metadata": {}, "hash": "57b82515bd1dedcf1569a3f2b1cfa4b4eeb5653ac7e6c02fc0b5ad135cf62f35", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 39 \n\nwashed with sterile n-saline (0.85%) and cut \n\ndown in 1 cm long pieces through sterile forceps \n\nwith the help of alcohol. The pieces were \n\ntransferred to sterile N-agar plate and incubated \n\nfor 24 hrs at 300C. \n \nMorphological and physiological \n\ncharacterization of endophytic isolates \n \nFor the present study, total 18 endophytic \n\nbacteria were isolated whose systemic \n\nmorphological characters done which includes: \n\nSize, Shape, Margin, Elevation, Consistency, \n\nOpacity, Pigmentation, was done by Systematic \n\nMicrobiology [3]. \n \nGram\u2019s staining bacterial suspension was \n\nprepared in sterile distilled water and from this \n\nsuspension a smear was prepared on clean & dry \n\nglass slide, air dried and then heat fixed. The \n\nsmear was treated with 1% crystal violet for 1-2 \n\nmin. Gram\u2019s iodine was applied for 30 sec. to 1 \n\nmin. Smears were then decolorized with 10% \n\nalcohol. The counter stain, saffranin was then \n\napplied for 45-60 seconds. The stained slide \n\nwere washed with tap water, air dried & \n\nobserved under oil immulsion.        \n \nFor motility test, the culture was inoculated into \n\nthe Edward\u2019s and Ewing motility agar stab \n\nmedium by stabbing the medium right into the \n\ncenter of agar. The entire depth of the medium \n\nwas punctured. The medium was incubated at 28 \n\n\u00b1 2\u00baC for 24 hrs. After incubation, it was \n\nobserved for the turbid growth across the line of \n\ninoculation, which indicates motile organisms. \n\n \nFor Antibiotic assay, top agar (1.5%) was \n\nprepared and autoclaved. It was cooled to 45\u00ba C, \n\n100\u00b5l of culture was added to this and overlaid \n\npreset N-agar plates. Using sterile forceps, disc \n\ncontaining the antibiotic of interest was placed \n\non the agar and incubated at 28\u00b12\u00baC for 48 hrs. \n\n \nEstimation of plant growth promoting \n\nproperties \n\n \n(i) Detection of siderophore production: This \n\nwas performed by a method described by \n\nSchwyn and Neilands [27], which involved the \n\nuse of chrome azurol S containing indicator \n\nplates. Siderophore detection was performed by \n\nmixing equal volumes of chrome azurol S (CAS) \n\nassay solution and the culture supernatant. \n\nColour change from blue to yellowish orange \n\nwas indicative of presence of Siderophore. Two \n\npercentage of overnight grown culture was \n\ninoculated in magnetotactic bacterium \n\nMagnetospirillum magneticum AMB-1 (AMB) \n\nand grown for 48 hrs. Then the culture was \n\ncentrifuge at 8000 rpm for 20 min and the \n\nsupernatant was examined for the presence of \n\nsiderophore by CAS solution. \n\n \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 5914, "end_char_idx": 8948, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1e6cec9a-1768-4e21-be67-bfbbbd67191a": {"__data__": {"id_": "1e6cec9a-1768-4e21-be67-bfbbbd67191a", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2ead232f-e83b-4ce2-8df1-e7a0b28653a4", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "1e423e74e4bfbafa729d7e6f8c588e68661cfe67530a8c6ff9ff79c445df5f18", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1b678420-2221-47e1-876a-155f97e2bcf4", "node_type": "1", "metadata": {}, "hash": "139f69763a0201a295eadb0873a80b1abd426caa8f532a0f9be0ed8cf45336fc", "class_name": "RelatedNodeInfo"}}, "text": "2012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 40 \n\n(ii) Evaluation of endophytes for chromium \n\ntolerance: Isolated strains were tested for \n\nresistance to Cr (VI) by plate dilution method \n\nusing yeast extract mannitol agar (YEMA) \n\nmedium. In a plate dilution method, agar plates \n\namended with K2Cr2O7 at 50-500 \u00b5g/ml were \n\ninoculated with 48 hrs grown cultures and \n\nincubated at 28 \u00b1 2\u00baC for 72hrs. The lowest \n\nconcentration of Cr (VI) inhibiting on YEMA \n\nplates was defined as minimum inhibitory \n\nconcentration [35]. \n\n \n(iii) Phosphate solubilization ability: The \n\nphosphate solubilizing ability of the cultures \n\nwere examined by growing the cultures on \n\nPikovskaya\u2019s agar plate and looking for the zone \n\nof clearance after incubating at 28 \u00b1 2\u00b0C for 48-\n\n72h. \n\n \n(iv) Antifungal activity: The spores of fungal \n\ncultures (Fusarium oxysporium, Alternaria,, \n\nTrichoderma and Rhizoctonia solani) grown on \n\nPotato dextrose agar (PDA) blocks were placed \n\nin the centre of PDA plates and the bacterial \n\ncultures were streaked at four ends of the plate. \n\nThis was incubated at 28 \u00b1 2\u00b0C for 48-96 hrs \n\nand examined for zone of growth inhibition. \n\n \n(v) Protease production: It is indicated by \n\ncasein degradation, which was determined by \n\nobserving clearing zones in Nutrient casein agar \n\nplate. All isolated culture was streak on Nutrient \n\ncasein agar plate and incubated at 28 \u00b1 2\u00b0C for \n\n24-48 hrs. Next day flood the plate with \n\nFrazier\u2019s reagent to detect clear zone around the \n\ncolony. \n\n \n(vi) Indole 3-acetic acid (IAA)  production \n\ntest: IAA in presence of FeCl3 develops pink \n\ncolor. This fact is utilized in determination of \n\nIAA. Different mineral acids like hydrochloric \n\nacid, perchloric acid, phosphoric acid, nitric acid \n\nand sulphuric acid can be used to develop the \n\ncolor. FeCl3 \u2013HClO4 reagent is the most \n\nsensitive and shows least interference by other \n\niodole compounds like, tryptophan, skatole, \n\nacetyletryptamine etc.  \n\n \nLoopful of each culture was inoculated in luria \n\nbroth (LB) 2ml containing 50\u00b5g/ml tryptophan \n\nand incubated at 28oC for 24 hrs on shaking \n\ncondition, centrifuged at 9000 rpm for 15min, \n\n2ml of supernatant was taken in fresh tube and \n\n2-3 drops of orthophosphoric acid was added. A \n\nquantity of 4ml of reagent (1ml of 0.5 M FeCl3 \n\nin 50 ml of 35% HClO4) was added to this \n\naliquot and incubated for 25 min at RT. \n\nAbsorbance was measured at 530 nm. Auxin \n\nquantification values were recorded by preparing \n\nstandard calibration curve made by using IAA \n\nstandard in the range of 10-100 \u00b5g/ml.", "start_char_idx": 8922, "end_char_idx": 11826, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1b678420-2221-47e1-876a-155f97e2bcf4": {"__data__": {"id_": "1b678420-2221-47e1-876a-155f97e2bcf4", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1e6cec9a-1768-4e21-be67-bfbbbd67191a", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "7fe24037268176dd177e23bf5c379ef3cd0ac4d470eeaa4f8b63ce46f406ef07", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c88aa95a-7957-4a57-96dd-471c442e567d", "node_type": "1", "metadata": {}, "hash": "e68b99204b2443ef6317048aa0d0ebeb883cdb0e9ff4fe95193d1b31821235dd", "class_name": "RelatedNodeInfo"}}, "text": "IAA \n\nstock solution was prepared as 100 \u00b5g/ml in \n\n50% ethanol. Standard graph of IAA \n\nconcentration was plotted against O.D 530 and \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 41 \n\nthe concentration of IAA in samples used was \n\ncalculated.  \n\n \nOrganic acid production: It was studied by \n\ngrowing the cultures in Calcium carbonate agar \n\nplate and observing for a clear zone around the \n\ncolony. \n\n \nChitinase production: It was observed by \n\nspotting the culture on chitin agar plate and \n\nobserving zone of clearance after incubating at \n\n28 \u00b1 2\u00b0C for 48- 72h. Chitinase activity \n\n(degradation of \u03b2- 1,4- N- acetylglucosamine \n\npolumer) were tested in a minimal medium. \n\nThere were clear zones were detected after 5 \n\ndays incubation period at 30\u00b0C.  \n\n \nPectinase production: It was detected by \n\nspotting the culture on pectin agar plate and \n\nobserving zone of clearance after incubating at \n\n28 \u00b1 2\u00b0C for 48- 72h. \n\n \n16S-rDNA  sequencing of PGPR isolates \n\n \nWell isolated colonies (2-3 colonies) of the \n\nculture whose 16S-rDNA had to be amplified \n\nwere suspended in 20\u00b5l-30\u00b5l of sterile distilled \n\nwater. The suspension was heated at 95\u00baC for 20 \n\nmin and centrifuged at 9000 rpm for 1min. The \n\nsupernatant was used as template DNA in the \n\nPCR system [26]. The 16S-rDNA gene fragment \n\nwas amplified using universal eubacterial full-\n\nlength primers. The amount of DNA taken for \n\namplification was 10ng.  \n\n \nPrimer sequences  \nForward Primer (PF)  5\u2019 AGA GTT TGA TCC TGG CTC AG 3\u2019\n        \nReverse Primer (PR 5\u2019 ACG GCT ACC TTG TTA CGA CTT 3\u2019\n  \nThe PCR components and conditions (to set a \n\nsystem of 30 \u00b5l) used for    amplification. \n\nAmplifications were performed in Eppendroff \n\ngradient thermal cyclers programmed for \n\n30cycles. The PCR thermal cycle consist of an \n\ninitial denaturation step of 3 min at 94\u00b0C, Then \n\n30 sec at 94\u00b0C for denaturation, 30 sec at 57\u00b0C \n\nfor primer annealing and in last step primer \n\nextension done by 2 min at 72\u00b0C. Steps 2, 3, 4 \n\nrepeated for 30 cycles followed by a final \n\nextension of 10 min at 72\u00b0C. The amplified \n\nproducts were then examined by an aliquot of \n\nthe DNA (2\u00b5l) was analyzed on a 1.0 % agarose \n\ngel along with 500bp ladder and stained with \n\nethidium bromide (0.5\u00b5g/ml). The gels were \n\nvisualized under UV light in a transilluminator \n\nand photographed subsequently. \n\n \nSequence Analysis: The product was sequenced \n\nand   matched with the already available \n\nsequences in the Gene Bank by uploading the \n\nobtained sequence in its FASTA format in \n\nnucleotide sequence match available at the \n\nonline tool of RDP Database Project II.", "start_char_idx": 11827, "end_char_idx": 14829, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c88aa95a-7957-4a57-96dd-471c442e567d": {"__data__": {"id_": "c88aa95a-7957-4a57-96dd-471c442e567d", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1b678420-2221-47e1-876a-155f97e2bcf4", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "7d96de8210b0d91c365832598e5d58e82d12cd09cfaa410565f7ede3c8ad2545", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "30b14678-e6fb-4f71-8ed4-a201266a8873", "node_type": "1", "metadata": {}, "hash": "730f2a0861e499778ea2a7052c122ab321ca3f6d21a5ca3750ce2957bbcaa615", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 42 \n\nResults and discussion \nIsolation of endophytic bacteria  \n\n \nWe have isolated endophytic bacteria from the \n\nLycopersicon esculentum (tomato) plants from \n\ndifferent field areas on the Nutrient agar (NA) \n\nmedium. Colonies showing different \n\nmorphological characteristics on the Nutrient \n\nagar plates were selected for further \n\ncharacterization. About 18 strains were isolated. \n\nThe number of isolates, the source of their plant \n\nand field from where the samples were procured \n\nare mentioned in the Table 1.  \n\n \nTable1: Bacterial endophytes isolates form Lycopersicon \n\nesculentum \n\n \n\nSample Location No. of \nisolate\ns \n\nName of the \nisolates \n\n \n1 \n\n \nAAU \n(Anand) \n\n \n \n5 \n\nHR 1 \nHR 2 \nHR 3 \nHR 4 \nHR 5 \n\n \n \n \n2 \n\n \n \n \nMansa \n(Gandhina\ngar) \n\n \n \n \n7 \n\nHR 6 \nHR 7 \nHR 8 \nHR 9 \nHR 10 \nHR 11 \nHR 12 \n\n \n \n3 \n\n \nGana \n(Anand) \n\n \n \n6 \n\nHR 13 \nHR 14 \nHR 15 \nHR 16 \nHR 17 \nHR 18 \n\n \n\nMorphological and physiological \n\nCharacterization  \n\n \nIn this work all the 18 isolates strains were  \n\npicked on the basis of different morphological \n\ncharacteristics. The morphological \n\ncharacteristics of the final four short-listed \n\nisolates are shown in Table 2. \n\n \nGram\u2019s staining and Motility \n\n \nResult showed that out of 18 isolates tested 9 \n\nwere gram negative coccobacilli, 5 were gram \n\npositive bacilli and only 4 were gram negative \n\ncocci. This indicated that majority (50%) of the \n\nbacteria in our studies belonged to gram \n\nnegative coccobacilli strains followed by \n\n22.22% gram positive bacilli and gram negative \n\ncocci seemed to be the most uncommon one \n\nconstituting only 27.77% of the total isolates. \n\nWhile in case of motility 66.66% were motile \n\nand remaining 43.44 % were non-motile (Table \n\n3). \n\n \nAntibiotic assay \n\n \nThe endophytic isolates were also checked for \n\ntheir sensitivity (S) and resistance (R) against \n\nantibiotics like Ampicillin, Gentamycin, \n\nSpectinomycin, Tetracycline. The result of the \n\nantibiotic assay of the rhizospheric isolates is \n\ntabulated (Table 4).", "start_char_idx": 14837, "end_char_idx": 17277, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "30b14678-e6fb-4f71-8ed4-a201266a8873": {"__data__": {"id_": "30b14678-e6fb-4f71-8ed4-a201266a8873", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c88aa95a-7957-4a57-96dd-471c442e567d", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "b0ffa8b8273b88e72d063c8474e3059b524e7043fc30d1198c7acd9ac6d4d550", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "daed2273-ff55-43d1-8090-1378b5fe2143", "node_type": "1", "metadata": {}, "hash": "ffa1d07ec43b3423f22c50711564e064631599ad5947af99ba4ad339ca3fca47", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 43 \n\nTable 2: Morphologyical and Physiological characteristics of 18 isolates \n\n \n\nColony \ncharacter \n\nSize Shape Margin Elevation Texture Opacity Pigmentation \n\nHR1 Medium Round Entire Raised Smooth Transparent No pigmentation \nHR2 Medium Round Entire Flat Smooth Transparent No pigmentation \nHR3 Small Round Entire Slightly \n\nRaised \nSmooth Transparent Yellow \n\npigmentation \nHR4 Small Round Entire Slightly \n\nRaised \nSmooth Opaque Yellow \n\npigmentation \nHR5 Small Round Entire Flat Rough Transparent No pigmentation \nHR6 Small Round Entire Slightly \n\nRaised \nSmooth Transparent Yellow \n\npigmentation \nHR7 Medium Round Entire Flat Smooth Opaque Pitch \n\npigmentation \nHR8 Medium Round Entire Raised Smooth Opaque Yellow \n\npigmentation \nHR9 Medium Irregular Irregular Flat Rough Opaque White \n\npigmentation \nHR10 Medium Round Entire Flat Smooth Transparent Yellow \n\npigmentation \nHR11 Small Round Entire Raised Smooth Transparent Yellow \n\npigmentation \nHR12 Medium Irregular Irregular Flat Rough Opaque White \n\npigmentation \nHR13 Small Round Entire Flat Rough Transparent No pigmentation \nHR14 Medium Round Entire Flat Smooth Transparent Yellow \n\npigmentation \nHR15 Medium Irregular Irregular Flat Rough Opaque White \n\npigmentation \nHR16 Medium Round Entire Flat Smooth Transparent Yellow \n\npigmentation \nHR17 Medium Irregular Irregular Raised Rough Transparent Golden yellow \n\npigmentation \nHR18 Small Irregular Irregular Flat Rough Opaque White \n\npigmentation \n \n\nSiderophore production \n\n \nAssay of siderophore production performed by \n\nCAS agar plate method in which following \n\nisolates HR1, HR3, HR4, HR7, HR18 showed \n\nproduction of siderophore. So further estimation \n\nof siderophore was performed to determine \n\nwhich types of siderophores are produced, either \n\ncathecolate or hydroxymates type of sideophore. \n\nUnfortunately we could not obtain the result. \n\n \nIAA production test   \n\n \nAll the isolates were tested for their IAA \n\nproduction. After 24 hrs of incubation with \n\ntryptophan all the strains exhibited a significant \n\namount of IAA production. The production of \n\nIAA by isolates indicated that the tested strains", "start_char_idx": 17287, "end_char_idx": 19849, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "daed2273-ff55-43d1-8090-1378b5fe2143": {"__data__": {"id_": "daed2273-ff55-43d1-8090-1378b5fe2143", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "30b14678-e6fb-4f71-8ed4-a201266a8873", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "08daf5828909a65cf6a408527299765472bd374de742e431757c05182fe87d3a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3c05b1dd-2d8d-4c34-ad82-a2de88f15238", "node_type": "1", "metadata": {}, "hash": "54ecc40e8559f248178b407b7891d82a2bc52f748c65f66c925dbe31fefa2a86", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 44 \n\nTable 3: The Gram nature and Motility of the 18 isolated strains.\n\nIsolates from Tomato  plant Gram\u2019s Nature Motility \nHR 1 Gram \u2013ve cocco bacilli + \nHR 2 Gram \u2013ve cocco bacilli + \nHR 3 Gram \u2013ve cocco bacilli + \nHR 4 Gram \u2013ve cocco bacilli + \nHR 5 Gram \u2013ve cocco bacilli + \nHR 6 Gram \u2013ve cocco bacilli + \nHR 7 Gram \u2013ve cocco bacilli + \nHR 8 Gram \u2013ve cocco bacilli + \nHR 9 Gram -ve cocci - \nHR 10 Gram -ve cocci - \nHR 11 Gram +ve  bacilli + \nHR 12 Gram +ve  bacilli - \nHR 13 Gram +ve  bacilli + \nHR 14 Gram +ve  bacilli - \nHR 15 Gram +ve bacilli - \nHR 16 Gram -ve cocci + \nHR 17 Gram \u2013ve cocco bacilli + \nHR 18 Gram \u2013ve cocco bacilli + \n\n+ : Indicates motile organism, - :  Indicates non-motile organism. \n\n \n\nutilized tryptophan as a precursor for growth and \n\nproduced IAA, the primary auxins in the \n\nmajority of plant species as a plant growth \n\npromoter. Data indicated that all the bacterial \n\nendophytes from plant were able to produce IAA \n\nin the presence of tryptophan (Table 5). \n\nProduction of IAA is widespread among \n\nbacteria-plant associated. Several bacteria \n\nhaving the ability to anabolise indole-3-acetic \n\nacid (IAA) with supplemented L- tryptophan \n\nhave been isolated from the plant surfaces. \n\nBacterial IAA producers (BIPs) have the \n\npotential to interfere with any of these processes \n\nby input of IAA into the plant's auxin pool [1]. \n\nPatten and Glick [20,21] have shown that \n\nbacterial IAA stimulates the development of the  \n\n \n\n \n\nroot system of the host plant and Brandi and \n\nLindow (1998) have studied the contribution of \n\nIAA for bacterial epiphytic fitness, observation \n\nsupported by the investigation of other workers \n\n[12,20,2,9,33]. \n\n \nChromium tolerance of the endophytic \n\nstrains \n\n \nAlmost 17 out of 18 isolates from Lycopersicon \n\nesculentum tolerated a chromium concentration \n\nupto 500\u00b5g/ml. One of the isolate HR11 \n\ntolerated upto 300\u00b5g/ml, wheres all the isolates \n\nshowing tolerance above 450\u00b5g/ml. There are \n\nreports of certain Bacillus spp. tolerating upto", "start_char_idx": 19855, "end_char_idx": 22300, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3c05b1dd-2d8d-4c34-ad82-a2de88f15238": {"__data__": {"id_": "3c05b1dd-2d8d-4c34-ad82-a2de88f15238", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "daed2273-ff55-43d1-8090-1378b5fe2143", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "7f26f84f67f92ab458c9e262969c2514b79605e565212474ec371fff34bfe08c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "30789272-ff76-4ee5-bd23-609ec06b5fb1", "node_type": "1", "metadata": {}, "hash": "362eb5129c911dbd923d539b9ad8501a252faffb7225d1abc9d8845294a45a03", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 45 \n\nTable 4: Antibiotic assay of isolated strains \n\n \nIsolates Ampicillin Streptomycin Tetracycline Chloramphenicol \n\nHR1 10 16 18 23 \nHR2 R 19 13 22 \nHR3 14 21 19 24 \nHR4 R 9 15 R \n\nHR5 R 17 R 14 \n\nHR6 R 9 R 13 \n\nHR7 R 18 R 17 \n\nHR8 R 11 R 8 \n\nHR9 28 29 27 38 \n\nHR10 R R 7 19 \n\nHR11 7 16 18 21 \n\nHR12 R 13 11 19 \n\nHR13 R R 9 16 \n\nHR14 13 21 15 14 \n\nHR15 8 R 11 14 \n\nHR16 R R 8 18 \n\nHR17 R 12 R R \n\nHR18 21 20 19 24 \n\nResistance microorganism- R, Number mentioned is zone of inhibition in mm\n\n \n\n550 \u00b5g/ml [35] and Bacilli spp. is a well known \n\nPGPR strain. All the standard strains except R. \n\nleguminosarum and S. meliloti showed very less \n\ntolerance to chromium. Both the strains R. \n\nleguminosarum and S. meliloti are well known \n\nfor their PGPR activity in leguminous plants. A \n\nRhodococcus erythropolis  \n\nMTCC 7905 strain has been shown to be \n\nresistant to 300 mg l-1 of Cr6+ isolated from \n\nmetal contaminated soil samples from a site near \n\nIndian Himalayan region has been reported to \n\nreduce substantial amounts of Cr6+ to Cr3+ as \n\nwell as showed to have plant growth promotion \n\nof pea (Pisum sativum) in the presence of toxic \n\nCr6+ concentration [30].", "start_char_idx": 22312, "end_char_idx": 23909, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "30789272-ff76-4ee5-bd23-609ec06b5fb1": {"__data__": {"id_": "30789272-ff76-4ee5-bd23-609ec06b5fb1", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3c05b1dd-2d8d-4c34-ad82-a2de88f15238", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "8223cb36601171fe22173d18449af425c41fa19b63f8af5fd9105af9d2fee161", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d15c82ae-ff9c-4d42-8ac0-d8c4fe0aaf71", "node_type": "1", "metadata": {}, "hash": "34733fdfe7b434940ca225a1d6ff34649eb861eec122cf9902fab3acac7e6ba2", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 46 \n\nTable 5: Indole Acetic Acid production by endophytic bacterial \n\nisolates \n\nIsolates OD at 530nm \nHR1 0.061 \nHR2 0.020 \nHR3 0.050 \nHR4 0.241 \nHR5 0.199 \nHR6 0.067 \nHR7 0.057 \nHR8 0.114 \nHR9 0.056 \nHR10 0.097 \nHR11 0.181 \nHR12 0.007 \nHR13 0.029 \nHR14 0.270 \nHR15 0.112 \nHR16 0.094 \nHR17 0.166 \nHR18 0.079 \n\n \n\nPhosphate solubilisation \n\n \nPhosphorous is one of the most important plant \n\nnutrient and a large portion of inorganic \n\nphosphate applied to soil as fertilizer is rapidly \n\nimmobilized [19,24]. Endophytic bacteria \n\npossess the capacity to solubilize immobilized \n\nmineral phosphates. In this study all the 18 \n\nisolates were tested for their phosphate \n\nsolubilizing activity on Pikovasky agar medium. \n\nIt was interesting to note that out of 18 \n\nendophytic isolates, 8 showed phosphate \n\nsolubilisation activity (Table 6). Result revealed \n\nthat majority of the PGPR strains do have \n\nphosphate solubilizing activity and such \n\norganisms play a major role in plant growth \n\npromotion [24].  \n\n \n\nTable 6: Phosphate Solubilization by endophytic bacterial isolates \n\n \n\nIsolate No. Growth on     PV Zone (mm) \n         HR 1 Full  growth    20                \n         HR 2 Full  growth     -  \n\n         HR 3 Full  growth    21 \n\n         HR 4 No growth     - \n\n         HR 5  No growth    - \n         HR 6 No growth    - \n\n         HR 7 Full  growth    17 \n\n         HR  8 No growth    - \n         HR 9 No growth    21 \n\n         HR10 No growth    - \n\n         HR 11 Less growth    8 \n\n         HR 12 Less growth   11 \n         HR 13 No growth    - \n\n         HR 14 No growth    - \n\n         HR 15    No growth    - \n         HR 16 No growth    - \n\n         HR17  Full  growth   31 \n\n         HR 18 Less growth   9 \n\nmm zone of clearance  (Pink colour Zone) \n\n \nOrganic acid production \n\n \nOut of the 8 endophytic isolates showing \n\nphosphate solubilization, all 8 showed organic \n\nacid production. The isolates number HR7, HR8, \n\nHR9, HR10, HR14, HR15   showed slight \n\norganic acid production by forming a very thin \n\nzone of clearance on the plates of Pikovasky \n\nwith methyl red as pH indicator dye. This gave \n\nthe pink coloured zone that indicated shift in pH \n\nchange from alkaline to acidic. Some isolates \n\nlike HR4, HR7, HR13, HR16 were unable to \n\nsolubilize phosphate and also did not produce \n\norganic acid. This could be because the amount", "start_char_idx": 23915, "end_char_idx": 26711, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d15c82ae-ff9c-4d42-8ac0-d8c4fe0aaf71": {"__data__": {"id_": "d15c82ae-ff9c-4d42-8ac0-d8c4fe0aaf71", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "30789272-ff76-4ee5-bd23-609ec06b5fb1", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "16dff4b71a0163e93a1882f78dda0600acaafd8eba5626237619970505356dff", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "85507da2-cbbf-4c0c-9f56-c37d52a920d6", "node_type": "1", "metadata": {}, "hash": "0c34eadc928b76aed981615dbc94bc63f38dce89d0c8f8f8b420962d59ff61ee", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 47 \n\nof organic acid produced might be very less to \n\ndo so (Table 7).  \n\n \nChitinase production \n\n \nIn the present study none of the strain revealed a \n\nclear zone, but 5 isolates out of 18 showed \n\ngrowth on the chitin agar plate, remaining 13 \n\nstrains did not show any growth (Table 8). \n\nBiological control of plant pests and diseases is \n\nmuch more attractive than chemical treatment \n\nmethods due to its greater specificity and less \n\nharmful impact on the environment [34,35]. \n\nMajor component of fungal cell is chitin. Thus \n\norganism having the ability to produce chitinase \n\nmight have antifungal property.  \n\n \nPectinase production \n\n \nFor pectinase production, 17 out of 18 isolates of \n\nLycopersicon esculentum revealed the \n\nproduction of pectinase. The strains showing  \n \n\nTable 7: Isolates showing organic acid production \n\nSample No. Isolate No. Production of \nOrganic acid \n\nZone (mm) Pink colour of \nZone \n\n \n \nSample 1. \n\n     HR  1 Medium   13    +++ \n     HR 2 Medium   10    ++ \n     HR  3 Very less    8    ++ \n     HR  4 No    -     - \n     HR 5  No    -     - \n\n \n \n \n \nSample 2. \n\n     HR 6 Very less    7     - \n      HR 7 Medium   11     ++ \n      HR 8 Medium    13     - \n      HR 9 Medium    10     - \n      HR 10 Very less     9     - \n      HR 11 Medium    12     +  \n      HR 12 Very less    8    ++ \n      HR 13 No     -     - \n\n \n \nSample 3. \n\n     HR 14 Medium    11     - \n     HR 15 Very less    8     - \n     HR 16 No    -     - \n     HR 17    Medium    11     +++ \n     HR 18 Medium    13     + \n\n+ : 1.0 mm ZOC (zone of clearance), ++: 1.2 mm ZOC, +++: 1.4 mm ZOC, -: No zone \n\n \n\nproduction of pectinase on pectin agar plate are \n\nlisted in Table 9. Maximum research indicated \n\nthat pectin methyl esterase (PME) (EC 3.1.1.11) \n\ncatalyzes the hydrolysis of methyl-ester groups \n\nof cell wall pectins. It has been found in all plant \n\ntissues and in some of plant cell wall-degrading \n\nmicroorganisms or insects [5,6] and has been \n\nimplicated in a number of processes including \n\ncell growth [18], fruit ripening [11, 29], \n\nabscission and senescence [17], pathogenesis [7] \n\nand cambial cell differentiation [13].", "start_char_idx": 26716, "end_char_idx": 29301, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "85507da2-cbbf-4c0c-9f56-c37d52a920d6": {"__data__": {"id_": "85507da2-cbbf-4c0c-9f56-c37d52a920d6", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d15c82ae-ff9c-4d42-8ac0-d8c4fe0aaf71", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "d55c0894d246eb8332826cb63f311396806253b96152c911da7c24ffbd14b667", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "330d727e-a43e-4aba-82c9-445132ffdb3e", "node_type": "1", "metadata": {}, "hash": "37ebcf75fc643aea9098b731b9ba3b64ac37784a16c2595a47623c0428df9344", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 48 \n\n \nTable 8: Isolates showing Chitinase production \n\nIsolates Chitinase \nProduction \n\nHR1 - \nHR2 + \nHR3 - \nHR4 + \nHR5 - \nHR6 - \nHR7 + \nHR8 - \n\n     HR9 - \nHR10 - \nHR11 - \nHR12 - \nHR13 - \nHR14 + \nHR15 - \nHR16 - \nHR17 - \nHR18 + \n\n                + : positive      - : negative  \n \n\nTable 9:  Isolates showing Pectinase production \n\nIsolates Pectinase \nproduction \n\nHR1 + \nHR2 + \nHR3 + \nHR4 + \nHR5 + \nHR6 + \nHR7 + \nHR8 + \n\n     HR9 + \nHR10 + \nHR11 + \nHR12 + \nHR13 + \nHR14 + \nHR15 + \nHR16 - \nHR17 + \nHR18 + \n\n                + : positive      - : negative  \n \n\n16S-rDNA sequencing of PGPR isolates \n\n \nColony PCR  \n\n \nAll the plant growth promoting results when \n\ncompiled together showed one strain (HR7) \n\nshowed maximum positive features and thus the \n\n16SrDNA of the strain was amplified using \n\nuniversal full length primers. An amplicon of \n\n1.5kb was obtained and sent for sequencing to \n\nBangalore Genei, Pvt, Ltd India. The sequence \n\nobtained was matched with the online available \n\nsequences in RDP (Ribosomal Database Project \n\nII) bioinformatics tool.   \n\n \nMultiple sequence alignment phylogenetic \n\nanalysis \n\n \nBLAST (Basic local alignment search tool) \n\nsearch was done for partial 16s rDNA of the \n\nisolates HR7 by submitting queries to NCBI-\n\nBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi.) \n\nand homologous sequences obtained by standard \n\nnucleotide-nucleotide BLAST (blastn) were \n\naligned with the different 16s rDNA isolates \n\nafter sequencing and various related sequence \n\nwere retrieve after blasting the partial sequence \n\nof the isolates obtained after sequencing. \n\nAccession No. of the related species  was \n\nretieved and Multiple sequence alignment (Fig \n\n1) was performed using CLC free protein \n\nworkbench 5.0. Evolutionary tree for the same", "start_char_idx": 29306, "end_char_idx": 31506, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "330d727e-a43e-4aba-82c9-445132ffdb3e": {"__data__": {"id_": "330d727e-a43e-4aba-82c9-445132ffdb3e", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "85507da2-cbbf-4c0c-9f56-c37d52a920d6", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "86b41aa36430f9f9a9541b10b4ce1fdac6238c76453e0093c39ddc519feb3ef9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "38bf25ca-5821-4057-b9a7-5c8fb8bf39d8", "node_type": "1", "metadata": {}, "hash": "0bab8dc83f933e806fc3e182b24bb754f56aab098a90bf8b0fd0c8ec2f92d4bf", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 49 \n\ndata was obtained by neighbor joining method \n\nwith Bootstrap values (expressed as percentages \n\nof 100 replications) as shown in (Fig 2). Except \n\nHR7 other do not give the sequencing results. \n\nAccession no. of some isolates used for multiple \n\nalignment with HARDIKSEQ-1(HR7) isolate \n\nwere,  JF423918, JF281099, HQ995502, \n\nHQ268732, HQ202541, HQ202540, HQ259948, \n\nFM995816, FM995815, FM995811, FM995802, \n\nFM995800, FM995798, FM995797, FM995796.\n\n \nFig. 1: Multiple sequence alignment for the partial 16s rDNA sequence of hardik seq-1 (HR7) isolate with other related species retrieve after \nBLAST, resulted in versatile coloring scheme that highlighted the conserved sequence in Aligned sequences. \n\n \nFig. 2: Phylogenetic tree of partial 16S rRNA genes of Hardik seq-1(HR7) islolates from closely related of resistant bacteria obtained after BLAST. \nThe tree was constructed based on partial 16S rRNA sequences of the isolates and the reference strains. Bootstrap values (expressed as percentages \nof 100 replications) are shown at branch points. Bootstrap values over 50% are shown. The scale bar 0.500 indicates 50% nucleotide sequence \nsubstitution\n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 50 \n\nPrecisely, the research concluded that \n\nendophytic bacteria isolated form Lycopersicon \n\nesculentum produced one or the other different \n\ncharacteristics involved in plant growth \n\npromotion. They either produced \n\nphytohormones like indole acetic acid, \n\nsiderophore, protease, pectinase, organic acid \n\nshowed antifungal activity, chromium tolerance \n\nand solubilized phosphate. Only HR7 endophyte \n\nof tomato turned out to be Pseudomonas \n\naeruginosa, It is a gram negative coccobacili, \n\nsporeforming motile bacilli,which showed \n\nmaximum PGPR activity. It may be concluded \n\nthat the above strains may be endophytic and \n\nwas associated with the plant probably because \n\nthey may benefit the plant by stimulating its \n\ngrowth.", "start_char_idx": 31511, "end_char_idx": 34266, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "38bf25ca-5821-4057-b9a7-5c8fb8bf39d8": {"__data__": {"id_": "38bf25ca-5821-4057-b9a7-5c8fb8bf39d8", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "330d727e-a43e-4aba-82c9-445132ffdb3e", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "98a32651ace839c9050cfd91aa368554c83c34e05e90022502b7eb7593b5e953", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "90fcea1b-7a53-447b-a323-d72b8c93d827", "node_type": "1", "metadata": {}, "hash": "2e4620367d483f70f7e22ec7b22c8c667e772eebafff6d695c69d62da9abb6cb", "class_name": "RelatedNodeInfo"}}, "text": "Acknowledgments   \n \nThis research was undertaken with support from \n\nthe Ashok and Rita Patel Institute of Integrated \n\nStudy and Research in Biotechnology and Allied \n\nSciences, New Vallabh Vidyanagar (managed by \n\nthe Charutar Vidya Mandal).  \n\n \n\nReferences \n \n1. Andreae WA, Van Ysselstein MWH: \n\nStudies on 3-indole acetic acid \nmetabolism. VI. 3-indole acetic acid \nuptake and metabolism pea roots and \n\nepicotyls. Plant Physiol.1960, 35: 225-\n232. \n \n\n2. Basti\u00e1n F, Cohen A, Piccoli P, Luna V, \nBaraldi and Bottini, V: Production of \nindole-3-acetic acid and gibberellin A1 \nand A3 by Acetobacter diazotrophicus \nand Herbaspirillum seropedicae in \nchemically-defined culture media. Plant \nGrowth Regul 1998 24: 7\u201311. \n \n\n3. Bergey's Manual of Systematic \nBacteriology 2009, 2nd Edition. Published \nby Springer, New York.  \n \n\n4. Brandi  MT, Lindow SE: Contribution of \nindole-3-acetic acid production to the \nepiphytic fitness of Erwinia herbicola. \nAppl Environ Microbiol 1998, 64: 3256\u2013\n3263. \n \n\n5. Campbell BC, Shea PJ: A simple staining \ntechnique for assessing feeding damage \nby Leptoglossus occidentalis Heidemann \non cones. Can Entomol 1990, 122: 963-\n968. \n \n\n6. Christgau S, Kofod LV, Halkier T, \nAndesen LN,  Hockauf M, Dorreich K,  \nDalboge H, Kauppienen S: Pectin methyl \nesterase from Aspergillus aculeatus: \nexpression cloning in yeast and \ncharacterization of the recombinant \nenzyme. Biochem. 1996,  J. 319:705-712. \n \n\n7. Collmer A, Keen, NT: The role of pectic \nenzyme in plant pathogenesis. Annual \nreview of phytopathology 1986, 24:383-\n409. \n\n8. Conrath U: Priming: getting ready for \nbattle. Mol. Plant Microbe Interact 2006, \n19: 1062\u20131071. \n \n\n9. Dobbelaere S, Croonenborghs A, Thys A, \nBroek AV,   Vanderleyden J: \nPhytostimulatory effect of Azospirillum \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 51 \n\nbrasiliense wild type and mutant strains \naltered in IAA production on wheat. \nPlant Soil 1999,  212: 155\u2013 164. \n\n10. Downing KJ,  Thomson JA:  Introduction \nof the Serratia marcescens chiA gene into \nan endophytic Pseudomonas fluorescens \nfor the biocontrol of phytopathogenic \nfungi. Can.", "start_char_idx": 34273, "end_char_idx": 36787, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "90fcea1b-7a53-447b-a323-d72b8c93d827": {"__data__": {"id_": "90fcea1b-7a53-447b-a323-d72b8c93d827", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "38bf25ca-5821-4057-b9a7-5c8fb8bf39d8", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "53929b6790a85c84694ffce9615a91f83877989448cf54bb98b09ff94b0303b9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fb42d213-b840-4775-8ccd-3879e2c9c35b", "node_type": "1", "metadata": {}, "hash": "a1f2ecdf2a0140ef9ccef084b979c370e01342a9e407e5316310c443a77a9a73", "class_name": "RelatedNodeInfo"}}, "text": "Can. J. Microbiol 2000, 46: 363-\n369. \n \n\n11. Gaffe JD, Tieman M, Handa AK: Pectin \nmethylestrase informs in tomato \n(Lycopersicon esculentum) tissue: effect \nof expression of methylestrase antisense \ngene. Plant Physiol 1994, 105:199-203. \n\n12. Glick BR: The enhancement of plant \ngrowth by free living bacteria. Can. J \nMicrobiol 1995, 41: 109-117. \n \n\n13. Guglielmino N, Liberman M, Catesson \nAM, Marek A, Prat R, Mutaftschiev S,  \nGoldberg R: Pectin methylesterases from \npoplar cambium and inner bark: \nlocalization, properties and seasonal \nchanges. Planta. 1997, 202: 70\u201375.  \n \n\n14. Hallmann J, Quadt-Hallmann A, Mahafee \nWF, Klopper JW: Bacterial endophytes \nin agricultural crops. Can. J. Microbiol. \n1997, 43: 895-914. \n\n15. Henning K, Villforth F: Experimentelle \nuntersuchungen zur frage der \nbacteriesymbiose in ho\u00a8heren pflanzen \nund ihre beeinflussung durch \n\u2018Leitemente\u2019. Biochem. Zeitschr. 1940, \n305: 299\u2013309. \n \n\n16. Kloepper JW: Plant growth promoting \nrhizobacteria as biological control \nagents. In: Soil Microbial Ecology\u2014\nApplications in Agricultural and \nEnvironmental Management, ed. F. B. \nMetting, Jr., 1993 pp. 255-274 (Marcel \nDekker, Inc., New York). \n \n\n17. Liners F, Van Custsem P: Distribution of \npectic polysaccharides throughout walls \nof suspension-cultured carrot cells. \nProtoplasma 1992, 170:10-21. \n \n\n18. Moustacas AM, Nari J, Borell M, Noat G,  \nRichard J: Pectin metylestarase: metal \nions and plant cell wall extention. The \nrole of metal ions in plant cell wall \nextention. J. Biochem. 1991, 279:351-354. \n \n\n19. Nautiyal CS, An efficient microbiological \ngrowth medium for screening phosphate \nsolubilizing microorganisms. FEMS \nMicrobiol Lett. 1999, 170: 265\u2013270. \n \n\n20. Patten CL, Glick BR, Bacterial \nbiosynthesis of indole-3-acetic acid. Can. \nJ. microbial 1996, 42: 207-220. \n \n\n21. Patten CL, Glick BR: Role of \nPseudomonas putida indole acetic acid in \ndevelopment of the host plant root \nsystem. Appl Environ Microbiol  2002, 68: \n3795\u20133801. \n \n\n22. Perotti R: On the limits of biological \ninquiry on soil science. Proc. Int. Soc. \nSoil Sci 1926, 2:146\u2013161. \n \n\n23. Reiter B, Pfeifer U, Schwab H,  Sessitsch \nA: Response of endophytic bacterial \ncommunities in potato plants to infection \nwith Erwinia carotovora subsp. \natroseptica. Appl Environ Microbiol 2002, \n68: 2261-2268. \n \n\n24. Rodriguez H, Fraga R:. Phosphate \nsolubilizing bacteria and their role in \nplant growth promotion. Biotechnol Adv \n1999, 17: 319\u2013339. \n \n\n25. Ryu CM, Kim JW, Choi OH, Park SY, \nPark SH, Park CS: Nature of a root-\nassociated Paenibacillus polymyxa from", "start_char_idx": 36783, "end_char_idx": 39379, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fb42d213-b840-4775-8ccd-3879e2c9c35b": {"__data__": {"id_": "fb42d213-b840-4775-8ccd-3879e2c9c35b", "embedding": null, "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-46", "node_type": "4", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "4b6490c4f2fc9db9c0d9d56c1bb9dead6798fc62d559a17d963e37b64ff892e6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "90fcea1b-7a53-447b-a323-d72b8c93d827", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "3d6ab77083629a5741d935fa6eed2dab1abfbf67dbb8f0405d5dc076dfc0ea62", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c82241db-1e07-48c8-9de8-02787779d60d", "node_type": "1", "metadata": {}, "hash": "40ffdaf8cad031d7f94f41733f97406fd728516965d05aa3937397b96fc18773", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a037\t\n \u00a0\u2013\t\n \u00a052\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n \n\n 52 \n\nfield-grown winter barley in Korea. J \nMicrobiol Biotechnol 2005, 15: 984-991. \n \n\n26. Sambrook J, Russell D W: Molecular \ncloning: A Laboratory Manual, 3rd edn \n2001, Vol.1, Cold Spring Harbor, New \nYork. \n \n\n27. Schwyn B  Neilands JB, Universal \nchemical assay for the detection and \ndetermination of siderophores. \nAnalytical Biochemistry 1987 160: 47-56. \n\n28. Sturz AV, Christie BR, Matheson BG, \nArsenault WJ, Buchanan NA, Endophytic \nbacterial communities in the periderm \nof potato tubers and their potential to \nimprove resistance to soil-borne plant \npathogens. Plant Pathol. 1999, 48:360-\n369. \n \n\n29. Tieman DM, Handa AK: Reduction in \npectin methylesterase activity modifies \ntissue integrity and action levels in \nripening tomato (Lycopersicon \nesculentum Mill.) fruits. Plant Physiol \n1994, 106: 429-436. \n \n\n30. Trivedi P, Pandey A, Tongmin S: \nChromate reducing and plant growth \npromoting activities of psychhrotrophic \nRhodococcus erythropolis MTCC 7905. J \nBasic Microbiol  2007, 47:513-517. \n\n31. Van Buren AM, Andre C, Ishimaru CA: \nBiological control of the bacterial ring \nrot pathogen by endophytic bacteria \nisolated from potato. Phytopathology \n1993, 83: 1406. \n\n32. Van Overbeek L, Van Elsas JD: Effects of \nplant genotype and growth stage on the \nstructure of bacterial communities \nassociated with potato (Solanum \ntuberosum L.). FEMS Microbiol Ecol \n2008, 64: 283\u2013296. \n \n\n33. Verma SC, Ladha JK, and Tripathi AK: \nEvaluation of plant growth promotion \nand colonization ability of endophytic \ndiazotrophs from deep water rice. J \nBiotechnol 2001, 91: 127\u2013141. \n \n\n34. Wang SL, Shih IL, Liang TW, Wang CH: \nPurification and characterization of two \nantifungal chitinases extracelluarly \nproduced by Bacillus amyloliquefaciens \nV656 in a shrimp and crab shell powder \nmedium. J Agric Food Chem 2002, 50: \n2241-2248.  \n \n\n35. Wani PA, Khan MS, Zaidi A:  Chromium \nreducing and plant growth promoting \nMesorhizobium improves chickpea \ngrowth in chromium amended soil. \nAgron Sustain Dev. 2007, 27: 145-153.", "start_char_idx": 39384, "end_char_idx": 41847, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c82241db-1e07-48c8-9de8-02787779d60d": {"__data__": {"id_": "c82241db-1e07-48c8-9de8-02787779d60d", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fb42d213-b840-4775-8ccd-3879e2c9c35b", "node_type": "1", "metadata": {"identifier": "njb-46", "author": "Patel, Hardik A; Patel, Rajesh K; Khristi, Sunil M; Parikh, Kruti; Rajendran, Geetha", "title": "Isolation and Characterization of Bacterial Endophytes from Lycopersicon esculentum Plant and their Plant Growth Promoting Characteristics", "date": "2022-07-11", "file": "njb-46.pdf"}, "hash": "80509955dfea4784418762aa48499a106721287c67f0afe40244a404ed9f1e19", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7ba97b17-5ea1-4ecc-9bc9-3c3e5ce9656b", "node_type": "1", "metadata": {}, "hash": "c49b0fa488ef26c8a0fe8ec743aeae1acb96e83b11e65d92ec48667977313ba0", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Paper 3 two Colum Final.docx\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n26\t\n \u00a0\n\n\t\n \u00a0 ORIGINAL\t\n \u00a0RESEARCH\t\n \u00a0ARTICLE\t\n \u00a0\n\n \nLovastatin\t\n \u00a0production\t\n \u00a0by\t\n \u00a0Aspergillus\t\n \u00a0fischeri\t\n \u00a0under\t\n \u00a0solid\t\n \u00a0state\t\n \u00a0fermentation\t\n \u00a0from\t\n \u00a0\ncoconut\t\n \u00a0oil\t\n \u00a0cake\t\n \u00a0\n\nParvatham Madhu Latha1*,, Pallem Chanakya2, Manipati Srikanth3 \n \n\n1,* NRI College of Pharmacy, Pothavarappadu, Agiripalli Mandal, Vijayawada Rural, Andhra Pradesh.  \n2Centre for Biotechnology, Department of Chemical Engineering, \nAU College of Engineering (A), Andhra University, Visakhapatnam, Andhra Pradesh. \n3NRI Academy of Medical Sciences, China Kakani, Mangalagiri Mandal, Guntur, Andhra Pradesh \n*Corresponding author: srimani.nri@gmail.com, chanakya.pallem@gmail.com \n\n \n\nAbstract: \n\nThe main aim of the present investigation was to optimize the fermentation parameters that \nenhance the maximum production of lovastatin by Aspergillus fischeri using coconut oil cake as \nthe solid substrate under solid state fermentation. The maximum yield of lovastatin (14.77 mg/g \ndry substrate) using coconut oil cake as the substrate was achieved with the following optimized \nprocess parameters: fermentation time (7 days), initial moisture content (60% v/w), inoculum \nvolume (2ml of five day old culture), initial pH (5.0), incubation temperature (30\u00baC), lactose (1% \nw/v) and malt extract (1% w/v).  \n\nKeywords: Lovastatin, Aspergillus fischeri, Coconut oil cake, Fermentation parameters, Optimization \n\n \n\nIntroduction:  \n\nLovastatin also called as Mevinolin, \n\nMonacolin K and Mevacor, a kind of fungal \n\nmetabolite, serves as one of the competitive \n\ninhibitors of enzyme hydroxymethylglutaryl \n\ncoenzyme A reductase (HMG-CoA), which \n\ncatalyzes the rate-limiting step in cholesterol \n\nbiosynthesis, resulting in lowered blood \n\ncholesterol [1,2]. In addition, lovastatin has \n\nbeen used in the biomedical applications such  \n\n \n\nas, in treating coronary heart diseases, renal \n\ndiseases, Alzheimer\u2019s disease, bone fractions \n\n[3]. Moreover, it has been indicated as a \n\npotential therapeutic agent for the treatment of \n\nvarious types of tumors because of its \n\ncapability to suppress tumor growth in vivo \n\nthrough inhibition of the synthesis of non-\n\nsterol isoprenoid compounds [4, 5]. \n\nCommercial production of lovastatin has been \n\ncarried out by submerged fermentation using \n\ndifferent microorganisms [6, 7]. In recent", "start_char_idx": 49, "end_char_idx": 2866, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7ba97b17-5ea1-4ecc-9bc9-3c3e5ce9656b": {"__data__": {"id_": "7ba97b17-5ea1-4ecc-9bc9-3c3e5ce9656b", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c82241db-1e07-48c8-9de8-02787779d60d", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "a64077a68f9405b033c2562684ed6a7ff96c2993048c0d55afa6e746951196e9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9de8baff-33a4-4366-bee0-57b66684013b", "node_type": "1", "metadata": {}, "hash": "07dd1a09540667ab14c3779e20030ae8edcc6c89e0c7f5f74d887bb7b29ce818", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n27\t\n \u00a0\n\nyears, researchers have shown an increasing \n\ninterest in solid state fermentation (SSF) as it \n\nis a potential alternative of submerged \n\nfermentation because economical and cheap \n\nsubstrates can be utilized in SSF for \n\nproduction of value-added products, and also \n\nrequires less energy, and low capital costs. \n\nNow days, SSF has been exploited for the \n\nproduction of highly potent drugs on large \n\nscale. But still, few reports are available on \n\nthe microbial production of lovastatin under \n\nSSF [8-10]. \n\nIn the present study, we evaluated the \n\nfeasibility of SSF process for lovastatin \n\nproduction by Aspergillus fischeri and \n\noptimized different process parameters that \n\nmaximize the lovastatin yield. \n\nMaterials and Methods \n\nMicroorganism and Inoculum preparation: \n\nThe fungal strain Aspergillus fischeri NCIM \n\n509 used in this study was procured from the \n\nculture collection centre of National \n\nCollection of Industrial Microorganisms \n\n(NCIM), NCL, Pune.  The microbial strain \n\nwas maintained on Potato Dextrose Agar \n\n(PDA) slants containing (g/l) dextrose 20.0 \n\nand agar 15.0. Inoculated slants were grown in \n\nan incubator at 28\u00b0C for 5 days. After that, the \n\nslants were preserved at 4\u00b0C and sub-cultured \n\nonce every four weeks (monthly). \n\nInoculum preparation: \n\nInoculum was prepared by adding 10 ml of \n\nsterilized 0.1% Tween 80 solution to a well-\n\nsporulated A. fischeri slant. The spore surface \n\nwas scrapped with an inoculating loop to \n\nsuspend the spores in the solution and the \n\nobtained spore suspension was used as the \n\ninoculum for the fermentation process. \n\nSolid state fermentation (SSF): \n\nCoconut oil cake obtained from the local \n\nmarket of Vijayawada, Andhra Pradesh was \n\nused as the substrate in the present study. Five \n\ngrams of coconut oil cake was dried and \n\ngrounded to required particle size having both \n\ncoarse and fine particles in 1:1 ratio (w/w). \n\nThe  substrate was taken in a 250ml \n\nErlenmeyer flask and moistened with distilled \n\nwater to maintain the moisture content of 50% \n\n(v/w) and autoclaved at 121\u00baC (15lb) for 15-\n\n20 min, cooled to room temperature and \n\ninoculated with 2 ml of the 5 day spore \n\nsuspension (spore concentration of about 107-108 \n\nper ml) of A. fischeri under aseptic conditions. \n\nThe contents of the inoculated flasks were \n\nmixed thoroughly and incubated at desired \n\ntemperature in an incubator for desired length \n\nof fermentation time.", "start_char_idx": 2871, "end_char_idx": 5765, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9de8baff-33a4-4366-bee0-57b66684013b": {"__data__": {"id_": "9de8baff-33a4-4366-bee0-57b66684013b", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7ba97b17-5ea1-4ecc-9bc9-3c3e5ce9656b", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "814ebac6b31f031f373579aa15bda5ab64d55a5d15a0022d40ffb7ea77e572ac", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "17dad699-8ddd-4c58-920d-a4aac88314f3", "node_type": "1", "metadata": {}, "hash": "b2a7a073817b613f89664b0e6a66c17acc10259e06cf99f67b998d2c75e1143b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n28\t\n \u00a0\n\nOptimization of Process Parameters: \n\nVarious process parameters that influence the \n\nlovastatin production during SSF were \n\noptimized over a wide range. The strategy \n\nadopted for standardization of fermentation \n\nparameters was to evaluate the effect of an \n\nindividual parameter and incorporate it at the \n\nstandard level before standardizing the next \n\nparameter. The process parameters optimized \n\nin the present study include fermentation time \n\n(24-192h), initial moisture content (40-100% \n\nv/w), inoculum volume (0.5-4ml), initial pH \n\n(4-10), incubation temperature (26-40\u02daC), \n\ncarbon sources (1% w/v) and nitrogen sources \n\n(1% w/v). All the experiments were done in \n\ntriplicate and the mean values of the lovastatin \n\nyield was reported. \n\nLovastatin extraction: \n\nAfter the fermentation time, the fermented \n\nflasks were dried at 60\u00b0C for 2 hrs. To this 20 ml \n\nof methanol was added to the flasks to extract \n\nlovastatin by keeping them in an orbital shaker at \n\n180 rpm for 2 h. The residue was filtered with \n\nfilter paper and then centrifuged at 1500 rpm for \n\n15 min. The supernatant obtained was \n\ncollected and analyzed for quantitative \n\ndetermination of lovastatin [10]. \n\nQuantitative analysis of lovastatin: \n\nTo 1 ml of the obtained supernatant, 1 ml of \n\n1% Trifloroacetic acid was added and \n\nincubated for 10 min (Lactonization of \n\nhydroxyl acid form of Lovastatin).From the  \n\nsolution, 0.5 ml was taken and diluted to 10-\n\n100 ml in methanol and its absorbance was \n\nread at 238 nm by using UV-Visible \n\nSpectrophotometer [11]. \n\n \n \n\nConcentration of Lovastatin (mg/g)   =  Concentration of Lovastatin (mg/ml) x Dilution factor (DF) \n                                                                       Amount of substrate taken (g) \n\n \n\nResults and Discussion \n\nSelection of solid substrates in SSF for \n\nlovastatin production: \n\nSolid substrates employed in SSF processes \n\nare heterogeneous natural raw-materials which \n\nare generally insoluble in water and play a  \n\n \n\n \n\ndual role-supply of nutrients to the microbial \n\nculture growing and anchorage for the \n\ngrowing cells. In the present study, coconut oil \n\ncake was used as the solid substrate for both \n\nthe fungal sporulation and lovastatin \n\nproduction by Aspergillus fischeri. Maximum", "start_char_idx": 5770, "end_char_idx": 8505, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "17dad699-8ddd-4c58-920d-a4aac88314f3": {"__data__": {"id_": "17dad699-8ddd-4c58-920d-a4aac88314f3", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9de8baff-33a4-4366-bee0-57b66684013b", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "81cc375bc9144d1bfc1766bdd2ffcd26a0a6cc40a1c133a067cd199161e7276d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ccf997f9-1536-4c67-bc7e-0f32b1d8b88d", "node_type": "1", "metadata": {}, "hash": "87e3be9ffea9ca9f7bad356fabbe13fb70f5876b543236e4cbc791d6a1b2cc43", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n29\t\n \u00a0\n\nlovastatin yield of 6.81 mg/g dry substrate was \n\nobtained.  \n\nEffect of Fermentation time: \n\nThe effect of fermentation time on lovastatin \n\nproduction was studied by incubating the \n\nfermentation flasks from 24-192 h. The \n\nmaximum lovastatin yield of 7.43 mg/g of dry \n\nsubstrate was achieved at 168 h of \n\nfermentation time (Fig.1). The lovastatin yield \n\nincreased from    24-168 h which explained \n\nthat lovastatin is a kind of secondary \n\nmetabolite and its accumulation in mycelia \n\nseems growth related and slightly decreased \n\nfrom 168-192h. The lovastatin yield decreased \n\nafter seven days (168 h) of fermentation which \n\nmay be due to the onset of death phase of \n\nmicroorganism and nutrient depletion.  \n\nFig.1: Effect of fermentation time on lovastatin production \n\nEffect of initial moisture content \n\nTo investigate the influence of initial \n\nmoisture content of the substrate, \n\nfermentation was carried out under various \n\ninitial moisture content levels (40, 50, 60, \n\n70, 80, 90 and 100(% v/w)) of coconut oil \n\ncake, which was adjusted with moistening \n\nmedia. Maximum lovastatin yield (8.04 \n\nmg/g dry substrate) was achieved at 60% \n\n(v/w) initial moisture content ( Fig. 2.).   \n\nAs the moisture content in the fermentation \n\nmedium increases, the air present in the void \n\nvolume decreases, resulting in poor oxygen \n\navailability for the process without forced \n\naeration and with low moisture content, the \n\navailable oxygen is sufficient but the water \n\ncontent is not enough to support good \n\nmetabolic activity and dissipation of heat \n\ngenerated and may account for lower \n\nlovastatin production. The same 60% (v/w) \n\nmoisture content was also observed for both \n\nAspergillus flavipes and Aspergillus terreus \n\nunder SSF [8, 10].  \n\n0\t\n \u00a0\n1\t\n \u00a0\n2\t\n \u00a0\n3\t\n \u00a0\n4\t\n \u00a0\n5\t\n \u00a0\n6\t\n \u00a0\n7\t\n \u00a0\n8\t\n \u00a0\n\n24\t\n \u00a0 48\t\n \u00a0 72\t\n \u00a0 96\t\n \u00a0 120\t\n \u00a0 144\t\n \u00a0 168\t\n \u00a0 192\t\n \u00a0\n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nFementation time (h)", "start_char_idx": 8510, "end_char_idx": 10909, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ccf997f9-1536-4c67-bc7e-0f32b1d8b88d": {"__data__": {"id_": "ccf997f9-1536-4c67-bc7e-0f32b1d8b88d", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "17dad699-8ddd-4c58-920d-a4aac88314f3", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "2322215c94ed5f2d13c8bc4c36a648500020be3cf0dada77aa1dce6084086a26", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c486735c-ed2b-4665-9f5b-5a46686606e2", "node_type": "1", "metadata": {}, "hash": "03482337f4b3a67400ef8ed0b01308a038d7f82350a1a61d6ecec2df0eca8961", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a084\t\n \u00a0\u2013\t\n \u00a0XX\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n. \n\nFig. 2: Effect of moisture content on lovastatin production \n\nEffect of Inoculum volume \n\nFermentation was carried out with different \n\ninoculum volumes (0.5-4ml) of five day old \n\nA. fischeri culture for a period of 144h to \n\nstudy its effect on lovastatin production. The \n\nmaximum yield of lovastation (8.97 mg/g \n\ndry substrate) was obtained with 2ml of \n\nA.fischeri culture as shown in Fig. 3. \n\n \n\n \n\nWith the further increase in inoculum \n\nvolume, the yield of lovastatin had \n\ndecreased which might be due to the \n\ndepletion of the available nutrients in the \n\nproduction medium, yielding poor mycelia \n\ngrowth, thus promoting less product \n\nformation. With low inoculum volume, the \n\nyield is also low due to the insufficient \n\nmicrobial culture to form mycelia and \n\nproduce lovastatin. \n\n0\t\n \u00a0\n\n1\t\n \u00a0\n\n2\t\n \u00a0\n\n3\t\n \u00a0\n\n4\t\n \u00a0\n\n5\t\n \u00a0\n\n6\t\n \u00a0\n\n7\t\n \u00a0\n\n8\t\n \u00a0\n\n9\t\n \u00a0\n\n40\t\n \u00a0 50\t\n \u00a0 60\t\n \u00a0 70\t\n \u00a0 80\t\n \u00a0 90\t\n \u00a0 100\t\n \u00a0\n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nMoisture content (% v/w)", "start_char_idx": 10914, "end_char_idx": 12335, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c486735c-ed2b-4665-9f5b-5a46686606e2": {"__data__": {"id_": "c486735c-ed2b-4665-9f5b-5a46686606e2", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ccf997f9-1536-4c67-bc7e-0f32b1d8b88d", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "58749d2c2b9327109490aa17e736d3186254ef3c8f8bab771c1e25ad623087a9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "440e3a58-a2df-4922-a48f-fa1cc8d0cfea", "node_type": "1", "metadata": {}, "hash": "34a72bff45b22d6c6a42a3b0e424477af7e1ec253e610d464eae6d675e02065d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a084\t\n \u00a0\u2013\t\n \u00a0XX\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n. \n\nFig. 3: Effect of inoculum volume on lovastatin production \n\n \n\nEffect of initial pH: \n\nExperiments were performed to the \n\noptimum pH in order to maintain the \n\nfavorable conditions for increased \n\nproduction of lovastatin. This was \n\nestablished by carrying out the fermentation \n\nby varying the pH from 4-10. \n\nThe profound effect of initial pH of the \n\nfermentation on lovastatin production was as \n\nshown in Fig. 4. Maximum lovastatin yield  \n\n \n\n \n\n(9.71 mg/g dry substrate) was recorded at \n\npH 5.0. A further increase in pH resulted in \n\ngradual decrease of lovastatin production \n\ndue to the denaturation or inactivation of the \n\nmicrobial strain, because pH strongly \n\ninfluences the transport of various components \n\nacross the cell membrane which support the cell \n\ngrowth and product formation, and most of the \n\nfungi are active in the pH range of 3.5-7 and also \n\nlower pH avoids the contamination by other \n\nmicrobes. The result was in accordance with \n\nthe lovastation production using Aspergillus \n\nterreus under SSF [8]. \n\n \n\n0 \n\n1 \n\n2 \n\n3 \n\n4 \n\n5 \n\n6 \n\n7 \n\n8 \n\n9 \n\n10 \n\n0.5 1 1.5 2 2.5 3 3.5 4 \n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nInoculum volume (ml)", "start_char_idx": 12340, "end_char_idx": 13953, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "440e3a58-a2df-4922-a48f-fa1cc8d0cfea": {"__data__": {"id_": "440e3a58-a2df-4922-a48f-fa1cc8d0cfea", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c486735c-ed2b-4665-9f5b-5a46686606e2", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "8a618b33677356d04756fbcfffd16ecbf0c17cc0148c519b50c821c4805a917d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b0b60f23-2b25-4f74-82c2-99701e7cb412", "node_type": "1", "metadata": {}, "hash": "ed22cb454f8aa2de2f388d52a976658d04fe29524e388ab78341c5bd84530f49", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a084\t\n \u00a0\u2013\t\n \u00a0XX\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n. \n\nFig. 4: Effect of pH on lovastatin production. \n\n \n\nEffect of temperature \n\nFermentation was carried out at various \n\ntemperatures from 26-40\u00b0C to study their \n\neffect on enzyme production (Fig. 5). \n\nResults indicated that maximum lovastatin \n\nproduction (10.88 mg/g dry substrate) was \n\nobtained when SSF was carried out at 30\u00b0C. \n\nHowever, lovastatin production reduced \n\ngradually above the optimal incubation \n\ntemperature of 30\u00b0C. With further increase  \n\n \n\nin temperature, more heat is accumulated in \n\nthe medium during mesophilic aerobic SSF, \n\nwhich leads to poor heat dissipation thus \n\nreducing the oxygen level and thereby \n\nreducing the growth of microorganism, as \n\nlovastatin is growth related product. These \n\nresults are coinciding with those previously \n\nreported for lovastatin production by \n\nAspergillus terreus and Monascus ruber \n\n[10,13]. \n\n0\t\n \u00a0\n\n2\t\n \u00a0\n\n4\t\n \u00a0\n\n6\t\n \u00a0\n\n8\t\n \u00a0\n\n10\t\n \u00a0\n\n12\t\n \u00a0\n\n4\t\n \u00a0 5\t\n \u00a0 6\t\n \u00a0 7\t\n \u00a0 8\t\n \u00a0 9\t\n \u00a0 10\t\n \u00a0\n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\npH", "start_char_idx": 13958, "end_char_idx": 15399, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b0b60f23-2b25-4f74-82c2-99701e7cb412": {"__data__": {"id_": "b0b60f23-2b25-4f74-82c2-99701e7cb412", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "440e3a58-a2df-4922-a48f-fa1cc8d0cfea", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "bb7063a5e96292f2527c75ad15b18df342ad913a3747e4adb03715b4ae291120", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8a4b1c11-59d7-40d8-befc-1652c1d627d6", "node_type": "1", "metadata": {}, "hash": "1d0cc77ad6afa39a9496b651a5788deb305c2595f6818591e076f8150a0a2d11", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Dec.\t\n \u00a02011,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a084\t\n \u00a0\u2013\t\n \u00a0XX\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n. \n\nFig. 5: Effect of temperature on lovastatin production \n\n \n\nEffect of carbon source  \n\nTo determine the effect of incorporation of \n\nadditional carbon sources on lovastatin \n\nyield, different carbon sources namely \n\nglucose, maltose, sucrose, xylose, lactose, \n\ngalactose and soluble starch at 1% (w/v) \n\nadded to the basal solid state fermentative \n\nmedium of A. fischeri exerted a considerable \n\neffect on the biosynthesis of lovastatin (Fig. \n\n6). Maximum lovastatin production was \n\npromoted by lactose (12.02 mg/g dry \n\nsubstrate) followed by maltose (11.11 mg/g \n\ndry substrate) and sucrose (10.47 mg/g dry \n\nsubstrate). The enhanced production of \n\nlovastatin by the incorporation of lactose to \n\nthe medium might be attributed to the \n\npositive influence of lactose as a co-\n\nmetabolic agent for enhanced microbial \n\nmetabolite biosynthesis.  \n\n \n\nFig. 6: Effect of different carbon sources on lovastatin \n\nproduction. \n\n \n\n0\t\n \u00a0\n\n2\t\n \u00a0\n\n4\t\n \u00a0\n\n6\t\n \u00a0\n\n8\t\n \u00a0\n\n10\t\n \u00a0\n\n12\t\n \u00a0\n\n26\t\n \u00a0 28\t\n \u00a0 30\t\n \u00a0 32\t\n \u00a0 34\t\n \u00a0 36\t\n \u00a0 38\t\n \u00a0 40\t\n \u00a0\n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nTemperature (\u02daC) \n\n0\t\n \u00a0\n\n2\t\n \u00a0\n\n4\t\n \u00a0\n\n6\t\n \u00a0\n\n8\t\n \u00a0\n\n10\t\n \u00a0\n\n12\t\n \u00a0\n\n14\t\n \u00a0\n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nCarbon source", "start_char_idx": 15404, "end_char_idx": 17060, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8a4b1c11-59d7-40d8-befc-1652c1d627d6": {"__data__": {"id_": "8a4b1c11-59d7-40d8-befc-1652c1d627d6", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b0b60f23-2b25-4f74-82c2-99701e7cb412", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "49ab5ced21152f89ed7c834077ae8fac6831d2cbd0a96a8ee9663bf350d916a2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "920c8546-dffc-415d-b8bd-57158d3b096b", "node_type": "1", "metadata": {}, "hash": "142bdb692aa896317d5248713bad633d3f81d7d3e4c290af9b4eabbc4ec784be", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n34\t\n \u00a0\n\nEffect of Nitrogen source \n\nNitrogen source can be an important limiting \n\nfactor in the production of microbial \n\nmetabolites [14]. The supplementation of \n\nadditional nitrogen sources (either organic or \n\ninorganic) such as sodium nitrate, ammonium \n\nsulphate, urea, peptone, tryptone, yeast \n\nextract, malt extract and beef extract at 1% \n\n(w/v) had shown a profound impact on the \n\nproduction of lovastatin by A. fischeri under \n\nSSF ( Fig. 7). \n\nAmong the various nitrogen sources \n\nevaluated, malt extract in the fermentation \n\nmedium promoted enhanced fungal growth \n\nand consequent lovastatin production of 14.77 \n\nmg/g dry substrate was achieved followed by \n\nyeast extract (12.36 mg/g dry substrate) and \n\nsodium nitrate (11.03 mg/g dry substrate).  \n\n \n\nFig. 7: Effect of different nitrogen sources on lovastatin \n\nproduction \n\n \n\nConclusion: \n\nThe current investigation was mainly \n\nfocused on the evaluation of the potentiality \n\nof Aspergillus fischeri for utilization of \n\ncoconut oil cake as the substrate for the \n\nproduction of lovastatin under solid state \n\nfermentation. The yields obtained in the \n\npresent study have to be further increased \n\nfor its industrial importance. It has proved \n\nthe feasibility of solid state fermentation as a \n\npromising technique in exploiting cheaply \n\navailable agro-residual wastes as substrates \n\nfor the large-scale production of microbial \n\nmetabolites of biotechnological importance \n\nultimately leading to an effective solid waste \n\nmanagement. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n0 \n2 \n4 \n6 \n8 \n\n10 \n12 \n14 \n16 \n\nY\nie\n\nld\n o\n\nf L\nov\n\nas\nta\n\nti\nn \n\n(m\ng/\n\ng)\n \n\nNitrogen source \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n35\t\n \u00a0\n\nReferences: \n\n1. Alberts AW:  Lovastatin and \nSimvastatin-inhibitors of HMG CoA \nreductase and cholesterol-\nbiosynthesis: Cardiology 1990, 77:14-\n21. \n \n\n2.", "start_char_idx": 17065, "end_char_idx": 19751, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "920c8546-dffc-415d-b8bd-57158d3b096b": {"__data__": {"id_": "920c8546-dffc-415d-b8bd-57158d3b096b", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8a4b1c11-59d7-40d8-befc-1652c1d627d6", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "3ba9eae046fa10573f4b49edee9b220d7f8d09019b8e528c97b3cca8e413f59b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e642427-550f-4122-9f67-88fa1c6a6147", "node_type": "1", "metadata": {}, "hash": "8df579b854d64df9dcdbd7dd45a83b926dfb053963935e41e0b3d99405010a9d", "class_name": "RelatedNodeInfo"}}, "text": "2. Alberts AW, Chen J, Kuron G,  Hunt V,  \nHuff J,  Hoffman C, Rothrock J, Lopez \nM, Joshu H, Harris E,  Patchett A,  \nMonaghans R,  Currie S, Stapley E, \nAlberts SG, Hensens O, Hirshfield T, \nHoogsteent J, Liescht J, Springeri J: \nMevinolin: A highly potent \ncompetitive inhibitor of \nhydroxymethylglutaryl-coenzyme A \nreductase and a cholesterol-lowering \nagent. Proc. Natl. Acad. Sci.  1980, 77 \n(Suppl. 7): 3957-3961. \n \n\n3. Seenivasan A, Subhagar S, Aravindan R, \nViruthagiri T: Microbial production \nand biomedical applications of \nlovastatin. Ind. J. of Pharm. Sciences \n2009, 701-709. \n \n\n4. Jones KD, Couldwell WT, Hinton DR, \nSu YH, He DK, Anker L, Law RE: \nLovastatin induces growth inhibition \nand apoptosis in human malignant \nglioma cells.  Biochem. Biophys. Res \n.Commun. 1994, 205:1681-1687. \n \n\n5. Newman A, Clutterbuck RD, Powles \nRL, Millar JL: Selective inhibition of \nprimary acute myeloid leukemia cell \ngrowth by lovastatin. Leukemia 1994, \n8:2022-2029. \n \n\n6. Casas Lopez JL, Sanchez Perez JA, \nFernandez Sevilla JM, Acien Fernandez \nFG, Molina Grima E, Chisti Y: \nProduction of lovastatin by \nAspergillus terreus: effects of the C: N \n\nratio and the principal nutrients on \ngrowth and metabolite production. \nEnzyme Microbiol Technol. 2003,  \n33(Suppl. 2-3): 270-277. \n \n\n7. Sitaram Kumar M, Jana SK, Senthil V, \nShashanka V, Vijayakumar S, \nSadhukhan AK: Repeated fed batch \nprocess for improving lovastatin \nproduction.  Process Biochem. 2000, 36 \n(Suppl. 4): 363-368. \n \n\n8. Valera HR, Gomes J, Lakshmi S, \nGurujara R, Suyanarayan S, Kumar D: \nLovastatin production by solid state \nfermentation using Aspergillus flavipes. \nEnzyme Microbiol Technol. 2005, 37 \n(Suppl. 5): 521-526. \n \n\n9. Xu BJ, Wang QJ, Jia XQ, Sung CK: \nEnhanced Lovastatin Production by \nSolid State Fermentation of Monascus \nruber. Biotechnol and Bioprocess Engg. \n2005, 10 (Suppl. 1):78-84. \n \n\n10. Pie-Lian WEI, Zhi-nan XU, Pei-Lin \nCEN: Lovastatin production by \nAspergillus terreus in Solid-State \nfermentation. J. Zheijan Univ. 2007, \n9:1521-1526. \n \n\n11. Mielcarek J, Naskreni M, Grobelny P: \nPhotochemical properties of \nsimvastatin and lovastatin by \nradiation. J. Thermal Analysis \nCalorimetry 2009, 96:301-305. \n \n\n12. Pansuriya RC, Singhal RS: Response \nsurface methodology for optimization \nof production of Lovastatin by solid \nstate fermentation.  Brazilian J. \nMicrobiol. 2010, 41:164-172. \n \n\n13. Panda BP, Javed S, Ali M: \nOptimization of fermentation \nParameters for higher lovastatin", "start_char_idx": 19749, "end_char_idx": 22249, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e642427-550f-4122-9f67-88fa1c6a6147": {"__data__": {"id_": "9e642427-550f-4122-9f67-88fa1c6a6147", "embedding": null, "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-47", "node_type": "4", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "f378829304973f02d824471673737f38a9665fef2f68edfbb19f7b7d7596e7bf", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "920c8546-dffc-415d-b8bd-57158d3b096b", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "59b68e7aad5a4ff3a7e70581bf9954de4b6ee4838bde9c7cc08d64e53452df2f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "945a5832-6359-47b6-b754-c3410aad353b", "node_type": "1", "metadata": {}, "hash": "7cd2367af481059e5972dc1e9ac64cd8266c1feb9de25fac165df5befb4f4930", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a026\t\n \u00a0\u2013\t\n \u00a036\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n\t\n \u00a0\n\n\t\n \u00a0\n\t\n \u00a0\n\n36\t\n \u00a0\n\nproduction in Red mold rice through \nco-culture of Monascus purpureus and \nMonascus ruber.  Food Bioprocess \nTechnol. 2008, 53:342-346. \n\n \n14. Chandrasekaran M, \n\nLashmanaperumalsamy P, \nChandramohan D: Combined effect of \n\nenvironmental factors on spoilage \nbacteria.  Fish Technology (India) 1991, \n28: 146-153.", "start_char_idx": 22254, "end_char_idx": 23018, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "945a5832-6359-47b6-b754-c3410aad353b": {"__data__": {"id_": "945a5832-6359-47b6-b754-c3410aad353b", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e642427-550f-4122-9f67-88fa1c6a6147", "node_type": "1", "metadata": {"identifier": "njb-47", "author": "Latha, Parvatham Madhu; Chanakya, Pallem; Srikanth, Manipati", "title": "Lovastatin production by Aspergillus fischeri under solid state fermentation from coconut oil cake", "date": "2022-07-11", "file": "njb-47.pdf"}, "hash": "dc4e2103e99673ee8e64151140e85e2d9c992d6b56ef27df268807686130d7cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d661606f-be13-4844-ad4f-45a01da9e2d5", "node_type": "1", "metadata": {}, "hash": "3a02b43ffca502948e2d12935f6e533e987b135b60f0ff3df86ca588643c505e", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - BSN RAPD rice_Joshi_Final two column.docx\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 16 \n\n ORIGINAL RESEARCH ARTICLE \n\n \nGenetic\t\n \u00a0Relationship\t\n \u00a0among\t\n \u00a0Nepalese\t\n \u00a0Rice\t\n \u00a0Landraces\t\n \u00a0and\t\n \u00a0Cultivars\t\n \u00a0based\t\n \u00a0on\t\n \u00a0\n\nRAPD\t\n \u00a0Markers \n\nBal K. Joshi*, Hari P. Bimb, David Kansakar and Ekta Ghimire \nBiotechnology Unit, NARC, PO Box 1136 Kathmandu, Nepal \n\n* Corresponding author: Email: joshibalak@rediffmail.com \n\n \n \n\nAbstract \n\nGenetic information of any genotype is necessary to manage and utilize them in conservation and \nbreeding program. A total of 28 RAPD markers were used to relate the genetic structure among 50 \nNepalese rice genotypes consisting of 29 landraces, 12 breeding lines and 9 released cultivars. \nSome of them are aromatic and blast resistance. Only four primers (P41, P60, P109 and P141) \namplified the DNA of these genotypes with scorable bands. Primer 60 produced the highest \nnumber of bands (8). The highest number of present bands (6) was shown by primer 41 in 10 rice \ngenotypes. Grouping of these genotypes based on the adaptation to agro-climatic zone was not \nobserved, probably due to low percentage coverage of genome by four primers.  Most of the \ngenotypes grouped in two clusters. Kali Marsi and IR-24 formed separate individual cluster. \nMansara and Jarneli were the most similar landraces (0.96). Churenodhan and Pranpyuri were the \nmost closely related with Masuli. Only one genotype NR-285-18 has fallen in the first quadrant by \nprincipal component (PC) analysis and the fourth quadrant was empty. The highest contribution in \nPC1 was from the second band of primer 41. This RAPD information can be used for selecting \nlines and for blast resistance breeding. \n\n \nKey words: Genetic distance, rice, RAPD \n\n \n\nIntroduction \n\nNepal is rich in rice genetic resources [1, 2]. \n\nKnowledge on genetic diversity contributes \n\nsignificantly for the better management and \n\nutilization of these resources. Diversity \n\nanalysis with the help of molecular markers \n\nprovides reliable information which can be \n\nutilized for breeding purposes. RAPD \n\n(Randomly Amplified Polymorphic DNA) [3] \n\nmarkers though dominant markers, provides \n\nfast, reliable and cost effective determination \n\nof genetic diversity in plant varieties, breeding \n\nlines and accessions [4-6]. In RAPD, a single \n\nrandom primer is added to the template DNA \n\nand subjected to polymerase chain reaction \n\n(PCR). This simple but effective method of \n\nrevealing polymorphism is cheap and", "start_char_idx": 49, "end_char_idx": 2950, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d661606f-be13-4844-ad4f-45a01da9e2d5": {"__data__": {"id_": "d661606f-be13-4844-ad4f-45a01da9e2d5", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "945a5832-6359-47b6-b754-c3410aad353b", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "62e3d259abee59022ed3574ad16e1a30c886014f55f843f091677596a4dd9815", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6034a4fc-0efb-4326-bc77-1f649f2b356c", "node_type": "1", "metadata": {}, "hash": "31efb9d85eda022960403cae41f777e404927ea06f46cdd7b882174e94e17f27", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 17 \n\nuniversally applicable [7, 8]. The indica and \n\njaponica cultivars are classified into separate \n\ngroups by cluster analysis using RAPD [5]. \n\nWe studied the genetic diversity of rice \n\nparticularly adapted to mid and high hills \n\nusing RAPD markers to support for effective \n\nmanagement and utilization of rice genetic \n\nresources.  \n\n \n\nMaterials and methods \n\n \na. Plant materials and plant DNA \n\nextraction \n\n \nThe rice genotypes analyzed are given in \n\nTable 1. A total of 50 rice samples consisting \n\nof landraces, breeding lines and released \n\ncultivars were used. DNA was extracted \n\nemploying the Modified CTAB method of [9].  \n\n \nb. DNA amplification \n\n \nFor RAPD analysis 28 decamer primers were \n\ntested (Table 2). Amplification was carried out \n\nin a 10 \u00b5l reaction volumes consisting of \n\n10mM Tris-HC1 pH 8.3, 2mM MgC12, \n\n0.2mM dNTPs, 1mM primer, 0.35 unit of Taq \n\nDNA polymerase and 1 ng of total DNA as \n\ntemplate. The amplification reaction was \n\ncarried out in PTC-100 thermocycler (MJ \n\nResearch, USA). The first cycle consisted of \n\ndenaturation of template DNA at 93.5oC for 1 \n\nmin, primer annealing at 36oC for 2 min and \n\nprimer extension at 72oC for 3 min. In the next \n\n44 cycles, the three steps of first cycle were \n\nrepeated. In the last cycle it is hold at 72oC for \n\n7 min and then at 4oC for 3 min. PCR products \n\nwere separated on a 1.8% agarose gel using \n\nTAE buffer. The gels were run for 2.5-3 hr at \n\n70 V and stained with ethidium bromide. \n\nDNA fragments were visualized under UV \n\nlight and photographed using Gel Doc system.  \n\n \nOnly the four primers amplified the DNA of \n\ntest lines. Polymorphisms were scored for the \n\npresence or absence of bands on a 1/0 basis \n\nand data analyzed using the NTSYS-pc \n\nsoftware [10].  \n\n  \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 18 \n\nTable 1.", "start_char_idx": 2955, "end_char_idx": 5602, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6034a4fc-0efb-4326-bc77-1f649f2b356c": {"__data__": {"id_": "6034a4fc-0efb-4326-bc77-1f649f2b356c", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d661606f-be13-4844-ad4f-45a01da9e2d5", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "e2c4ed98f7d1a5783879c281d9926d8435ff7a76f3245a65ebdda20db0d8d11b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "31d7e8a6-8cc5-4f8f-a353-59cce8c74fd6", "node_type": "1", "metadata": {}, "hash": "6f04fa4ac30cea16f7f4ba2ce8e06ab1d08f887ef586c7e4b5b49c1a398bfa22", "class_name": "RelatedNodeInfo"}}, "text": "Rice landraces, cultivars and breeding lines used in this study. \n  \n\nS.N. Genotype Collection site Altitude, m Collection \nyear \n\nRemarks \n\n1 Krishnabhog Achham 1000 1985 Landrace \n2 Thapachini Bajura 1768 1995 Landrace \n3 Tauli Bhojpur 1219 1987 Landrace \n4 Tunde dhan Dailekh 1400 1995 Landrace \n5 Rato dhan Dadeldhura 1585 1995 Landrace \n6 Hansraj Dadeldhura 1128 1995 Landrace \n7 Mansara Dadeldhura 1128 1995 Landrace \n8 Chureno dhan Dang 2120 1985 Landrace \n9 Anpjhutte Gorkha 1981 1988 Landrace \n10 Jarneli Gulmi 2000 1998 Landrace \n11 Bhuwa dhan Humla 1970 1985 Landrace \n12 Jhul dhan  Humla 1350 1985 Landrace \n13 Pahele Kaski 1075 1998 Landrace \n14 Radha-7 Kaski 1040 1998 Released \n15 Pakhe Lamjung 1920 1988 Landrace \n16 Pranpyuri Lamjuing 1996 1988 Landrace \n17 Madise Lamjung 1524 1988 Landrace \n18 Kali marsi Mugu 2600 1985 Landrace \n19 Ghaiya dhan Mugu 2380 1985 Landrace \n20 Dhokro Mugu 2350 1985 Landrace \n21 Maine pokhreli Mustang 1400 1985 Landrace \n22 Lekali dhan Myagdi 1800 1985 Landrace \n23 Hanse Sallyan 1200 1992 Landrace \n24 Pale dhan Sindupalchok 1500 1985 Landrace \n25 Bageri dhan Solukhumbu 1707 1989 Landrace \n26 Jethobor Tanahun 1250 1988 Landrace \n27 Pokhara masino Tanahun 1250 1988 Landrace \n28 Chananchur Udaypur 1829 1989 Landrace \n29 Lalshar Udaypur 1829 1989 Landrace \n30 NR10315-145 ABD, Khumaltar   Breeding line \n31 NR10286-6 ABD, Khumaltar   Breeding line \n32 Manjushree-2 ABD, Khumaltar   Released \n33 NR10375-20 ABD, Khumaltar   Breeding line \n34 Khumal-11 ABD, Khumaltar   Released  \n35 NR10353-8 ABD, Khumaltar   Breeding line \n36 NR285-18 ABD, Khumaltar   Breeding line \n37 NR10276-15 ABD, Khumaltar   Breeding line \n38 NR10414-25 ABD, Khumaltar   Breeding line \n39 NR10414-34 ABD, Khumaltar   Breeding line \n40 Taichung-176 ABD, Khumaltar   Released  \n41 Jumli White ABD, Khumaltar   Landrace \n42 Chandan nath-1 ABD, Khumaltar   Released  \n43 Chandan nath-3 ABD, Khumaltar   Released  \n44 NR10276-9 ABD, Khumaltar   Breeding line \n45 NR10285-29 ABD, Khumaltar   Breeding line", "start_char_idx": 5603, "end_char_idx": 7627, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "31d7e8a6-8cc5-4f8f-a353-59cce8c74fd6": {"__data__": {"id_": "31d7e8a6-8cc5-4f8f-a353-59cce8c74fd6", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6034a4fc-0efb-4326-bc77-1f649f2b356c", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "de8593c01b77f61fb59fb6e745042f907c76564b51eadad5850d97d56e294807", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fb269854-c50a-4e16-8036-3bc3a7ee5f06", "node_type": "1", "metadata": {}, "hash": "4283f7c9621b2adf6ad1ae771e7599cf5ee4a643df123759dd9e0febb32a868a", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 19 \n\nS.N. Genotype Collection site Altitude, m Collection \nyear \n\nRemarks \n\n46 Sabitri NRRP, Hardinath   Released, BR  \n47 IR-24 NRRP, Hardinath   Released, BR \n48 A57-115-8  NRRP, Hardinath   Breeding line, BDI (3 \n\ngene pyramid) \n49 CO39 NRRP, Hardinath   Breeding line, BS  \n50 Masuli NRRP, Hardinath   Released, BS  \n \nNote: ABD , Agriculture Botany Division. NRRP, National Rice Research Program. B, Blast. R, Resistant. S, Susceptible. DI, Differential \nline. \n \n\nTable 2. Details of RAPD primers used in this study. \n \n\nS.N. Primer Sequence  Band scored Remarks  \n1 P36 GGGGGTCGTT -  \n2 P40 GGCGGACTGT -  \n3 P41 GAGTGCGCAG 6 Rice genome \n4 P42 CCGGACTGAG -  \n5 P48 GAAGGCGCGT -  \n6 P52 GGCACCACCA -  \n7 P60 CATCGGCCCT 8  \n8 P109 TGGCCACTGA 3  \n9 P141 GTGATCGCAG 7 Operon Tech \n10 P142 CAATCGCCGT -  \n11 P144 CAGCACCCAC -  \n12 P165 CTGACGTCAC -  \n13 P169 AGTCGACGCC -  \n14 P181 ACGGACGTCA -  \n15 P189 TGGGTCCCTC - Operon Tech  \n16 P191 CTGCGCTGGA -  \n17 P194 AGGCCCGATG -  \n18 P197 GACCCCGGCA -  \n19 P198 GCCTGGTTAC -  \n20 P202 CGCAGACTTG - TAG 91:65-667.\u201995. Lentil \n21 P205 GCCGTGAAGT - TAG 91:65-667.\u201995. Lentil \n22 P209 GGCGTCGGGG - TAG 91:65-667.\u201995. Lentil \n23 P217 GGGTTGCCGT - TAG 85:937-95.\u201993.Vicia faba \n24 P222 GTCACCCGGA - TAG 85:937-95.\u201993.Vicia faba \n25 P225 AGTGGTCGCG -  \n26 P232 GCGCATTAGA - Bio/Tec 10:686-690. Conifer \n27 P270 AGCCAGTTTC - TAG 85:190-196.\u201992. Brassica \n28 P292 CAAACGGCAC - TAG 86:788-794.\u201995. Alfalfa", "start_char_idx": 7632, "end_char_idx": 9502, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fb269854-c50a-4e16-8036-3bc3a7ee5f06": {"__data__": {"id_": "fb269854-c50a-4e16-8036-3bc3a7ee5f06", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "31d7e8a6-8cc5-4f8f-a353-59cce8c74fd6", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "f304fce4f6f886bdea86f0d77fa7830cfee0d69874d07f9ef08228c37dfa0794", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d11052ad-a458-4f6b-b552-dda490811d82", "node_type": "1", "metadata": {}, "hash": "1d910ccf6eff47aacc08d37108bbeaa1da7f399c13ba5d4f5b5def914deff8f4", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 20 \n\nResults and discussion  \n \n\na. Primers and genetic similarity \n \n\nAmong the 28 RAPD primers, only four \n\nprimers (P41, P60, P109 and P141) amplified \n\nthe genomic DNA of test lines (Table 2). The \n\npercentage of primers that amplified the DNA \n\nwas very low. These four primers showed \n\npolymorphism. We considered only those \n\nprimers that could amplify the DNA of all \n\nsamples with scorable bands (Figure 1, 2). \n\nMost of the primers did not work probably due \n\nto the old or not related to rice genome or poor \n\nquality of template DNA. Polymorphism \n\npercentage of the tested RAPD primers are \n\n90.0 in the study of [11] and 67 in [12]. In \n\ntheir study, with selected primers, sufficient \n\npolymorphism is detected to allow \n\nidentification of individual varieties. RAPD \n\nanalyses offer the greatest chance of detecting  \n\nsmall genetic differences, since a larger \n\ncomponent of the genome can be scanned than \n\nin other systems [8, 13]. Primer 60 produced \n\nthe highest number of bands (8). The highest \n\nnumber of present bands (6) was shown by \n\nprimer 41 in 10 rice genotypes. The genetic \n\nsimilarity ranged from 0.00 to 0.96. Mansara \n\nand Jarneli were the most similar landraces \n\n(0.96). The second most similar landraces \n\nwere Tunde dhan and Krishnabhog. IR-24 \n\nshowed the zero similarity coefficients with all \n\ngenotypes. The zero similarity coefficient of \n\nKali Marshi with Thapachini, Krishnabhog \n\nand Tauli indicates the most genetic \n\ndissimilarity. The similarity index between \n\nChandannath-1 and Lalshar was also zero. \n\nA57-115-8 showed the zero similarity index \n\nwith Chandannath-3. Two blast susceptible \n\nvarieties, Mansuli and CO-39 have mostly the \n\nsimilar coefficients with all tested genotypes.  \n\n \n\n  \nFig.1. RAPD polymorphism of different rice genotypes with primer 141 \n\nM\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a01\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a02\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a03\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a04\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a05\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a06\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a07\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a08\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a09\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a010\t\n \u00a0\t\n \u00a0\t\n \u00a011\t\n \u00a0\t\n \u00a0\t\n \u00a012\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a013\t\n \u00a0\t\n \u00a0\t\n \u00a014\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a016\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a017\t\n \u00a0\n\nA\n\nB\n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 9516, "end_char_idx": 12188, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d11052ad-a458-4f6b-b552-dda490811d82": {"__data__": {"id_": "d11052ad-a458-4f6b-b552-dda490811d82", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fb269854-c50a-4e16-8036-3bc3a7ee5f06", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "dc69797ab5a5d8a63c0cc49fe2b585b63a9c3d1b78e3e39fd3030a1f52f5a66f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "938fbf2e-3404-4702-af4e-3fb26fc5133d", "node_type": "1", "metadata": {}, "hash": "edba35723d6c58d48979d5c3165b72e7e3303e465c4c6802f61035c6e618fe8d", "class_name": "RelatedNodeInfo"}}, "text": "2012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 21 \n\n \n(M, marker; Sample A: 1, Kali Marshi; 2, Ghaiya dhan; 3, Dhokro dhan; 4, Maine Pokhreli; 5, Lekali dhan; 6, Hanse; 7, Pale dhan; 8, Bageri \ndhan; 9, Jethobor; 10, Pokhara Masino; 11, Chananchur; 12, Lalshar; 13, NR10315-145-2-3; 14, NR10286-6-3-2-2; 15, Manjushree-2 ; 16, \nNR10375-20-1-2; 17, Khumal 11. Sample B: 1, NR10353-8-2-1; 2, NR28518-3-2-3-1; 3, NR10276-15-2-3-3-2; 4, NR10414-25-2; 5, NR10414-\n34-2-3; 6, Taichung-176; 7, Jumli White; 8, Chandhannath-1; 9, Chandhannath-3; 10, NR10276-9-3-3-3-2; 11, NR10285-29-3-1; 12, Sabitri; 13, \nIR-24; 14, A57-115-8; 15, CO 39; 16, Masuli;17, Check3 from Jumla, 2 from Humla and 3 Mugu).  \n  \n   \n\n  \nFig.2. RAPD polymorphism of different rice genotypes with primer 41 \n\n(M, marker; Sample A: 1, *Krishnabhog; 2, *Thapachini; 3,Tauli; 4,Tunde dhan; 5,Rato dhan; 6, *Hansraj; 7, Mansara; 8,Chureno dhan ; 9, \nAnpjhutte; 10,Jarneli ; 11, Bhuwa dhan; 12, Jhuldhan; 13, *Pahele; 14,Radha-7; 15,Pakhe ; 16, Pranpyuri; 17, Madise. Sample B: 1, Kali \nMarshi; 2, Ghaiya dhan; 3, Dhokro dhan; 4, Maine Pokhreli; 5, Lekali dhan; 6, Hanse; 7, Pale dhan; 8, Bageri dhan; 9, *Jethobor; 10, \n*Pokhara Masino; 11, Chananchur; 12, Lalshar; 13, NR10315-145-2-3; 14, NR10286-6-3-2-2; 15, Manjushree-2 ; 16, NR10375-20-1-2; 17, \nKhumal 11.", "start_char_idx": 12162, "end_char_idx": 13809, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "938fbf2e-3404-4702-af4e-3fb26fc5133d": {"__data__": {"id_": "938fbf2e-3404-4702-af4e-3fb26fc5133d", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d11052ad-a458-4f6b-b552-dda490811d82", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "13e5146cdb18007ff23bfdb78f342b7772bec312afae9ca82a249043dfb65d9a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5b5abf04-dd1f-47e9-8ae6-1bd91202fc53", "node_type": "1", "metadata": {}, "hash": "2bc635cd15c37cae3030e2929a6a04bc66a760bc155baddaad870af469fac563", "class_name": "RelatedNodeInfo"}}, "text": "Sample C:1, NR10353-8-2-1; 2, NR28518-3-2-3-1; 3, NR10276-15-2-3-3-2; 4, NR10414-25-2; 5, NR10414-34-2-3; 6, Taichung-\n176; 7, Jumli White; 8, Chandhannath-1; 9, Chandhannath-3; 10, NR10276-9-3-3-3-2; 11, NR10285-29-3-1; 12, Sabitri; 13, IR-24; 14, A57-115-\n8; 15, CO 39; 16, Masuli;17, Check3 from Jumla, 2 from Humla and 3 Mugu). * Aromatic rice. \n \n \nb. Cluster analysis \n\n \nThe dendrogram generated by the RAPD \n\nanalysis showed four distinct groups (Figure \n\n3). IR-24 and Kali Marsi formed the \n\nseparate individual cluster. Most of the \n\ngenotypes fell in two clusters. Grouping of  \n\n \n\nthese genotypes based on the adaptation to \n\nagro-climatic zone was not observed,  \n\nprobably due to low percentage coverage of \n\ngenome by four primers.  Mansara and \n\nJarneli were the most similar landraces \n\nfollowed by NR-10276-9 and NR-10285-20. \n\nChurenodhan and Pranpyuri were the most \n\nM     1     2      3      4       5      6     7     8     9    10   11     12    13   14   15    16   17 \n\n1     2     3      4     5     6     7      8     9     10  11    12    13    14     15   16    17\n\nA\n\nB\n\nC", "start_char_idx": 13810, "end_char_idx": 14915, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5b5abf04-dd1f-47e9-8ae6-1bd91202fc53": {"__data__": {"id_": "5b5abf04-dd1f-47e9-8ae6-1bd91202fc53", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "938fbf2e-3404-4702-af4e-3fb26fc5133d", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "7c84715173ee81b91acb64affa6da5e40e891d9133eb625dc85e87ff2425e93f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0619a1e1-f8f9-4ada-b34e-fae0046b4503", "node_type": "1", "metadata": {}, "hash": "82ddcfcd5818bf0c50e2a0769d3f08822b1e21aab0475643e99c268da97e657d", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 22 \n\nclosely related with Masuli. The three blast \n\nresistance genes pyramided rice genotype, \n\nA57-115-8 was genetically near with \n\nAnpjutte, Tauli and Thapachini.  \n\n \n\n \nFig.3. Clustering of 50 rice genotypes based on RAPD markers. \n\n \n \n\nc. Principal component analysis \n \n\nA scatter plot was drawn based on the \n\nsimilarity coefficients among the 50 rice \n\ngenotypes (Figure 4). All genotypes except \n\nNR-285-18 fell in the second and third \n\nquadrant. Only one genotype NR-285-18 has \n\nfallen in the first quadrant by principal \n\ncomponent analysis and the fourth quadrant \n\nwas empty. The highest contribution in PC1  \n\n \n\nwas from the second band of primer 41 \n\n(Table 3). Considerable overlapping among \n\nthe various samples is evident, which \n\nsuggests that genetic variation among them \n\nis rather narrow. Nevertheless, some rice \n\nsamples appeared separate from the \n\noverlapping ones e.g. aromatic rice like \n\nPahele, Jethobor, Maine Pokhreli, Pokhara \n\nMasino, and Hansraj. The level of \n\nCoefficient\n0.00 0.24 0.48 0.72 0.96\n\n          \n\n Krishnabhog \n Tundedhan \n JumliWhite \n PokharaMasino \n Jhuldhan \n ChandanNath-1 \n NR10276-15 \n Sabitri \n Hansraj \n Mansara \n Jarneli \n Bhuwadhan \n NR10315-145 \n Pahele \n NR10414-25 \n Taichung-176 \n RatoDhan \n NR10276-9 \n NR10285-29 \n ChandanNath-3 \n Radha-7 \n Madise \n Pakhe \n Ghaiyadhan \n MainePokhreli \n Paledhan \n Dhokrodhan \n Hanse \n Chananchur \n Lekalidhan \n Jethobor \n Bageridhan \n Manjshree-2 \n NR10353-8 \n NR10414-34 \n Thapachini \n Tauli \n Anpjhutte \n A57-115-8 \n Churenodhan \n Pranpyuri \n Masuli \n Lalshar \n NR10286-6 \n NR10375-20 \n Khumal-11 \n NR285-18 \n CO39 \n KaliMarsi \n IR-24", "start_char_idx": 14919, "end_char_idx": 16989, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0619a1e1-f8f9-4ada-b34e-fae0046b4503": {"__data__": {"id_": "0619a1e1-f8f9-4ada-b34e-fae0046b4503", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5b5abf04-dd1f-47e9-8ae6-1bd91202fc53", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "9363452fb7bcd4d7d77b7e70d85870adcb587ea2d98a0b51c7837e4cc4426c11", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e792b3f4-491b-472e-9d79-836c5f8c7536", "node_type": "1", "metadata": {}, "hash": "7776d8a9d9cdbca61419c75489eaa715c26eb193a5bbef7180e2e95481a61aac", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 23 \n\ndistinctness versus overlapping was in good \n\nconcordance with that of the cluster. \n\n \nThis preliminary genetic information could \n\nsupplement for breeding and conservation \n\nworks based on morphological markers. For \n\nincreasing the value of genetic information \n\nderived from RAPD markers, number of \n\nprimers should be increased. Choudhury et \n\nal. [14] suggest that a set of 10 primers can \n\nbe employed for an initial assessment of \n\ngenetic diversity in a large number of \n\ncollections. Because of multilocus nature of \n\nRAPD, its use is considered more suitable \n\nfor fingerprinting and genetic diversity \n\nmeasurement.  \n\n \n\n \nTable 3. Eigen vectors of RAPD primers based on 50 rice genotypes. \n\n \nPrimer Band PC1 PC2 PC3 \nP41 1 -0.363 0.203 -0.123 \n\n2 -0.374 0.217 0.019 \n3 -0.300 0.370 0.046 \n4 -0.371 0.247 -0.012 \n5 -0.299 0.240 0.078 \n6 -0.148 0.174 0.536 \n\nP60 1 -0.225 -0.284 -0.022 \n2 -0.252 -0.357 -0.071 \n3 -0.167 -0.200 -0.327 \n4 -0.096 -0.053 -0.172 \n5 -0.097 -0.153 0.029 \n6 0.197 0.268 -0.034 \n7 0.124 0.071 0.083 \n8 0.065 0.063 -0.037 \n\nP141 1 0.085 0.124 -0.060 \n2 -0.157 -0.257 0.472 \n3 -0.112 -0.321 0.372 \n4 -0.083 -0.143 0.198 \n5 0.068 0.016 0.048 \n6 -0.045 -0.090 0.059 \n7 -0.009 0.003 0.032 \n\nP109 1 -0.202 -0.097 -0.221 \n2 -0.199 -0.135 -0.230 \n3 -0.180 -0.175 -0.157 \n\nEigenvalue  1.062 0.629 0.396 \nProportion  0.243 0.144 0.090 \nCumulative  0.243 0.386 0.477", "start_char_idx": 16994, "end_char_idx": 18818, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e792b3f4-491b-472e-9d79-836c5f8c7536": {"__data__": {"id_": "e792b3f4-491b-472e-9d79-836c5f8c7536", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0619a1e1-f8f9-4ada-b34e-fae0046b4503", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "c469ce62413528c4870fc329b442f59ae05cbf28ce558f472a4c1e5413dffe6e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c42f32b4-f11a-4f18-beae-3d7274c458f1", "node_type": "1", "metadata": {}, "hash": "a5b1902b5055fde866a69d49138079b83b049fe57fa0d586780f61c49231eb04", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 24 \n\nFig.4. Scatter plotting of 50 rice genotypes based on four RAPD markers. \n \n \n  \n\n0-1-2-3-4\n\n2\n\n1\n\n0\n\n-1\n\n-2\n\nPC I\n\nP\nC\n\n II Masu liCO 3 9A 5 7 -1 1 5 -8\n\nIR-2 4\n\nSab itri\n\nN R1 0 2 8 5 -2 9\nN R1 0 2 7 6 -9\n\nCh an d an N ath -3\n\nCh an d an N ath -1Ju mliWh ite\n\nTaich u n g -1 7 6\n\nN R1 0 4 1 4 -3 4\n\nN R1 0 4 1 4 -2 5\n\nN R1 0 2 7 6 -1 5\n\nN R2 8 5 -1 8\n\nN R1 0 3 5 3 -8\n\nK h u mal- 1 1\n\nN R1 0 3 7 5 -2 0\n\nMan ju sh ree-2\n\nN R1 0 2 8 6 -6\n\nN R1 0 3 1 5 -1 4 5\n\nLalsh ar\n\nCh an an ch u r\n\nPo k h araMasin o\n\nJeth o b o r\n\nBag erid h an\n\nPaled h an\n\nH an se\n\nLek alid h an\n\nMain ePo k h reli\n\nD h o k ro d h an G h aiy ad h an\n\nK aliMarsi\nMad ise\n\nPran p y u riPak h e Rad h a-7\n\nPah ele\n\nJh u ld h an\n\nBh u w ad h an\n\nJarn eli\n\nA n p jh u tte\n\nCh u ren o d h an\n\nMan sara H an sraj\n\nRato D h an\nTu n d ed h an\n\nTau li\n\nTh ap ach in i\n\nK rish n ab h o g\n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a016\t\n \u00a0\u2013\t\n \u00a025\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 25 \n\nReferences \n\n1. Joshi BK: Rice gene pool for mid and \nhigh hills and its conservation in Nepal.", "start_char_idx": 18831, "end_char_idx": 20643, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c42f32b4-f11a-4f18-beae-3d7274c458f1": {"__data__": {"id_": "c42f32b4-f11a-4f18-beae-3d7274c458f1", "embedding": null, "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-48", "node_type": "4", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "907cf69ed1225444d00b7632dcda1ec6ad450347c2b9196cc00623b02bab8e7f", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e792b3f4-491b-472e-9d79-836c5f8c7536", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "efd92001d6240d1c1a91fddda807bd70791ed31356d102757070a6b116368969", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9f03331f-804e-4c42-abb2-0e050ae85b39", "node_type": "1", "metadata": {}, "hash": "1f4d7b20a29153fe11a79df7af2d8cb61988a8fa5c524d3b76b5d23c90e0e440", "class_name": "RelatedNodeInfo"}}, "text": "Joshi BK: Rice gene pool for mid and \nhigh hills and its conservation in Nepal. In \nAgricultural Research for Enhancing \nLivelihood of Nepalese People. Proceedings \nof 2nd SAS-N Convention, 30 July - 1 Aug \n2003; Kathmandu. edited by Joshi BK, Joshi \nSL, Paudyal KP2004:252-264. \n\n2. Joshi BK: Rice gene pool for Tarai and \nInner Tarai areas of Nepal. Nepal Agric. \nRes. J. 2005, 6:10-23. \n\n3. Williams JGK, Kubelik AR, Livak KJ, \nRafalski JA, Tingey SV: DNA \npolymorophisms amplified by arbitrary \nprimers are useful as genetic markers. \nNucleic Acids Res. 1990, 18:6531-6535. \n\n4. Fuentes JL, Escobar F, Alvarez A, et al. \nAnalysis of genetic diversity in Cuban rice \nvarieties using isozyme, RAPD and AFLP \nmarkers. Euphytica 1999, 109:107-115. \n\n5. Mackill DJ: Classifying Japonica rice \ncultivars with RAPD markers. Crop Sci. \n1995, 35:889-894. \n\n6. Virk PS, Newbury HJ, Jackson MT, Pord-\nLloyd BV: The identification of duplicate \naccessions within a rice germplasm \ncollection using RAPD analysis. Theor. \nAppl. Genet. 1995, 90:109-155. \n\n7. Karp A, Kresovich S, Bhat KV, Ayad W, \nHodgkin T: Molecular tools in plant genetic \nresources conservation: A guide to the \ntechnologies. IPGRI Technical Bulletin No. \n2. IPGRI, Rome; 1997. \n\n \n\n \n\n \n\n \n\n8. Ren F, Lu BR, Li S, Hunag J, Zhu Y: A \ncomparative study of genetic relationships \namong the AA-genome Oryza species \nusing RAPD and SSR markers. . Theor. \nAppl. Genet. 2003, 108:113-120. \n\n9. Sul IW, Korban SS: A highly efficient \nmethod for isolating genomic DNA from \nplant tissues. Plant Tissue Culture \nBiotechnol. 1996, 2:113-116. \n\n10. Rohlf FJ: NTSYS-pc. Numerical \ntaxonomy and multivariate analysis system. \nVersion 1.8. New York: Exerter Software; \n1993. \n\n11. Ravi M, Geethanjali S, Sameeyafarheen \nF, Maheswaran M: Molecular marker \nbased genetic diversity analysis in rice \n(Oryza sativa L.) using RAPD and SSR \nmarkers. Euphytica 2003, 133:243-252. \n\n12. Ko HL, Cowan DC, Henry RJ, et al. \nRandom amplified polymorphic DNA \nanalysis of Australian rice (Oryza sativa \nL.) varieties. Euphytica 1994, 80:179-189. \n\n13. Morell MK, Peakall R, Appels R, \nPreston LR, Lloyd HL: DNA profiling \ntechniques for plant variety identification. \n. Aust. J. Exp. Agric. 1995, 35:807-819. \n\n14. Choudhury PR, Kohli S, Srinivasan K, \nMohapatra T, Sharma RP: Identification \nand classification of aromatic rice based \non DNA fingerprinting. Euphytica 2001, \n118:243-251.", "start_char_idx": 20564, "end_char_idx": 22987, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9f03331f-804e-4c42-abb2-0e050ae85b39": {"__data__": {"id_": "9f03331f-804e-4c42-abb2-0e050ae85b39", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c42f32b4-f11a-4f18-beae-3d7274c458f1", "node_type": "1", "metadata": {"identifier": "njb-48", "author": "Joshi, Bal K; Bimb, Hari P; Kansakar, David; Ghimire, Ekta", "title": "Genetic Relationship among Nepalese Rice Landraces and Cultivars based on RAPD Markers", "date": "2022-07-11", "file": "njb-48.pdf"}, "hash": "68982066b2fb245bb0204e851e8bfcf0c43df8ceb35d49e668565d5e27727105", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a6af4ec3-4291-4191-8f64-c5749a8e414f", "node_type": "1", "metadata": {}, "hash": "ff21fe7026a72dddf97fec2f4d7a0dabf6e7874b42129566363ec025ea130f0e", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Latest one.doc\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 1 \n\n \n ORIGINAL RESEARCH ARTICLE \n\n \nCloning,\t\n \u00a0Expression,\t\n \u00a0Purification,\t\n \u00a0and\t\n \u00a0Characterization\t\n \u00a0of\t\n \u00a0Clostridium\t\n \u00a0botulinum\t\n \u00a0Neurotoxin\t\n \u00a0\n\nSerotype\t\n \u00a0F\t\n \u00a0Domains\t\n \u00a0\n \n\nSantosh Dulal1*, Bhupal Ban2, Gi Hyoek Yang2, and Hyun Ho Jung1, 2 \n\n \n1Department of Applied Molecular Biology, Sun Moon University, Asan-si, Chung-nam, 336-708, The Republic of Korea \n2Medy-Tox Inc., Cheongwon-gun, Chungbuk-do, 363-883, The Republic of Korea \n*Correspondence Author recent address:  Microbiology Graduate Program, Department of Biology, New Mexico State     \nUniversity, Las Cruces, NM 88003, United States of America.  \nE-mail: santoshd@nmsu.edu \n \n\nAbstract \n\nThe use of recombinant BoNT domains has been proposed as a means to develop strategies to treat \nand prevent botulism. Here, details on the molecular cloning, protein expression, purification, and \nimmunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-\nlength synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and \nreceptor binding) were designed and cloned into Escherichia coli for expression. Recombinant \nproteins were then purified through GST affinity chromatography preceding elution of GST-free \nrecombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light \nchain and translocation N-terminal heavy chain were subsequently used to perform in vivo \nimmunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by \nWestern blot analysis and elevated immunoreactivity was identified through indirect ELISA. In \nconclusion, availability of the recombinant protein provides an effective system to study the \nimmunological aspects of BoNT/F and corresponding applications in pathogen detection and \nvaccine candidacy.  \n \nKeywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant \nprotein expression; immunoreactivity \n\n \n \n\nIntroduction \nBotulinum neurotoxins (BoNTs), the most \n\npotent of all biological substances known to \n\ndate, are produced by several species of the \n\ngenera Clostridia (C. botulinum, C. butyricum, \n\nand C. baratii) [1-3]. Seven immunologically \n\ndistinct botulinum neurotoxin serotypes \n\nBoNTs/A-G are produced and implicated in \n\nbotulism poisoning [4]. Botulism is a serious \n\nneuroparalytic disease [5], which generally \n\noccurs through ingestion of preformed toxin \n\nor rarely, through infection of wounds. The \n\nCenter for Disease Control and Prevention \n\n(CDC) classifies BoNTs, among the six \n\nhighest risk threat agents for bioterrorism \n\n\u201cclass A biological warfare agent\u201d [6, 7]. \n\nDespite their potential to be used for \n\ndeleterious purposes, BoNTs have increasing", "start_char_idx": 49, "end_char_idx": 3254, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a6af4ec3-4291-4191-8f64-c5749a8e414f": {"__data__": {"id_": "a6af4ec3-4291-4191-8f64-c5749a8e414f", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9f03331f-804e-4c42-abb2-0e050ae85b39", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "cd67bb54108c88849336e093c209268fd5b632677b173c573726e41633845a56", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "55b93f37-a118-4038-8c8a-79352676b028", "node_type": "1", "metadata": {}, "hash": "bbd0cf4298cc75436ca3e34a23b1b3160eb891d4589ebe6ea4cc7bb667b7a7bf", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 2 \n\napplications in cosmetics [8] and therapeutics \n\nfor the treatment of numerous dystonias, \n\ninflammation, and chronic pain [9-11]. \n\nBotulinum neurotoxin serotype F (BoNT/F), is \n\na member of the botulinum neurotoxin family \n\nas a single ~150 kDa inactive polypeptide \n\nchain post-translationally nicked, forming a \n\ndichain consisting of a C-terminal ~100 kDa \n\nheavy chain (HC) and a N-terminal ~50 kDa \n\nlight chain (LC) linked by a disulphide bond \n\n[12, 13]. BoNT/F cleaves its substrate vesicle \n\nassociated membrane protein (VAMP) at \n\nposition (Gln58-Lys59) [14, 15], one of three \n\nneuronal proteins associated with exocytosis; \n\nsubsequently inhibiting acetylcholine release, \n\nresulting in death by flaccid paralysis [16, 17]. \n\nEach BoNT/F partial fragment; light chain \n\ncatalytic domain (rF-LC), N-terminal half of \n\nthe heavy chain translocation domain (rF-\n\nHN), the C-terminal half of the heavy chain \n\nreceptor binding domain (rF-HC), and C-\n\nterminal quarter part of the heavy chain \n\nreceptor binding domain (rF-HCc) plays a \n\nspecific role in the toxicity mechanism [18, \n\n19]. Botulinum neurotoxin LCs operate by \n\nzinc dependent proteolysis involved in \n\nneurotransmitter exocytosis from presynaptic \n\ntermini [20]. Botulinum neurotoxin HNs \n\npossesses channel-forming capability in the \n\nacidic environment of the endosome, allowing \n\ninternalization of the toxin, while HCs are \n\ninvolved in specific binding to the presynaptic \n\nmembrane via gangliosides and a protein co-\n\nreceptor [21]. Lastly, the HCc region of \n\nBoNTs are known to harbor the receptor \n\nbinding neutralizing epitopes which are targets \n\nfor antibodies that can specifically bind to the \n\nreceptor, and show neutralizing activity \n\nagainst BoNT toxicity [22]. \n\nThus, it is imperative to construct each \n\ndomain for the analysis of their molecular and \n\nbiochemical activities as well as for the \n\ndevelopment of potential neutralizing \n\nantibodies specific to each. To achieve this \n\ngoal, BoNT/F domains from C. botulinum \n\nwere cloned and expressed using a high \n\nexpression vector and compatible host E. coli \n\nstrains to obtain high quality of recombinant \n\nproteins suitable for administration into mice. \n\n \n\nMethods \nChemicals, Buffers, and Reagents \n\nComponents related to DNA manipulation, \n\nincluding Ex-Taq polymerase, dNTP, and \n\nrestriction enzymes were purchased from \n\nTakara Bio. Inc., (Shiga, Japan). Luria Bertani \n\n(LB) media and cell culture media were \n\npurchased from Becton Dickinson and \n\nCompany (MD, USA) and Hyclone (UT, \n\nUSA) respectively. Additional chemicals \n\nincluding ampicillin, IPTG, Freund\u2019s \n\ncomplete and incomplete adjuvant, and \n\nbuffers were purchased from Sigma Aldrich \n\n(MO, USA).  \n\n \n\nBacterial strains, plasmids, and purification \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.", "start_char_idx": 3259, "end_char_idx": 6515, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "55b93f37-a118-4038-8c8a-79352676b028": {"__data__": {"id_": "55b93f37-a118-4038-8c8a-79352676b028", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a6af4ec3-4291-4191-8f64-c5749a8e414f", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "b6832cf5528d87d0f7bf9bb022425303841714f2242a29caf7c1e2c98e491112", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4440d54-59a8-41f3-a9e4-c7c837139994", "node_type": "1", "metadata": {}, "hash": "d535625efd6144ccf89d0fea520e7f0ef39063c1be9fd1883233c340b6ab0501", "class_name": "RelatedNodeInfo"}}, "text": "Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 3 \n\nsystems \n\nC. botulinum type F strain Langeland [23] and \n\nBoNT/F (Wako Pure Chemicals Ind., Osaka, \n\nJapan) was kindly supplied by Medy-Tox Inc. \n\nPlasmids pGEX-4T-1 (Amersham Pharmacia \n\nBiotech acquired by GE Healthcare, Uppsala, \n\nSweden) and pET-32a(+) (Novagen, EMD \n\nChemicals Inc., affiliate of Merck KGaA, \n\nDarmstadt, Germany) were used for the \n\nconstruction of expression vectors. E. coli \n\nstrains DH5\u03b1 (Takara Bio Inc.), BL21-\n\nCodonPlus-RIL and BL21-CodonPlus(DE3)-\n\nRIL (Stratagene, CA, USA) were used as host \n\nstrains for propagation and expression of \n\nrecombinant proteins, respectively. \n\nGlutathione-S-Transferase (GST) purification \n\nmodules and thrombin protease were \n\npurchased from Amersham Pharmacia \n\nBiotech.  \n\n \n\nConstruction of BoNT/F domains  \n\nChromosomal DNA was isolated from C. \n\nbotulinum F str. Langeland, which was used as \n\nthe template for polymerase chain reaction \n\n(PCR) for all domains (Fig. 1). Forward and \n\nreverse primers for each domain were \n\ndesigned (Table 1) based on the published \n\nNCBI sequence of the Langeland genome \n\n(NC_009699). \n\n \n \n\n \n\n \n\nSynthetic genes encoding BoNT/F LC (light chain), catalytic \n\ndomain (1308 bp); HN (N-terminal 1/2 of heavy chain), \n\ntranslocation domain (1266 bp); HC (C-terminal 1/2 of heavy \n\nchain), receptor binding domain (1263 bp); and HCc (far C-\n\nterminal quarter part of heavy chain), (501 bp) were designed \n\nbased on the published BoNT/F sequence (ORF: 883635-887471) \n\nLangeland str. (NCBI GenBank Accession: NC_009699) for the \n\nplasmid construction of each domain. \n\nbp indicates base pair of DNA. \n\n1kb marker is provided for scale. \n\n \n\nTABLE 1. Primers used for amplification of BoNT/F domains. \n\naPrimer direction: F, forward; R, reverse. \nbDirection of each sequence is in 5' to 3' orientation (underlined) \n\nincluding restriction endonuclease sequence (bold) and inserted \n\nstop codon sequences (blue) for LC and HN reverse primers. \ncNucleotide positions based on published C. botulinum F str. \n\nLangeland genome sequence (NCBI Genbank accession: \n\nNC_009699). \ndRestriction endonucleases utilized to digest PCR amplified \n\ndomain and vector for plasmid construction. \n\n \n\nIsolation of genomic DNA was done utilizing \n\nprotocols and chemicals mentioned by Wizard \n\nGenomic DNA Purification Kit (Promega, WI, \n\nUSA). PCR was performed using a Bio-Rad \n\niCycler (CA, USA).", "start_char_idx": 6511, "end_char_idx": 9269, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4440d54-59a8-41f3-a9e4-c7c837139994": {"__data__": {"id_": "d4440d54-59a8-41f3-a9e4-c7c837139994", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "55b93f37-a118-4038-8c8a-79352676b028", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "6509a40ef006af25cd8051cca46bc0a8d6770953deb4b3d923d415ac899a2b45", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "19563e72-afc7-45a8-95f0-d10428accfc8", "node_type": "1", "metadata": {}, "hash": "aaf58b2b537f5debaf2af842c5230cc4cd399b4327c85cb9e1e5a2ecfb7d4d6f", "class_name": "RelatedNodeInfo"}}, "text": "PCR was performed using a Bio-Rad \n\niCycler (CA, USA). Reaction mixtures were \n\npreheated for 5 min at 94 \u00b0C for initial \n\ndenaturation and then 30 cycles of PCR \n\n(denaturation, annealing, and elongation) were \n\nperformed for amplification of domains as \n\nfollows: 1 min at 94 \u00b0C, 1 min at 57 \u00b0C, and 1 \n\nmin 30 sec at 72 \u00b0C for F-LC; 1 min at 94 \u00b0C, \n\n1 min at 50 \u00b0C, and 1 min 30 sec at 72 \u00b0C for \n\nF-HN and F-HC; 1 min at 94 \u00b0C, 1 min at 54.5 \n\n\u00b0C, and 40 sec at 72 \u00b0C for F-HCc. Following \n\ncompletion of cycles, a final extension was \n\nLC \n\n1 kb \nScale \n\nS      S  \n\nHN HC \n\n1308 bp 1266 bp \n\n1263 bp \n\nHCc \n\n       501 bp   \n\n Native BoNT/F (3837 bp) \n883635                                                                                                              887471     \n\n_____________________________________________________________________________________________________________________________ \nPrimera  Sequence 5' ! 3'b       Nucleotide positionsc Restriction enzymesd  \n_____________________________________________________________________________________________________________________________ \nF-LC-F  CGTGTCGACATGCCAGTTGTAATAAATAGTTTT   883635-883658  SalI        \nF-LC-R  CCGGCGGCCGCTTATTTTCTAGGAATAACGCTCTT  884942-884922  NotI    \n \nF-HN-F  CGTGGATCCGGTACAAAGGCGCCACCGCGACTA  884943-884966  BamHI    \nF-HN-R   CCGCTCGAGTTAATATAAAATTAGAATTTTATC  886209-886188  XhoI     \n \nF-HC-F   CGTGGATCCTTTAATAAATTATATAAAAAAATT  886209-886232  BamHI    \nF-HC-R   CCGCTCGAGTTAGTTTTCTTGCCATCCATGCTC  887471-887448  XhoI    \n \nF-HCc-F CGTGGATCCGATAAGTCTATTACTCAGAATTCA  886971-886994  BamHI    \nF-HCc-R CCGCTCGAGTTAGTTTTCTTGCCATCCATGCTC  887471-887448  XhoI     \n_____________________________________________________________________________________________________________________________\n\n \n\nFig.1: Schematic diagram demonstrating BoNT/F domains for cloning.", "start_char_idx": 9215, "end_char_idx": 11088, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "19563e72-afc7-45a8-95f0-d10428accfc8": {"__data__": {"id_": "19563e72-afc7-45a8-95f0-d10428accfc8", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4440d54-59a8-41f3-a9e4-c7c837139994", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "afe28331a7850adc0bb67551195bd57c31d33fa83b925efe18f8ed3973d119c3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0e7a337b-84fa-40cf-8312-05997c0a309a", "node_type": "1", "metadata": {}, "hash": "7a472deb637d5fbc513ff8e211112b2a649c4cfdd304c9a9d27d85f84585e47e", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 4 \n\ncarried out for an additional 7 min at 72 \u00b0C. \n\nAmplified PCR products were purified by \n\nagarose gel elution kit (Intron Biotechnology, \n\nThe Republic of Korea), and resultant PCR \n\nproducts were digested with restriction \n\nenzymes (Table 1) and subcloned into pGEX-\n\n4T-1 vector using T4 ligase kit (Promega) for \n\novernight at 16 \u00b0C, so that the correct reading \n\nframe was incorporated along the thrombin \n\ncleavage site under the GST gene \n\n(representative constructs made from \n\nMacVector 10.0.2 (NC, USA) software \n\ncontaining F-LC and F-HN domains are \n\nshown in Fig. 2). Ligated samples were \n\ntransformed into DH5\u03b1 by heat-shock (42 \u00b0C \n\nfor 50 sec) method and positive clones with \n\nappropriate insert were screened out by use of \n\nLB agar plates containing 100 \u00b5g/ml \n\nampicillin. Positive colonies containing the \n\nligated constructs were transformed into \n\nBL21-RIL for the expression of type F \n\nrecombinant domains. \n\n \n\n \n\n \n\n \n\n \n \n\n \n\nExpression and purification of \n\nrecombinant proteins \n \n\nTo monitor the induction of recombinant \n\ndomains in BL21-RIL, 2 ml of transformant \n\ncultures were inoculated and grown in LB \n\nbroth and induced at various time intervals \n\nusing different concentrations of isopropyl \n\n\u03b2-D-1-thiogalactopyranoside (IPTG). In \n\norder to purify recombinant proteins, mass \n\nproduction was performed by inoculating 2 \n\nml induced culture in 1 liter followed by \n\ncentrifugation (12,000 xg) at 4 \u00b0C for 20 \n\nmin. Pellet was then resuspended in 10 ml of \n\n50 mM phosphate buffered saline (PBS) and \n\ncells were then lysed by sonication, with 10 \n\nshort bursts of 30 sec followed by intervals \n\nof placing samples on ice for 1 min cooling.  \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n2 (a) 2 (b) \n\nFig.2: Schematic diagram of constructed plasmids generated by MacVector 10.0.2 software (Symantec Corporation). \n\nPlasmids pGEX-4T-1-F-LC (a) and pGEX-4T-1-F-HN (b) encoding catalytic and translocation domains.", "start_char_idx": 11093, "end_char_idx": 13470, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0e7a337b-84fa-40cf-8312-05997c0a309a": {"__data__": {"id_": "0e7a337b-84fa-40cf-8312-05997c0a309a", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "19563e72-afc7-45a8-95f0-d10428accfc8", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "de2a604b7d104f00a7cb941638b0301b74c4ca82e7c2a70c5f526dd864998970", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0b403a57-8c64-4dfe-a1ea-f767ba69db3e", "node_type": "1", "metadata": {}, "hash": "792e2f5adca9fff2420e41d4a4cc642d72bd26181bf1f69e8099556e7a7f163b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 5 \n\nAfter centrifugation at 12,000 xg for 30 min, \n\nthe pellet was resuspended in 10 ml PBS \n\novernight at 4 \u00b0C and supernatant was placed \n\nin a GST affinity chromatography column \n\nafter column was initially washed multiple \n\ntimes with deionized water and PBS, followed \n\nby normalization with GST resin. The large \n\nscale of GST-fusion proteins was eluted by \n\nsingle step affinity chromatography \n\n(containing sepharose 4B beads). Fractions \n\ncontaining desired proteins were pooled and \n\ndialyzed for 2 h at 4 \u00b0C against PBS. \n\nFor thrombin protease treatment, resin bound \n\nGST-fusion proteins were cleaved with 20 \n\nunits thrombin incubating for 30 min at 37 \u00b0C \n\nfollowed by 15 min at room temp (20 - 25 \u00b0C) \n\nand final elution with 500 \u00b5l PBS. The eluted \n\nproteins were further dialyzed to maintain salt \n\nconcentrations and pH. Protein concentrations \n\nwere measured according to the Bradford \n\nmethod using a Bio-Rad model 550 microtiter \n\nplate reader. All proteins were labeled, \n\naliquoted and stored at \u2013 70 \u00b0C prior to use. \n\nLastly, sodium dodecyl sulfate polyacrylamide \n\ngel electrophoresis (SDS-PAGE) was carried \n\nout on a 12% gel under reducing conditions in \n\norder to determine solubility of the eluted \n\nproteins. \n\n \n\nProduction and analysis of polyclonal \n\nantibodies against rF-LC and rF-HN \n\n \n\nFor immunization, three female Balb/c mice \n\n(four weeks old having around 18-22 gram \n\nbody weight) were immunized \n\nintraperitoneally with 10 \u00b5g of antigen \n\nmixtures containing recombinant proteins (25 \n\n\u00b5g GST free F-LC or 25 \u00b5g GST free F-HN) \n\nin PBS. Additionally, an equal volume of \n\nFreund\u2019s complete adjuvant was separately \n\ninjected into each mouse. After two weeks \n\nlapse, the mice were immunized with the \n\nmixture containing 20 \u00b5g of recombinant \n\nprotein and equal volume of Freund\u2019s \n\nincomplete adjuvant. At the start of the fourth \n\nweek following the initial immunization, the \n\nmice were finally boosted by intravenous \n\ninjection of the recombinant proteins (30 \u00b5g) \n\nwithout adjuvant. Three days following final \n\nboost, mice were then bled from the tail and \n\ntested by indirect ELISA.  \n\n \n\nPreparation of monoclonal antibodies \n\n(hybridoma technology) \n\n \n\nCell fusion and culture of hybridomas was \n\ncarried out according to the protocol by \n\nKohler et al. [24]. Briefly, cultures were \n\nincubated at 37 \u00b0C in an incubator with 5% \n\nCO2-in-air and 98% relative humidity. Four \n\ndays following final boost, the mouse showing \n\nthe highest antibody titer by ELISA was \n\nsacrificed by cervical dislocation and its \n\nspleen was removed aseptically. Spleen cells \n\n(1x108 viable cells) were mixed with SP2/0-\n\nAg 14 myeloma cells (1x107viable cells) and", "start_char_idx": 13478, "end_char_idx": 16602, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0b403a57-8c64-4dfe-a1ea-f767ba69db3e": {"__data__": {"id_": "0b403a57-8c64-4dfe-a1ea-f767ba69db3e", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0e7a337b-84fa-40cf-8312-05997c0a309a", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "70cf1b0a1c3d6b46797bd4f6f4d1581e3759d2f2cea9e66e5778dbe98b135b4e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "748823cf-0932-42bf-9159-07378ebbd2c6", "node_type": "1", "metadata": {}, "hash": "efc2c063a3b9f48b944e977e9571111dbf171ae5cc493b0eefb1e6ffdc1f5624", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 6 \n\ngrown in Dulbecco\u2019s modified Eagle\u2019s \n\nmedium (DMEM) supplemented with 10% \n\nfetal bovine serum (FBS) (Hyclone). The \n\naddition of 50% polyethylene glycol 4000 \n\n(PEG) (Sigma Aldrich) allowed for the fusion \n\nof mixed cells; resulting in hybrid cells. \n\nHybrid cells were screened by incubation in \n\n96-well plates (Nunc) containing \n\nhypoxanthine-aminopterin-thymidine (HAT) \n\nselection medium in combination with mouse \n\nfeeder cells for one week. Antibody \n\nproduction from hybrid cells was monitored \n\nthrough indirect ELISA; positive cells were \n\nexpanded into 24-well plates (Nunc) \n\ncontaining hypoxanthine-thymidine (HT) \n\nmedia supplemented with dilute aminopterin \n\naccording to the standard protocol by Harlow \n\nand Lane [25]. ELISA-positive hybridomas \n\nwere selected and cloned twice via limiting \n\ndilution; one cell per well into 96-well plates \n\nsupplemented with HT media and feeder cells. \n\nFinally, cloned cell lines were grown in \n\nDMEM supplemented with 10% FBS and \n\nstored in liquid nitrogen. \n\n \n\nIndirect enzyme linked immunosorbent \n\nassay (ELISA)  \n\n \n\nFor the measurement of serum antibody titers, \n\nELISA was performed as a standard protocol \n\nmentioned by Sigma Aldrich, with only minor \n\nmodifications. Each well of 96 well plates \n\n(Nunc, Copenhagen, Denmark) was coated with \n\nantigen (100 \u00b5l containing 100 ng of either \n\nGST free recombinant protein or 100 ng \n\nBoNT/F) and incubated at 4 \u00b0C overnight. \n\nEach well was washed once with 200 \u00b5l of \n\nPBS containing 0.05% Tween 20 (PBST) and \n\nblocking solution (100 \u00b5l 1% skim milk) was \n\nadded and incubated for 30 min at 37 \u00b0C. \n\nFollowing two more washes, the serum (1: \n\n1000 dilution) in PBS was added as the \n\nprimary antibody. The plates were incubated \n\nat 37 \u00b0C for 90 min, and then washed three \n\ntimes as described above. Goat anti-mouse \n\nIgG conjugated with alkaline phosphatase was \n\nadded as the secondary antibody (diluted \n\n1:2000 with PBS) and incubated for 2 h. After \n\nincubation with IgG, plates were washed four \n\ntimes prior to substrate treatment and \n\nvisualization. Immobilized antigens were \n\nvisualized with the p-Nitrophenyl phosphate \n\n(disodium) in substrate buffer containing 9.7% \n\ndiethanolamine, and 1 mM MgCl2 and the \n\nresultant absorbance was measured at 415 nm \n\nwith microplate reader (Model 550 Bio-Rad) \n\nfor 5 - 10 min at 37 \u00b0C. \n\n \n\nWestern blot analysis \n\n \n\nWestern blot experimentation was adapted \n\nfrom previous methodology [26]. The protein \n\nsamples were separated by SDS-PAGE and \n\nelectroblotted for ~70 min by using 230 mA \n\ncurrent onto a nitrocellulose membrane \n\n(Schleicher and Schuell Inc., NH, USA).", "start_char_idx": 16607, "end_char_idx": 19674, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "748823cf-0932-42bf-9159-07378ebbd2c6": {"__data__": {"id_": "748823cf-0932-42bf-9159-07378ebbd2c6", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0b403a57-8c64-4dfe-a1ea-f767ba69db3e", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "93692aec94bddb2ae7f4f17771bba61cc0be526514b3f5afe7ba7b9f7db8f50d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e6418b2-3ab3-4ce4-8275-6357abacfd6b", "node_type": "1", "metadata": {}, "hash": "523b087eefe31d59a9c7bc1ab5da8d3a5ccd2402e371fdaa6b003db631ceedac", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 7 \n\nSubsequently, the membrane was blocked for \n\nnonspecific binding incubating with blocking \n\nsolution for 1 h and then incubated with \n\nprimary antibody overnight at room temp. The \n\nmembrane was washed two times with PBST \n\nand incubated with alkaline phosphate \n\nconjugated goat anti-mouse IgG as a \n\nsecondary antibody for 2 h. After washing \n\nthree more times with PBST, the membrane \n\nwas color developed by using 10 ml alkaline \n\nphosphate substrate solution, 50 \u00b5l nitro blue \n\ntetrazolium chloride, and 50 \u00b5l 5-bromo-4-\n\nchloro-3-indolyl phosphate. \n\n \n\nResults and Discussion \n\n \nThe cloning of both light and heavy chain \n\ndomains using synthetic genes has been \n\nreported for various BoNTs [27-33]. However, \n\nthere exist few studies on the cloning and \n\nexpression of BoNT/F domains [34, 35]. \n\nConstruction of recombinant plasmids \n\ncontaining pGEX-4T-1-F-LC, -HN, -HC, -\n\nHCc was made for subsequent GST-tagged \n\nexpression in E. coli. Initially, sequences of \n\nBoNT/F DNA domains were PCR amplified \n\nfrom genomic DNA of C. botulinum F str. \n\nLangeland and flanked by restriction sites \n\n(Table 1). Specifically, the amplified products \n\nwere cloned into pGEX-4T-1 after digestion \n\nwith respective restriction enzymes. Ligated \n\nconstructs (for reference see schematic \n\ndiagrams of constructed plasmids made by \n\nMacVector software 10.0.2, Fig. 2) were \n\ntransformed into DH5\u03b1 for propagation and \n\npositive colonies were identified after \n\nscreening on plates containing ampicillin. \n\nSubsequently the recombinant plasmids were \n\ndigested with restriction enzymes (Table 1) \n\nand appropriate band sizes were identified for \n\nboth pGEX-4T-1 (4.9 kb) and all individual \n\nfragment sizes (Fig. 3). For confirmation of \n\ndomain homology incorporated into our \n\nvector, sequencing was performed. After \n\nsequence analysis, plasmid DNA of positive \n\nclones had 100% identity with native \n\nsequences (data not shown).  \n\nFor protein expression, the four recombinant \n\nclones were transformed into E. coli BL21-\n\nCodonPlus-RIL (strain used for protein \n\nexpression with vectors driven by non-T7 \n\npromoters). The BL21-CodonPlus-RIL was \n\nchosen because of its capability to express rare \n\ncodons which allows for the high-level \n\nexpression of recombinants [36]. Thus, BL21-\n\nCodonPlus-RIL was a compatible host strain \n\nfor our pGEX-4T-1 plasmid systems \n\n(containing a tac promoter). All domains were \n\nexpressed in the form of fusion proteins with \n\nGST localized at the N-terminal of the fusion \n\nprotein, enabling the  \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.", "start_char_idx": 19679, "end_char_idx": 22723, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e6418b2-3ab3-4ce4-8275-6357abacfd6b": {"__data__": {"id_": "9e6418b2-3ab3-4ce4-8275-6357abacfd6b", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "748823cf-0932-42bf-9159-07378ebbd2c6", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "44b0faad2e9ecde3f8eaa39d4caad17c6a5f084abdf5690fd3ecd8ea59c1e330", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "af54b7d4-adb3-45e2-9fe9-730099cb351b", "node_type": "1", "metadata": {}, "hash": "230612295aa45d1cce381e9a3a755b801735b6dd0f01c9d3eea7c1be092b7341", "class_name": "RelatedNodeInfo"}}, "text": "2012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 8 \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n (i) Visible bands of amplified F-LC product (3a), F-HN (3b), F-HC (3c), and F-HCc (3d); (ii) Endonuclease restricted bands of purified pGEX-\n\n4T-1-F-LC with SalI/NotI (3a), pGEX-4T-1-F-HN (3b), pGEX-4T-1-F-HC (3c), and pGEX-4T-1-F-HCc (3d) with BamI/XhoI. \n\nM denotes DNA marker and Lane 1, DNA sample loaded. \n \n\nease of purification using GST affinity \n\nchromatography. Expression of clostridial \n\nproteins at 37 \u00b0C has been shown to increase \n\nprotein degradation [37], thus we chose an \n\noptimal temperature range of 20 - 25 \u00b0C for our \n\nexpression studies. Conditions for expression \n\nof recombinant proteins were as follows: 7 h at \n\n24 \u00b0C using 0.5 mM IPTG concentration for \n\nGST-F-LC, 8 h at 22 \u00b0C using 0.4 mM IPTG \n\nconcentration for GST-F-HN, 8 h at 20 \u00b0C \n\nusing 0.25 mM IPTG concentration for GST-F-\n\nHC, and 8 h at 20 \u00b0C using 0.10 mM IPTG \n\nconcentration for GST-F-HCc. GST-F-LC and \n\nGST-F-HN induction was monitored by SDS-\n\nPAGE (Fig. 4) and were found to be highly \n\nover-expressed in soluble form and purified at \n\nhigh concentrations.  \n\nDespite induction at low temperature and IPTG \n\nconcentrations, GST-F-HC and GST-F-HCc \n\nwere expressed in inclusion bodies. Change in \n\nexpression vectors from pGEX-4T-1 to pET-\n\n32a(+) was then carried out in attempt to obtain \n\nsoluble forms of GST-F-HC and GST-F-HCc. \n\npET-32a(+) was subsequently transformed into \n\nBL21-CodonPlus(DE3)-RIL host cells, that \n\nutilizes the T7 RNA polymerase promoter.", "start_char_idx": 22697, "end_char_idx": 24567, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "af54b7d4-adb3-45e2-9fe9-730099cb351b": {"__data__": {"id_": "af54b7d4-adb3-45e2-9fe9-730099cb351b", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e6418b2-3ab3-4ce4-8275-6357abacfd6b", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "c3fcf7e8a8a40432a308d54eee094efb51812e78f0f69028651004cc9bde2cf7", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ad53c634-48b4-4ae8-bc44-9512201e8656", "node_type": "1", "metadata": {}, "hash": "fdf495893d401ca2bdf83c5e822f82d74dded65e5595d658d327ee6fcdd3a242", "class_name": "RelatedNodeInfo"}}, "text": "M    1 M   1 \n\nInsert  \nF-LC  \n(1308 bp) \n\nVector  \npGEX-4T-1  \n(4.9 kb) \n\nbp \n\n300 \n400 \n\n850 \n650 \n\n1650 \n\n3000 \n\n2000 \n\n4000 \n5000 \n\n500 \n\n1000 \n\n100 \n\n400 \n500 \n\n3000 \n\n2000 \n\n5000 \n\n1000 \n\n300 \n200 \n\n650 \n850 \n\n1650 \n\n4000 \n\nAmplified  \nF-LC \nPCR product \n(1308 bp) \n\na (i)  \n\nbp \n\na (ii)  \n\nAmplified  \nF-HN \nPCR product \n(1266 bp) \n\n3000 \n\nM    1 \n\n500 \n\n1000 \n\n850 \n\n650 \n\n1650 \n2000 \n\nbp \n\nb (i)  \n\nM    1 \n\nInsert  \nF-HN  \n(1266 bp) \n\n500 \n650 \n\n1000 \n850 \n\n1650 \n\n3000 \n\n2000 \n\n4000 \n5000 \nbp Vector  \n\npGEX-4T-1  \n(4.9 kb) \n\nb (ii)  \n\nM     1 \n\nAmplified  \nF-HC \nPCR product \n(1263 bp) \n\nbp \n\n100 \n\n400 \n500 \n\n3000 \n\n2000 \n\n5000 \n\n1000 \n\n300 \n200 \n\n650 \n850 \n\n1650 \n\n4000 \n\nc (i)  \n\nM     1 \n\nInsert  \nF-HC  \n(1263 bp) \n\nVector  \npGEX-4T-1  \n(4.9 kb) \n\n300 \n400 \n\n850 \n650 \n\n1650 \n\n3000 \n\n2000 \n\n4000 \n5000 \n\n500 \n\n1000 \n\nbp \n\nc (ii)  \n\n2000 \n\n1000 \n\n1650 \n\n4000 \n\nM     1 \n\nAmplified  \nF-HCc \nPCR product \n(501 bp) \n\nbp \n\n100 \n\n400 \n500 \n\n300 \n\n200 \n\n650 \n850 \n\nd (i)  \n\nM     1 \n\nInsert  \nF-HCc  \n(501 bp) \n\nVector  \npGEX-4T-1  \n(4.9 kb) \n\nd (ii)  \n\n2000 \n\n1000 \n\n1650 \n\n4000 \nbp \n\n100 \n\n400 \n500 \n\n300 \n\n200 \n\n650 \n850 \n\nFig.3: Agarose gel (1%) electrophoresis of PCR product and restriction digested plasmid stained with ethidium bromide and visualized by UV", "start_char_idx": 24573, "end_char_idx": 25847, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ad53c634-48b4-4ae8-bc44-9512201e8656": {"__data__": {"id_": "ad53c634-48b4-4ae8-bc44-9512201e8656", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "af54b7d4-adb3-45e2-9fe9-730099cb351b", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "e1a693efb77d8ceea8a38c6ad988f40585e448f001d762d597804c7d236a4d46", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "669dcdae-33ad-4d71-a7ab-a23cefba7745", "node_type": "1", "metadata": {}, "hash": "52bacdf0f5d77dacbaa709bd64fe85c9d36d668c6141f6de1ac15775d7f80099", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 9 \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n \n\n \n\n \n\n \n\n \n\n \n\n \nIPTG induction was carried out in a time dependent manner and SDS\u2013PAGE (12 %) analysis of total lysate of E. coli BL21- (pGEX-4T-1-F-LC \n\n(a) and pGEX-4T-1-F-HN (b) with or without IPTG induction stained with Coomassie blue.  \n\nLane M: protein marker (Bio-Rad); Lanes 1 - 4: (0, 2, 5, and 7 h for F-LC and 0, 2, 5, and 8 h for F-HN) after IPTG induction. Lanes 2 - 4 \n\ndemonstrate expressed GST-F-LC a) and GST-F-HN (b) with treatment of IPTG. \n\nkDa, protein size expressed in kiloDalton. \n\n \n\nAlthough both vectors contain thrombin \n\ncleavage sites and are ideal for production of \n\nsoluble proteins, neither one was able to alter \n\nthe formation of inclusion bodies. Protein-\n\nrefolding procedures were performed using \n\nreducing reagents including various molar \n\nconcentrations of urea or guanidine \u2013 HCl to \n\nsolubilize the inclusion bodies but attempts \n\nwere unsuccessful. As a result, only LC and \n\nHN domains were used for protein analysis \n\nand collection of polyclonal serum from \n\nimmunized mice. GST affinity \n\nchromatography was then carried out to \n\ncollect GST tagged LC and HN. After GST \n\naffinity chromatography and cleavage with \n\nthrombin protease treatment, recombinant \n\nGST free F-LC and GST free F-HN were \n\nanalyzed on SDS-PAGE to monitor expected \n\nprotein bands. SDS-PAGE revealed the \n\nappropriate size of both recombinants; GST \n\nfused 75 kDa and GST free 50 kDa (Fig. 5). \n\nFinal recombinant protein concentrations were \n\ndetermined to be 1.0 mg/l. In order to analyze \n\nspecificity of mice polyclonal sera against F-\n\nLC and F-HN, mice were immunized \n\nintraperitoneally with antigens; GST free F-\n\nLC and F-HN. To analyze the specificity of \n\nthe polyclonal serums against F-LC and F-\n\nHN, mouse polyclonal serum was collected \n\nfrom the tail vain and tested by Western \n\nblotting and indirect ELISA (both of these \n\ntechniques being highly valuable in the \n\ndetection of candidate biomarkers). The \n\nmouse anti-F-LC polyclonal antibodies were \n\nfound to specifically recognize F-LC and \n\nBoNT/F (Figs. 5a and 6a). Similarly, anti-F-\n\nHN polyclonal antibodies demonstrated a \n\nM   1      2     3      4       kDa \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n250 \n\n15 \n\n10 \n\nM   1      2     3      4       kDa \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n250 \n\n15 \n\nGST-F-LC \nGST-F-HN \n\nSDS-PAGE (12%)  \n   4 (a)   \n\nSDS-PAGE (12%)  \n  4 (b)   \n\nFig.4: Induction and expression analysis of recombinant proteins", "start_char_idx": 25853, "end_char_idx": 28744, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "669dcdae-33ad-4d71-a7ab-a23cefba7745": {"__data__": {"id_": "669dcdae-33ad-4d71-a7ab-a23cefba7745", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ad53c634-48b4-4ae8-bc44-9512201e8656", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "3e69d87dd705ac350be411be1a045f38932836a299730332a32b0aeca6a65ec2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b401a904-3c3c-473b-b180-54f55db504e3", "node_type": "1", "metadata": {}, "hash": "805230a734652c670e8bb0547208dfd68e82310b46f9c00842e8f4c7ce852c96", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 10 \n\nstrong affinity towards F-HN and native type \n\nF toxin (Figs. 5b and 6b). However, neither \n\nanti-F-LC nor anti-F-HN polyclonal \n\nantibodies were able to detect the HC domain \n\n(Fig. 5), validating the specificity of the \n\npolyclonal serums towards their respective \n\nregion of BoNT/F. The designated \n\nrecombinant domains have native structure as \n\nholotoxin and it was not unexpected for them \n\nto produce anti-sera against native toxin \n\n(BoNT/F). \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\nMouse showing highest immunoreactivity \n\nwas then sacrificed in attempt to isolate a \n\nmonoclonal antibody specific to F-LC or F-\n\nHN domains. This could allow further \n\nresearch into the development of a highly \n\neffective neutralizing monoclonal antibody \n\nfor a subunit vaccine and/or passive \n\nimmunotherapy against BoNT/F. \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n (i) SDS\u2013PAGE of purified GST F-LC and GST free F-LC (a) Purified GST F-HN and GST free F-HN (b) by single step GST affinity \n\nchromatography separated on a 12% gel and visualized by Coomassie blue stain; (ii) Western blot analysis performed by raising anti-F-LC (a) \n\nand anti-F-HN (b) mouse polyclonal antibody. \n\nLane M: protein marker (Bio-Rad); Lane 1, GST-F-LC or HN; Lane 2, GST free-F-LC or HN; and Lane 3, unpurified GST-F-HC. \n\nFig.5: Purification and characterization of recombinant BoNT/F domain proteins \n\nSDS-PAGE \n5b (i)  \n\nM     1        2      3       M    1       2        3       kDa \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n250 \n\nSDS-PAGE \n5a (i)  \n\nkDa \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n250 \n\nWestern blot \n5a (ii)  \n\nGST-F-LC \n\nGST free-F-LC \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n\n250 \n\nM     1        2      3       M    1       2        3       kDa kDa \n\nWestern blot \n5b (ii)  \n\n25 \n\n75 \n\n50 \n\n37 \n\n100 \n150 \n\n250 \n\nGST-F-HN \n\nGST free-F-HN", "start_char_idx": 28749, "end_char_idx": 31019, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b401a904-3c3c-473b-b180-54f55db504e3": {"__data__": {"id_": "b401a904-3c3c-473b-b180-54f55db504e3", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "669dcdae-33ad-4d71-a7ab-a23cefba7745", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "f3a1e63b90d6ed0ab8e0cef14eb88ae73ff537c184a6cc47f9624f1ef8ac34cb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "caed5cf1-3193-4fd4-b11d-498816a8101b", "node_type": "1", "metadata": {}, "hash": "2190378f50dde5375bce6a7fdd92e630bd59710237570f1f2161e7171ac51396", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 11 \n\n \n\n \n\nBar graph values represent interaction of anti F-LC (a) and anti-F-HN (b) mouse polyclonal serum antibody with: PC = BoNT/F as a coating \n\nantigen; S. 1 - 3 = samples of GST free F-LC (a) and GST free F-HN (b) coating antigens respectively & interaction of non-immunized mouse \n\nserum with: NC= GST free F-LC (a) and GST free F-HN (b) used as coating antigens respectively. \nMouse polyclonal serum was harvested from three different mice (from different batches) immunized with GST free F-LC or GST free F-HN. \n\nPC- positive control, NC- negative control. n = 3 wells, two-tailed students t-test, S. 1 - 3 compared to NC, p < 0.05. \n\n \n\nAttempts were unsuccessful in isolating a \n\nmonoclonal antibody specific towards our \n\nconstructed BoNT/F domains (data not \n\nshown). However, we have successfully \n\nisolated a mouse monoclonal antibody that \n\ndemonstrates neutralizing activity against \n\nnative BoNT/F toxin resulting from BoNT/F \n\ntoxiod in vivo immunization and hybridoma \n\ntechnology [38]. \n \n\nConclusion \n\n \nThis paper describes molecular and \n\nimmunological studies on C. botulinum \n\nneurotoxin type F domains. Synthetic genes \n\nencoding BoNT/F partial fragments: catalytic \n\ndomain (rF-LC), translocation domain (rF-\n\nHN), receptor binding domain (rF-HC) and \n\nquarter part of HC (rF-HCc) were designed \n\nand cloned into E. coli. Additionally, \n\nexpression, purification, and \n\nimmunoreactivites were analyzed by Western \n\nblotting and indirect ELISA for LC and HN \n\ndomains. The use of GST tagged recombinant \n\nprotein technology was chosen in order to \n\noptimize detection and acquisition of high \n\npurity, stable, and soluble proteins through use \n\nof single step affinity chromatography. \n\nAlthough host strains, vector types, and \n\ninduction conditions for expression were \n\noptimized to recover recombinant proteins in \n\nthe soluble fraction, we were unable to purify \n\nHC and HCc. Immunoreactivity was accessed \n\nthrough GST free F-LC and F-HN through in \n\nvivo immunization and polyclonal serum \n\nantibody collection, proceeded by Western \n\nblotting and indirect ELISA.  \n\nThe anti-F-LC and anti-F-HN mouse \n\n6 (a)  6 (b)  \n\nFig.6:Immunoreactivity analysis by Indirect ELISA", "start_char_idx": 31024, "end_char_idx": 33648, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "caed5cf1-3193-4fd4-b11d-498816a8101b": {"__data__": {"id_": "caed5cf1-3193-4fd4-b11d-498816a8101b", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b401a904-3c3c-473b-b180-54f55db504e3", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "4fd26e154f9987161fbff9be1346030ab0bd8131d6313d8bd5e2fdff8e469af8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d01606d9-5c4e-4af6-9670-ff47a14eb5c2", "node_type": "1", "metadata": {}, "hash": "d256e2db5cc1e940b2833eddc78a8571e2a35ce766893d7d561dc5a28dc8b10f", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 12 \n\npolyclonal antibodies exhibited a strong \n\naffinity towards GST free and GST tagged F-\n\nLC and F-HN respectively, while both \n\nrecognized the native type F toxin. From this \n\nwork, we conclude that purified BoNT/F LC \n\nand HN domains are capable of producing \n\nhighly effective immunogens. Moreover, if \n\nfuture attempts prove fruitful in the isolation \n\nof monoclonal antibodies (that possess \n\nneutralizing activity), towards BoNT/F \n\nfragments, this may lead to more efficient \n\nprotection against a high neurotoxin dose. \n\nImplications of this research could span across \n\nthe development of therapeutic approaches, \n\ndiagnostic detection systems, and vaccine \n\ncandidacy for the protection and treatment of \n\nbotulism as has been elucidated in previous \n\nwork [34, 37, 39-42]. In summary, we have \n\nexpressed both recombinant LC and HN \n\ndomains of BoNT/F. The recombinant \n\nproteins are soluble; elicit an active immune \n\nresponse (polyclonal antibodies) in mice, \n\nmaking them ideal for investigators to process \n\npotential subunit vaccines towards BoNT/F. \n\n \n\nAcknowledgements \n\n \nAll authors wish to acknowledge the financial \n\nsupport from Medy-Tox Inc. and for their help \n\nin the acquisition of C. botulinum F str. \n\nLangeland and BoNT/F (Wako Pure \n\nChemicals Ind., Osaka, Japan). We would also \n\nlike to express our gratitude to Jesus Andres \n\nCuaron for his contributions on the drafting of \n\nthis article.  \n\n \n\nReferences \n\n \n1. McCroskey LM, Hatheway CL, \n\nFenicia L, Pasolini B, Aureli P: \nCharacterization of an organism \nthat produces type E botulinal \ntoxin but which resembles C. \nbutyricum from the feces of an \ninfant with type E botulism. J Clin \nMicrobiol 1986, 23(1):201-202. \n\n \n2. McCroskey LM, Hatheway CL, \n\nWoodruff BA, Greenberg JA, \nJurgenson P: Type F botulism due \nto neurotoxigenic C. baratii from \nan unknown source in an adult. J \nClin Microbiol 1991, 29(11):2618-\n2620. \n\n \n3. Sakaguchi G: C. botulinum toxins. \n\nPharmacol Ther 1982, 19(2):165-\n194. \n\n \n4. Simpson LL (Ed): Botulinum \n\nNeurotoxin and Tetanus Toxin. \nSan Diego, CA: Academic Press; \n1989. \n\n \n5. Burgen AS, Dickens F, Zatman LJ: \n\nThe action of botulinum toxin on \nthe neuro-muscular junction. J \nPhysiol 1949, 109(1-2):10-24. \n\n \n6. Atlas RM: Bioterrorism and \n\nbiodefence research: changing the \nfocus of microbiology. Nat Rev \nMicrobiol 2003, 1(1):70-74. \n\n \n7. Bossi P, Bricaire F: [Botulism toxin, \n\nbioterrorist weapon]. Presse Med \n2003, 32(10):463-465.", "start_char_idx": 33653, "end_char_idx": 36547, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d01606d9-5c4e-4af6-9670-ff47a14eb5c2": {"__data__": {"id_": "d01606d9-5c4e-4af6-9670-ff47a14eb5c2", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "caed5cf1-3193-4fd4-b11d-498816a8101b", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "82f324a1dfc5f7a3a1747b55301be9d2067598d19837962f3cf9d315ef36b705", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0e5acaf6-e7ad-45f5-afb2-3c72ddf57996", "node_type": "1", "metadata": {}, "hash": "c2c85b8a7a97b74b0d612bdfae945a3780b40cd3040ea5ca6902f5617cd759a1", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 13 \n\n \n8. Moore A: The biochemistry of \n\nbeauty. The science and pseudo-\nscience of beautiful skin. EMBO \nRep 2002, 3(8):714-717. \n\n \n9. Brin MF: Botulinum toxin: \n\nchemistry, pharmacology, toxicity, \nand immunology. Muscle Nerve \nSuppl 1997, 6:S146-168. \n\n \n10. Dressler D: Electromyographic \n\nevaluation of cervical dystonia for \nplanning of botulinum toxin \ntherapy. Eur J Neurol 2000, \n7(6):713-718. \n\n \n11. Jankovic J: Botulinum toxin in \n\nmovement disorders. Curr Opin \nNeurol 1994, 7(4):358-366. \n\n \n12. Sugiyama H, Das Gupta R, Yang \n\nKH: Disulfide-toxicity relationship \nof botulinal toxin types A, E, and \nF. Proc Soc Exp Biol Med 1973, \n143(3):589-591. \n\n \n13. DasGupta BR, Sugiyama H: A \n\ncommon subunit structure in C. \nbotulinum type A, B and E toxins. \nBiochem Biophys Res Commun \n1972, 48(1):108-112. \n\n \n14. Schmidt JJ, Stafford RG: Botulinum \n\nneurotoxin serotype F: \nidentification of substrate \nrecognition requirements and \ndevelopment of inhibitors with low \nnanomolar affinity. Biochemistry \n2005, 44(10):4067-4073. \n\n \n15. Schiavo G, Rossetto O, Catsicas S, \n\nPolverino de Laureto P, DasGupta \nBR, Benfenati F, Montecucco C: \nIdentification of the nerve terminal \ntargets of botulinum neurotoxin \nserotypes A, D, and E. J Biol Chem \n1993, 268(32):23784-23787. \n\n16. Chen S, Wan HY: Molecular \n\nmechanisms of substrate \nrecognition and specificity of \nbotulinum neurotoxin serotype F. \nBiochem J 2011, 433(2):277-284. \n\n \n17. Agarwal R, Schmidt JJ, Stafford RG, \n\nSwaminathan S: Mode of VAMP \nsubstrate recognition and \ninhibition of C. botulinum \nneurotoxin F. Nat Struct Mol Biol \n2009, 16(7):789-794. \n\n \n18. Montecucco C, Schiavo G: \n\nMechanism of action of tetanus \nand botulinum neurotoxins. Mol \nMicrobiol 1994, 13(1):1-8. \n\n \n19. Schiavo G, Matteoli M, Montecucco \n\nC: Neurotoxins affecting \nneuroexocytosis. Physiol Rev 2000, \n80(2):717-766. \n\n \n20. Lacy DB, Tepp W, Cohen AC, \n\nDasGupta BR, Stevens RC: Crystal \nstructure of botulinum neurotoxin \ntype A and implications for \ntoxicity.", "start_char_idx": 36552, "end_char_idx": 38991, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0e5acaf6-e7ad-45f5-afb2-3c72ddf57996": {"__data__": {"id_": "0e5acaf6-e7ad-45f5-afb2-3c72ddf57996", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d01606d9-5c4e-4af6-9670-ff47a14eb5c2", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "abc856365a1091c1a9baddd7bfd8d67993afc3c398de5fd9c0f1ab7af5864f76", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "57ef0396-198e-4926-bb08-8602acbff515", "node_type": "1", "metadata": {}, "hash": "d1d886cd02af612ea32d2bd368ad289eb6819fcceac13dbb20e653d0db57df7a", "class_name": "RelatedNodeInfo"}}, "text": "Nat Struct Biol 1998, \n5(10):898-902. \n\n \n21. Montecucco C, Schiavo G, Gao Z, \n\nBauerlein E, Boquet P, DasGupta \nBR: Interaction of botulinum and \ntetanus toxins with the lipid \nbilayer surface. Biochem J 1988, \n251(2):379-383. \n\n \n22. Yu YZ, Sun ZW, Wang S, Yu WY: \n\nHigh-level expression of the Hcc \ndomain of C. botulinum neurotoxin \nserotype A in E. coli and its \nimmunogenicity as an antigen. \nSheng Wu Gong Cheng Xue Bao \n2007, 23(5):812-817. \n\n \n23. Moller V, Scheibel I: Preliminary \n\nreport on the isolation of an \napparently new type of C. \nbotulinum. Acta Pathol Microbiol \nScand 1960, 48:80. \n\n\n\nNepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 14 \n\n \n24. Kohler G, Howe SC, Milstein C: \n\nFusion between immunoglobulin-\nsecreting and nonsecreting \nmyeloma cell lines. Eur J Immunol \n1976, 6(4):292-295. \n\n \n25. Harlow E, Lane D: Antibodies: A \n\nLaboratory Manual: Cold Spring \nHarbor Laboratory Press; 1988. \n\n \n26. Towbin H, Staehelin T, Gordon J: \n\nElectrophoretic transfer of \nproteins from polyacrylamide gels \nto nitrocellulose sheets: procedure \nand some applications. Proc Natl \nAcad Sci U S A 1979, 76(9):4350-\n4354. \n\n \n27. Agarwal R, Eswaramoorthy S, \n\nKumaran D, Dunn JJ, Swaminathan \nS: Cloning, high level expression, \npurification, and crystallization of \nthe full length C. botulinum \nneurotoxin type E light chain. \nProtein Expr Purif 2004, 34(1):95-\n102. \n\n \n28. Zhou Y, Singh BR: Cloning, high-\n\nlevel expression, single-step \npurification, and binding activity \nof His6-tagged recombinant type B \nbotulinum neurotoxin heavy chain \ntransmembrane and binding \ndomain. Protein Expr Purif 2004, \n34(1):8-16. \n\n \n29. Sharma S, Zhou Y, Singh BR: \n\nCloning, expression, and \npurification of C-terminal quarter \nof the heavy chain of botulinum \nneurotoxin type A. Protein Expr \nPurif 2006, 45(2):288-295. \n\n \n30. Mansour AA, Mousavi SL, Rasooli \n\nI, Nazarian S, Amani J, Farhadi N: \nCloning, high level expression and \nimmunogenicity of 1163-1256 \nresidues of C-terminal heavy chain \n\nof C. botulinum neurotoxin type E. \nBiologicals 2010, 38(2):260-264. \n\n \n31.", "start_char_idx": 38992, "end_char_idx": 41448, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "57ef0396-198e-4926-bb08-8602acbff515": {"__data__": {"id_": "57ef0396-198e-4926-bb08-8602acbff515", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0e5acaf6-e7ad-45f5-afb2-3c72ddf57996", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "7e12d52143bb3470e379be0475ec1a84524fe87dc6f84f42bfcfbe29b8ccb54f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "65c1ba99-a395-49bb-99f9-59ff78620eb7", "node_type": "1", "metadata": {}, "hash": "7fa0452075d44a18e9f36341aa3ee6d19237510bc0033e03828d87c7a4f4294c", "class_name": "RelatedNodeInfo"}}, "text": "31. Whelan SM, Elmore MJ, Bodsworth \n\nNJ, Brehm JK, Atkinson T, Minton \nNP: Molecular cloning of the C. \nbotulinum structural gene \nencoding the type B neurotoxin \nand determination of its entire \nnucleotide sequence. Appl Environ \nMicrobiol 1992, 58(8):2345-2354. \n\n \n32. Yang GH, Rhee SD, Jung HH, Jhee \n\nOH, Yang KH: Cloning and \ncharacterization of the upstream \nregion of C. botulinum type B \nneurotoxin gene. Biochem Mol Biol \nInt 1998, 45(2):401-407. \n\n \n33. Zhou Y, Paturi S, Lindo P, \n\nShoesmith SM, Singh BR: Cloning, \nexpression, purification, and \ncharacterization of biologically \nactive recombinant hemagglutinin-\n33, type A botulinum neurotoxin \nassociated protein. Protein J 2007, \n26(1):29-37. \n\n \n34. Yu YZ, Li N, Wang RL, Zhu HQ, \n\nWang S, Yu WY, Sun ZW: \nEvaluation of a recombinant Hc of \nC. botulinum neurotoxin serotype \nF as an effective subunit vaccine. \nClin Vaccine Immunol 2008, \n15(12):1819-1823. \n\n \n35. Agarwal R, Binz T, Swaminathan S: \n\nStructural analysis of botulinum \nneurotoxin serotype F light chain: \nimplications on substrate binding \nand inhibitor design. Biochemistry \n2005, 44(35):11758-11765. \n\n \n36. Zdanovsky AG, Zdanovskaia MV: \n\nSimple and efficient method for \nheterologous expression of \nclostridial proteins. Appl Environ \nMicrobiol 2000, 66(8):3166-3173.", "start_char_idx": 41445, "end_char_idx": 42751, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "65c1ba99-a395-49bb-99f9-59ff78620eb7": {"__data__": {"id_": "65c1ba99-a395-49bb-99f9-59ff78620eb7", "embedding": null, "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-49", "node_type": "4", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "64132c487b0d7c8f23e5ef3371e5c03f2cdad12f3d563a1ade488ea171a75692", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "57ef0396-198e-4926-bb08-8602acbff515", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "c85c19fab9327e354c7e42f0b529ab25ed417913d54ecf595d4cc01a03e58f59", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "30d36423-0594-4220-88c7-572628b25ee3", "node_type": "1", "metadata": {}, "hash": "c9c16969e3ed2216b7f66bf950184079ab886f5224de78ce298fed58d4a32c12", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\t\n \u00a0Journal\t\n \u00a0of\t\n \u00a0Biotechnology.\t\n \u00a0\t\n \u00a0Jan.\t\n \u00a02012,\t\n \u00a0Vol.\t\n \u00a02,\t\n \u00a0No.\t\n \u00a01:\t\n \u00a01\t\n \u00a0\u2013\t\n \u00a015\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0\t\n \u00a0Biotechnology\t\n \u00a0Society\t\n \u00a0of\t\n \u00a0Nepal\t\n \u00a0(BSN),\t\n \u00a0All\t\n \u00a0rights\t\n \u00a0reserved\t\n \u00a0\n\n 15 \n\n37. Woodward LA, Arimitsu H, Hirst R, \nOguma K: Expression of HC \nsubunits from C. botulinum types \nC and D and their evaluation as \ncandidate vaccine antigens in mice. \nInfect Immun 2003, 71(5):2941-\n2944. \n\n \n38. Dulal S, Ban B, Kim KS, Yang GH, \n\nJung HH. : Neutralizing antibody \nspecific to C. botulinum neurotoxin \nserotype F (BoNT/F). Abstracts of \nthe 110th General Meeting of the \nAmerican Society for Microbiology \n(American Society for Microbiology, \nSan Diego , CA, 23rd - 27th May) , p \n110 2010. \n\n \n39. Yang GH, Kim KS, Kim HW, Jeong \n\nST, Huh GH, Kim JC, Jung HH: \nIsolation and characterization of a \nneutralizing antibody specific to \ninternalization domain of C. \nbotulinum neurotoxin type B. \nToxicon 2004, 44(1):19-25. \n\n \n40. Baldwin MR, Tepp WH, Przedpelski \n\nA, Pier CL, Bradshaw M, Johnson \nEA, Barbieri JT: Subunit vaccine \nagainst the seven serotypes of \nbotulism. Infect Immun 2008, \n76(3):1314-1318. \n\n \n41. Webb RP, Smith TJ, Wright PM, \n\nMontgomery VA, Meagher MM, \nSmith LA: Protection with \nrecombinant C. botulinum C1 and \nD binding domain subunit (Hc) \nvaccines against C and D \nneurotoxins. Vaccine 2007, \n25(21):4273-4282. \n\n \n42. Webb RP, Smith TJ, Wright P, \n\nBrown J, Smith LA: Production of \ncatalytically inactive BoNT/A1 \nholoprotein and comparison with \nBoNT/A1 subunit vaccines against \ntoxin subtypes A1, A2, and A3. \nVaccine 2009, 27(33):4490-4497.", "start_char_idx": 42759, "end_char_idx": 44580, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "30d36423-0594-4220-88c7-572628b25ee3": {"__data__": {"id_": "30d36423-0594-4220-88c7-572628b25ee3", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "65c1ba99-a395-49bb-99f9-59ff78620eb7", "node_type": "1", "metadata": {"identifier": "njb-49", "author": "Dulal, Santosh; Ban, Bhupal; Yang, Gi Hyoek; Jung, Hyun Ho", "title": "Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains", "date": "2022-07-11", "file": "njb-49.pdf"}, "hash": "6fb5a7a4a92a69a6821c1218c25d1606607d9a69c4f047d8ed9f4820a1a5765c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7261d17f-115e-4a54-89be-cb72b215ee11", "node_type": "1", "metadata": {}, "hash": "8f461238668fce68c0f13c0a6204d88ba8b61b40d8aea97a403808b9c9d5a60b", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 1  D e c . 9 (2):14-20       Research article  DOI: https://www.doi.org/10.54796/njb.v9i2.41909 \n  \n\n  \n\n\u00a9NJB, BSN   14 \n\nPhytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and \nMature Leaves of Cinnamomum tamala  \nRasna Maharjan1 , Prakash Thapa1 , Karan Khadayat 1, 2  , Surya Kant Kalauni1    \n1Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal \n 2Department of Biotechnology, National College, Tribhuvan University, Naya Bazar, Kathmandu, Nepal \n\nReceived: 5th Nov 2020; Revised: 22nd Sep 2021; Accepted: 6th Dec 2021; Published online: 31st Dec 2021 \n\nAbstract \nThe bioactive chemical components of the plant's origin have been used as primary remedies for a wide array of human diseases \nincluding diabetes. The present research deal to evaluate and compare anti-diabetic potential of ethanolic and methanolic, \nyoung and mature leaves of medicinally valuable Cinnamomum tamala. Total phenolic and flavonoid contents of young and \nmature leaves were determined. In vitro \u03b1-amylase inhibition was carried out using 2-chloro-4-nitrophenyl-\u03b1-D-maltotrioside \n(CNPG3) as substrate. Phytochemical screening revealed the presence of polyphenols, flavonoids, terpenoids, quinones, \ncarbohydrates, glycosides, diterpenes, tannins, and reducing sugars. The highest total phenolic content and flavonoid content \nwere observed in methanolic extract of mature leaves (13.725 \u00b1 0.54 mg GAE/g) and young leaves (12.591 \u00b1 0.71 mg QE/g) \nrespectively. Methanolic young leaves extract showed \u03b1-amylase inhibition with IC50 value 224.6 \u00b1 2.76 \u03bcg/mL as compared to \nacarbose with IC50 value 5.93 \u00b1 0.14 \u03bcg/mL. The result suggests that young leaves of C. tamala had anti-diabetic activity so \nfurther work should be carried out.  \n\nKeywords: \u03b1-Amylase, Anti-diabetic, Cinnamomum tamala, Phytochemicals  \n\n Corresponding author, email: karankhadayat55@gmail.com; skkalauni@gmail.com \n\nIntroduction \nDiabetes mellitus is one of the most common health \n\nissues among the serious health concerns around the \n\nworld. It is a chronic multifactorial endocrine disorder of \n\nglucose intolerance. Approximately 463 million \n\nadults (20-79 years) suffered from diabetes worldwide in \n\n2019 and may escalate to 700 million by 2045. In Nepal, \n\nabout 696,900 adults are living with diabetes [1]. Among \n\nthree type of diabetes, Type 1 diabetes occur due to \n\ninsulin deficiency, type 2 diabetes occur due to inability \n\nto utilize insulin and gestational diabetes occur during \n\npregnancy condition [2]. Among them, diabetes of type 2 \n\nis the most severe and fast-growing in most countries \n\nmajorly due to rapid urbanization, diet, lifestyles [3,4]. In \n\nhyperglycemia, the rise in blood glucose levels generates \n\nexcessive superoxide anions which produce hydroxyl \n\nradicals through Haber Weiss reaction that can lead to the \n\nperoxidation of membrane lipids, oxidative protein \n\ndamage to cell membranes, and also affects other \n\nbiomolecules including carbohydrates, proteins, and \n\nDNA [5]. Consequently, long-term diabetes leads to \n\nstroke, blindness, heart attack, kidney failure, \n\namputation, and the menace of dying prematurely [2].", "start_char_idx": 48, "end_char_idx": 3240, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7261d17f-115e-4a54-89be-cb72b215ee11": {"__data__": {"id_": "7261d17f-115e-4a54-89be-cb72b215ee11", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "30d36423-0594-4220-88c7-572628b25ee3", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "df365e9b7aa821902ab5a609f516f492f1a10d45c35708b5d0a647ee2d0853da", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "99629377-2fcb-4a62-b5f1-4595fb5ec016", "node_type": "1", "metadata": {}, "hash": "8dc325080734873cd28de00869e49355677e22a57c048e3ff1ea348870e08ecc", "class_name": "RelatedNodeInfo"}}, "text": "In \n\nrecent years, the therapeutic approach for diabetes \n\npatients is diet control, exercise, and hypoglycemic drugs \n\nsuch as sulphonylureas, acarbose, and insulin with \n\nundesirable side effects such as liver toxicity, lactic \n\nacidosis, and diarrhea [6].   \n\nIn this regard, inhibition of key carbohydrate digestive \n\nenzymes (\u03b1-amylase) is considered one of the best \n\ntherapeutic techniques for the treatment of diabetes and \n\nits associated diseases. Pancreatic \u03b1-amylase is also \n\nknown as 1, 4-\u03b1-D-glucan glucanohydrolase EC (3.2.1.1) \n\nplays a vital role in the digestion of starch molecules in \n\nthe human body. At the first stage, there is partial \n\ndigestion of starch by salivary amylase enzyme. The \n\nenzyme cleaves polymeric substrate (Starch) into shorter \n\noligomers. At the second stage, these shorter oligomers \n\nare further split into maltose, maltotriose, and small \n\nmalto-oligosaccharides with the help of pancreatic \u03b1-\n\namylase in the gut [7]. In this present study, \u03b1-amylase \n\ncatalyzes the endohydrolysis of \u03b1-1, 4 glycosidic linkages \n\nof 2-chloro-4-nitrophenyl-\u03b1-D-maltotrioside (CNPG3) to \n\nyield free chromophore, 2-chloro-nitrophenol (CNP) and \n\nrelease 2-chloro-4-nitrophenyl-\u03b1-D-maltoside (CNPG2), \n\nmaltotriose (G3), and glucose (G). The rate of increase in \n\nchromophore absorbance is related to the \u03b1-amylase \n\nactivity of the sample [8,9]. In recent years, the scientist \n\nhas been so much attracted on the exploration of the \n\nsecondary metabolites of plants as inhibitors for most \n\neffective formulation aspect of drugs with few or hardly \n\nnegative effects. The medicinal plant contains various \n\nnatural antioxidants such as tocopherols, vitamin C, and \n\nNepal Journal of Biotechnology  \nPublisher: Biotechnology Society of Nepal  ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb  ISSN (Print): 2091-1130 \n\n \n\nhttps://orcid.org/0000-0003-4460-137X\nhttps://orcid.org/0000-0002-3559-9215\nhttps://orcid.org/0000-0002-8125-9756\nmailto:karankhadayat55@gmail.com\nhttps://orcid.org/0000-0002-4882-4864\nmailto:skkalauni@gmail.com\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20                 Maharjan et al. \n \n\n\u00a9NJB, BSN   15 \n\nphenolic compounds which are capable to neutralize \n\noxidative damage and protecting from various diseases \n\nincluding diabetes [10]. \n\nPlants have been the primary medicinal source for \n\ncenturies to cure a wide array of human diseases, \n\nparticularly in developing countries like Nepal due to \n\nscarce resources, affordability, and insufficient access to \n\nconventional treatment. Thus, considering the \n\nextraordinary biodiversity of Nepal, there is a need for \n\nscientific investigations of plant species. Among the \n\nvarious medicinal plants, C. tamala is the focus of this \n\nresearch. C. tamala (Buch.-Ham.) T. Nees and Eberm \n\n(Luraceace) is one of several traditional remedies used \n\nunder the Ayurvedic system in Nepal. Its leaves are \n\ncommonly called tejpat and are used as a common \n\ningredient in cooking. This species is a perennial or \n\nevergreen tree up to 7.5 m in height and a girth of about \n\n1.4 cm [11]. Young leaves red-brown, smooth and mature \n\nleaves sericeous, glabrescent, and rarely glaucous [12]. \n\nLeaves are natural food preservatives for pineapple juice \n\n[13]. Besides, tejpat is also a traditional dye-yielding plant \n\n[14].", "start_char_idx": 3241, "end_char_idx": 6588, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "99629377-2fcb-4a62-b5f1-4595fb5ec016": {"__data__": {"id_": "99629377-2fcb-4a62-b5f1-4595fb5ec016", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7261d17f-115e-4a54-89be-cb72b215ee11", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "5e912dddf2c1fb977035130630b98c3cb2f39ef0d7d6e1a30430ed0bccdb757d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9ec3957a-ecea-4fe2-93d6-f905ae632ef3", "node_type": "1", "metadata": {}, "hash": "0fc39fa2f05bf81d8d5b36d41687a135befecf0999c7d316e69956ecefcd8d1c", "class_name": "RelatedNodeInfo"}}, "text": "Besides, tejpat is also a traditional dye-yielding plant \n\n[14]. It has been traditionally used as an astringent, \n\nstimulant, and carminative [15]. C. tamala leaves \n\nexhibited significant biological properties with scientific \n\nvalidation including antioxidant [16,17], antimicrobial \n\n[18,19,20], anti-diabetic [21,22,23], anticancer [24], and \n\nanti-inflammatory [25]. \n\nTo the best of the authors\u2019 knowledge, no scientific \n\nliterature was published regarding the comparison of \n\nyoung and mature leaves extracts of C. tamala to evaluate \n\ntheir bioactivities. The aim of this research was therefore \n\nto estimate total phenolic and total flavonoid contents \n\nalong with the evaluation of in vitro anti-diabetic activity \n\nof the extracts of C. tamala by the microplate-based \n\nmethod. \n\nMaterials and Methods \nChemicals and reagent   \nMethanol, ethanol, folin-ciocalteu (FC) reagent were \n\npurchased from Qualigens, sodium carbonate, dimethyl \n\nsulphoxide (DMSO), potassium acetate, disodium \n\nhydrogen phosphate dihydrate, sodium dihydrogen \n\nphosphate dihydrate, and sodium chloride were \n\nobtained from Fisher Scientific, gallic acid, and quercetin \n\nfrom Hi-media, aluminum chloride from Merck and \n\nacarbose, \u03b1-amylase from porcine pancreas, and 2-\n\nchloro-4-nitrophenyl-\u03b1-D-maltotrioside were purchased \n\nfrom Sigma.  \n\nCollection and authentication of plant \nmaterials  \nFresh young and mature leaves of C. tamala were \n\ncollected separately from Machhegaun, Kathmandu in \n\nJuly 2019. A herbarium specimen was deposited at the \n\nCentral Department of Botany, Tribhuvan University, \n\nand voucher code RM001 was provided.  \n\nExtraction  \nThe collected young and mature leaves were washed \n\nproperly with water, shade dried, and ground into \n\npowder. The extraction was done by cold percolation \n\nmethod. In short, the powder of young and mature leaves \n\nwas soaked in methanol and ethanol for 72 hrs with \n\noccasional shaking at room temperature. After 72 hrs, the \n\nsolvent was filtered and the filtrate was evaporated using \n\na rotary evaporator under vacuum at 400 C [26].  The \n\npercentage yield was calculated by the given formula: \n\n%\u2004Yield =\nDry weight of extract\n\nDry  weight of a plant\n\u00d7 100 \n\nPhytochemical screening  \nThe presence of phytochemicals in the young and mature \n\nleaves of C. tamala was analyzed through the following \n\nstandard protocol [27, 28, 29]. \n\nEstimation of Total Phenolic Content (TPC) \nCrude extract of young and mature leaves of C. tamala \n\nwas estimated for TPC by using FC reagent [30,31,32]. In \n\nbrief, 20 \u03bcL sample was added with 100 \u03bcL Folin-\n\nciocalteu (2 N), accompanied by 80 \u03bcL Na2CO3 (1N). The \n\nreaction mixture was incubated in dark at room \n\ntemperature for 15 minutes until the dark blue color was \n\nobserved. Finally, the absorbance was taken at 765 nm \n\nusing a spectrophotometer (Synergy LX, BioTek, \n\nInstruments, Inc., USA). A standard calibration curve for \n\ngallic acid (10-100 \u03bcg/mL) was prepared and phenolic \n\ncontent was expressed as milligrams of gallic acid \n\nequivalent per gram of dry weight of the extract (mg \n\nGAE/g). All experiments were performed in triplicate \n\nand expressed as mean \u00b1 standard error of mean. \n\nEstimation of Total Flavonoid Content (TFC) \nCrude extract of young and mature leaves of C. tamala \n\nwas estimated for TFC by using the aluminum trichloride \n\n(AlCl3) method [32,33]. Briefly, 20 \u03bcL sample with 110 \u03bcL \n\ndistilled water was added with 60 \u03bcL ethanol, 5 \u03bcL AlCl3 \n\n(10%), and 5 \u03bcL potassium acetate (1 M).  The reaction \n\nmixture was incubated in dark at room temperature for \n\n30 minutes.", "start_char_idx": 6524, "end_char_idx": 10121, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9ec3957a-ecea-4fe2-93d6-f905ae632ef3": {"__data__": {"id_": "9ec3957a-ecea-4fe2-93d6-f905ae632ef3", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "99629377-2fcb-4a62-b5f1-4595fb5ec016", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "5e066e2036e70582dc20e4fe3e4f77045f8c11d22316b703b872263e9d806882", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fe046281-d9b1-4739-926d-79fcd3c46bd5", "node_type": "1", "metadata": {}, "hash": "634aed68643ca286e90b64989c12b7789c7aef5209c1edd60a2c3d6d66652f4a", "class_name": "RelatedNodeInfo"}}, "text": "The reaction \n\nmixture was incubated in dark at room temperature for \n\n30 minutes. After incubation, absorbance was measured \n\nat 415 nm using a spectrophotometer (Synergy LX, \n\nBioTek, Instruments, Inc., USA). A standard calibration \n\ncurve for quercetin (10 - 100 \u00b5g/mL) was prepared and \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20                 Maharjan et al. \n \n\n\u00a9NJB, BSN   16 \n\nflavonoid content was expressed as milligrams of \n\nquercetin equivalent per gram of dry weight of the \n\nextract (mg QE/g). All experiments were done in \n\ntriplicate and expressed as mean \u00b1 standard error of \n\nmean. \n\nIn vitro \u03b1-amylase inhibition assay  \nThe in vitro \u03b1-amylase inhibition was performed in a \n\nmicroplate reader as previously described method [34]. \n\nFirstly, young and mature leaves extract was prepared \n\nusing 50% DMSO, and the reaction was carried out using \n\n50 mM sodium phosphate buffer (pH 7) with 0.9% NaCl. \n\nConcisely, 20 \u00b5L of extract of different concentrations \n\n(31.25 - 1000 \u00b5g/mL) was mixed with 80 \u00b5L of the enzyme \n\nat a final concentration of 1.5 U/mL. Then, it was pre-\n\nincubated at 37 \u00b0C for 10 min and after pre-incubation, \n\n100 \u00b5L CNPG3 substrate at a final concentration of 1 mM \n\nwas added and left for 15 minutes at the same \n\ntemperature. The absorbance was measured at 405 nm. \n\nDMSO (Not more than 5% was taken as final \n\nconcentration) was taken as negative control and \n\nacarbose as a positive control. All experiments were \n\ncarried out in triplicate.  \n\n%\u2004Inhibition =\nA(control) \u2212 A(sample)\n\nA(control)\n\u00d7 100 \n\n A (control) = Absorbance of enzyme-substrate with 50% \n\nDMSO  \n\n A (sample) = Absorbance of enzyme-substrate with \n\nextract as inhibitor. \n\nStatistical analysis \nThe data were analyzed by using Gen 5 Microplate Data \n\nCollection and Analysis Software of microplate reader \n\nand then by MS Excel. The IC50 value of crude extract was \n\ncalculated by using Graph Pad Prism version 8 software \n\nand the results were expressed as mean \u00b1 Standard error \n\nmean (SEM).  \n\nResults  \nPhytochemical screening \nThe percentage yield was found to be higher in methanol \n\nas compared to ethanol and are given in Table 1. The \n\nrange of percentage yield of mature and young leaves \n\nvaries from 7.36 to 18.22%.  The young leaf had the \n\nhighest percentage yield (EEY 10.22 % and MEY 18.22 %) \n\nand the mature leaf had the lowest percentage yield \n\n(EEM 7.36 % and MEM 12.24 %). \n\nThe different phytochemical tests had shown the \n\npresence of polyphenols, flavonoids, tannins, quinones, \n\ncarbohydrates, glycosides, terpenoids, and reducing \n\nsugar whereas basic alkaloids are absent in both extracts \n\nof C. tamala (Table 2). \n\nTable 1. Percentage yield of different extract of C. tamala \n\nS. No. Sample Percentage yield (%) \n\n1 Ethanolic extract of \nmature leaf (EEM) \n\n7.36 \n\n2 Ethanolic extract of \nyoung leaf (EEY) \n\n10.22 \n\n3 Methanolic extract of \nmature leaf (MEM) \n\n12.24 \n\n4 Methanolic extract of \nyoung leaf (MEY) \n\n18.22 \n\nTable 2. Preliminary phytochemical screening of different \nextract of C. tamala \n\nS. No.", "start_char_idx": 10039, "end_char_idx": 13098, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fe046281-d9b1-4739-926d-79fcd3c46bd5": {"__data__": {"id_": "fe046281-d9b1-4739-926d-79fcd3c46bd5", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9ec3957a-ecea-4fe2-93d6-f905ae632ef3", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "bc0aa4ac1591002d83b9d6655cac080cc1929ac039d5f6ff9b4dea0cc59e6737", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "01c022a3-5ad0-47a9-92bb-b7bbd6888945", "node_type": "1", "metadata": {}, "hash": "7c524ef7dbfbcee030e6f8880b23860a06077ea0c21812ff12230250f83914ac", "class_name": "RelatedNodeInfo"}}, "text": "Phytochemicals MEM MEY EEM EEY \n\n1 Polyphenols + + + + \n\n2 Flavonoids + + + + \n\n3 Tannins + + + + \n4 Quinones + + + + \n5 Carbohydrates + + + + \n6 Glycosides + + + + \n7 Terpenoids + + + + \n8 Diterpenes + + + + \n9 Reducing sugars + + + + \n\n10 Basic alkaloids - - - - \n\u201c+\u201d indicates presence, \u201c-\u201d indicates absence \n\nTotal Phenolic and Flavonoid Content  \nThe TPC value of ethanolic young and mature leaves \n\nextract was 9.83 \u00b1 0.30 mg GAE/g and 8.91 \u00b1 0.39 mg \n\nGAE/g respectively whereas, the TPC value of \n\nmethanolic young and mature leaves extract was 11.26 \u00b1 \n\n0.17 mg GAE/g and 13.73 \u00b1 0.55 mg GAE/g. The TFC \n\nvalue of ethanolic young and mature leaves extract was \n\n6.82 \u00b1 0.67 mg QE/g and 7.02 \u00b1 0.32 mg QE/g \n\nrespectively whereas, the TFC value of methanolic young \n\nand mature leaves extract was 12.59 \u00b1 0.71 mg QE/g and \n\n11.84 \u00b1 0.66 mg QE/g as shown in Table 3. \n\nTable 3. Total phenolic and flavonoid content of different \nextract of C.  tamala \n\nS. No. Sample TPC mg GAE/g TFC mg QE/g \n\n1 MEM 13.73 \u00b1 0.55 11.84 \u00b1 0.66 \n2 MEY 11.26 \u00b1 0.17 12.59 \u00b1 0.71 \n3 EEM 8.91 \u00b1 0.39 7.02 \u00b1 0.32 \n\n4 EEY 9.83 \u00b1 0.30 6.82 \u00b1 0.67 \nMean \u00b1 SEM (n = 3) \n\nIn vitro \u03b1-amylase inhibition assay \nTable 4. Screening for \u03b1-amylase inhibition of different extract \n\nS. No. Sample Concentration % Inhibition \n\n1 MEM 500 \u00b5g/mL < 50 \n2 MEY 500 \u00b5g/mL 70.33 \u00b1 0.47 \n3 EEM 500 \u00b5g/mL < 50 \n4 EEY 500 \u00b5g/mL < 50 \n5 Acarbose 50 \u00b5g/mL 96.77\u00b1 0.028 \n\nMean \u00b1 SEM (n = 3) \n\nThe percentage inhibition of young, and  mature leaves \n\nof C. tamala using methnol and ethanol as solvent were \n\nscreened for \ud835\udefc-amylase inhibitory activity at 500 \u00b5g/mL \n\n(Table 4). Young leaf had the highest inhibition of \ud835\udefc-\n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20                 Maharjan et al. \n \n\n\u00a9NJB, BSN   17 \n\namylase enzyme whereas mature leaf showed the lowest \n\ninhibition at the same concentration in our study.  \n\nOnly methanolic extract of young leaves had shown \n\n70.33% inhibition while screening. So, the methanolic \n\nextract of young leaves was further assessed for IC50 \n\nvalue at different concentrations (Table 5) and was found \n\nto be 224.6 \u00b1 2.76 \u03bcg/mL. However, compared to \n\nstandard drug acarbose, the methanolic extract of young \n\nleaves showed moderate \ud835\udefc-amylase inhibitory activity \n\n(Table 6) and was found to be 5.93\u00b1 0.14 as shown in \n\nTable 7.", "start_char_idx": 13099, "end_char_idx": 15432, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "01c022a3-5ad0-47a9-92bb-b7bbd6888945": {"__data__": {"id_": "01c022a3-5ad0-47a9-92bb-b7bbd6888945", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fe046281-d9b1-4739-926d-79fcd3c46bd5", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "044712e142f948b3c5d6714ac84fcf4604d69393c4908c25a572543aa9eb8164", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "aeccd1f0-76b0-4408-9581-39200c6b9df2", "node_type": "1", "metadata": {}, "hash": "4bbd7caad9aaea1b32b743ce97e29d9f5716b28b543b99f63525b8781bc100c5", "class_name": "RelatedNodeInfo"}}, "text": "Table 5. \u03b1-Amylase inhibition of methanolic extract of young \nleaves (MEY) at different concentration \n\nConcentration Inhibition \n1 \n\nInhibition \n2 \n\nInhibition \n3 \n\nMean\n\u00b1 \n\nSEM \n\n500 \u00b5g/mL 70.15 69.62 71.23 70.33 \n\u00b1 0.47 \n\n250 \u00b5g/mL 54.80 58.59 58.38 57.26 \n\u00b1 1.23 \n\n125 \u00b5g/mL 33.37 35.45 31.89 33.57 \n\u00b1 1.03 \n\n62.50 \u00b5g/mL 14.89 9.46 10.04 11.46 \n\u00b1 1.72 \n\n31.25 \u00b5g/mL 2.83 3.78 6.78 4.46 \n\u00b1 1.19 \n\nMean \u00b1 SEM (n = 3) \n\n \n\n0 200 400 600\n\n0\n\n20\n\n40\n\n60\n\n80\n\nMEY\n\nConcentration (\u00b5g/mL)\n\n%\n I\nn\n\nh\nib\n\nit\nio\n\nn\n\nInhibition 1\n\nInhibition 2\n\nInhibition 3\n\n \n\n \n\nTable 6. \u03b1-Amylase inhibition of acarbose at different concentration \n\nConcentration Inhibition \n1 \n\nInhibition \n2 \n\nInhibition \n3 \n\nMean\u00b1 \nSEM \n\n50 \u00b5g/mL 96.82 96.75 96.72 96.76\u00b1 0.02 \n\n25 \u00b5g/mL 90.08 89.93 89.80 89.94\u00b1 0.08 \n\n12.5 \u00b5g/mL 77.72 77.07 76.88 77.22\u00b1 0.25 \n\n6.25 \u00b5g/mL 55.44 51.92 53.38 53.58\u00b1 1.02 \n\n3.125 \u00b5g/mL 24.27 23.77 33.52 27.19\u00b1 3.16 \n\n1.563 \u00b5g/mL 5.33 6.64 4.01 5.33\u00b1 0.75 \nMean \u00b1 SEM (n = 3) \n\n0 20 40 60\n\n0\n\n50\n\n100\n\n150\n\nAcarbose\n\nConcentration (\u00b5g/mL)\n\n%\n I\nn\n\nh\nib\n\nit\nio\n\nn\n\nInhibition 1\n\nInhibition 2\n\nInhibition 3\n\n \n\nTable 7. IC50 values of acarbose and potent sample (MEY) \n\nS. No. Sample IC50 (\u00b5g/mL) \n\n1 Acarbose 5.93\u00b1 0.14 \n2 MEY 224.6 \u00b1 2.76 \n\nMean \u00b1 SEM (n = 3) \n\nDiscussion \nDiabetes has been stated as the primary cause of global \n\nmorbidity and mortality [35]. Retarding the absorption \n\nand digestion of carbohydrates in the intestine through \n\ninhibition of carbohydrate digestive enzymes such as \u03b1-\n\namylase is one of the therapeutic strategies to minimizing \n\npostprandial hyperglycemia [36, 37, 38]. This study \n\nreported the potential in vitro amylase inhibitory \n\nproperties of C. tamala mature and young leaf by \n\nidentifying its main phytochemical constituents. \n\nExtraction of phytochemicals is the preliminary process \n\nfor recovering and isolating compounds from the \n\npulverization of plant materials [39]. The extraction yield \n\ndepends upon different parameters such as solvent with \n\nvarying polarity, pH, temperature, extraction time, and \n\nsample composition [40]. In our study methanolic \n\nextracts showed (MEM, MEY) higher yield than that of \n\nethanolic extracts (EEY, EEM). The difference might be \n\ndue to the high polarity of methanol that may cause \n\nhigher solubility of phenolics, flavonoids, alkaloids, and \n\nterpenoids compounds in methanol as compared to other \n\nsolvents including ethanol [40, 41,42].  \n\nThe phytochemicals obtained from C. tamala are shown \n\nin Table 2 that was compared with the previously \n\nreported study and found similar phytochemical \n\nprofiling [43].", "start_char_idx": 15436, "end_char_idx": 18040, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "aeccd1f0-76b0-4408-9581-39200c6b9df2": {"__data__": {"id_": "aeccd1f0-76b0-4408-9581-39200c6b9df2", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "01c022a3-5ad0-47a9-92bb-b7bbd6888945", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "63affdf0105966a356711db252a6a943245ed720bcee5e6893b0c922eb90336e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "31777f0f-8104-409b-a976-03c13210ca4f", "node_type": "1", "metadata": {}, "hash": "876ccb3cdeb5dbff6189baf5d2942c87cd1fac349478a5c1dff52618b17499f0", "class_name": "RelatedNodeInfo"}}, "text": "Our findings exhibited that methanolic \n\nextracts have the higher TPC and TFC relative to \n\nethanolic extracts as seen in Table 3. Aryal et al. \n\nsuggested that numerous phenolic compounds are \n\npresent in plant extracts that are responsible for enzyme \n\ninhibition supported by TPC (mg GAE/g) and TFC (mg \n\nQE/g) of MEM (13.73 \u00b1 0.55, 11.84 \u00b1 0.66), MEY (11.26 \u00b1 \n\n0.17, 12.59 \u00b1 0.71), EEM (8.91 \u00b1 0.39, 7.02 \u00b1 0.32), and EEY \n\n(9.83 \u00b1 0.30, 6.82 \u00b1 0.67) respectively [44]. Phenolics and \n\nflavonoids compounds such as quercetin, ferulic acid, \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20                 Maharjan et al. \n \n\n\u00a9NJB, BSN   18 \n\nanthocyanins, catechin, and resveratrol are the reason \n\nbehind controlling glycemia via \u03b1-glucosidase, \u03b1-\n\namylase, and lipid peroxidation inhibition, insulin \n\nsecretion, and accelerating glucose uptake [45,46]. The \n\ndifference in TPC and TFC is due to different nature of \n\nextracting solvents [47]. Previously, it was revealed that \n\nyounger leaves are most active in biosynthesis and \n\naccumulation of phenolic, flavonoid, and \n\nproanthocyanidins than the mature leaves of Lantana \n\ncamara [48]. Similarly in our study, among young and \n\nmature leaf extracts, only methanolic extract of young \n\nleaf showed significant inhibition toward the \u03b1-amylase \n\nenzyme. The ethanolic extract of young leaf failed to \n\nshow significant inhibition that might be due to its less \n\npolar nature causing lower yield of phenolics and \n\nflavonoids compounds. \n\nBesides that, high flavonoid content of MEY might be \n\nresponsible for high inhibition as compared to other \n\nextracts. Several potential \u03b1-amylase and \u03b1-glucosidase \n\ninhibitors from medicinal plants belongs to flavonoid \n\nclass as reported by earlier study [49]. Phenolic \n\ncompounds including flavonoids and tannins have been \n\nreported to play a vital role in controlling diabetes [50, 51, \n\n52]. Phenolic compounds mainly flavonoids interact \n\nstrongly with proteins and could inhibit their enzymatic \n\nactivities by making complexes and changing \n\nconformation [53]. The IC50 value of acarbose as positive \n\ncontrol showed around 40 times more potency than MEY \n\nas shown in Table 5. This might be due to plant extract \n\ncontains a jumble of multiple compounds as compared to \n\npure inhibitor compound acarbose [34, 54]. \n\nPrevious studies reported that polyphenol compounds \n\n(proanthocyanidins) derived from different plant sources \n\nshowed potent amylase inhibitors [55, 56]. A-type \n\nprocyanidin oligomer of cinnamon led to potent \n\nantioxidant properties and acts as an insulin sensitizer \n\n[57]. Besides that, cinnamtannin D-1 (CD1) isolated from \n\nC. tamala was reported to shield pancreatic \u03b2-cells from \n\npalmitic acid-induced apoptosis and oral consumption of \n\ncinnamaldehyde had hypoglycemic and hypolipidemic \n\nactivity in STZ-induced diabetic rats respectively [23, 58]. \n\nThese compounds might be present in the extract of \n\nyoung leaves, so further isolation and molecular \n\ncharacterization are needed.  \n\nConclusion \nThe present study evaluated phytochemical constituents \n\nas well as \u03b1-amylase inhibition of methanolic and \n\nethanolic extracts from young and mature leaves of C. \n\ntamala. The in vitro results revealed that methanolic \n\nextract and young leaves possess higher antidiabetic \n\nactivity. Thus, it was suggested that further investigation \n\nfor isolation and characterization of a bioactive \n\ncompound and its kinetic study from MEY should be \n\ncarried out that can act as an anti-diabetic agent.", "start_char_idx": 18041, "end_char_idx": 21569, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "31777f0f-8104-409b-a976-03c13210ca4f": {"__data__": {"id_": "31777f0f-8104-409b-a976-03c13210ca4f", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "aeccd1f0-76b0-4408-9581-39200c6b9df2", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "f9816221fb8088f286dd7c3ee57b4018bceba54aa71b45b139bc9bf64902305e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6d33203b-97f1-45ac-a798-c105c89b3798", "node_type": "1", "metadata": {}, "hash": "a6ba4244f1922e0f595d03ca6071fde0cca808b09d16630511d2e46663179056", "class_name": "RelatedNodeInfo"}}, "text": "Abbreviations \nGAE (Gallic Acid Equivalent), QE (Quercetin \n\nEquivalent), CNPG3 (2-chloro-4-nitrophenyl-\u03b1-D-\n\nmaltotrioside), MEY (Methanolic Extract of Young leaf), \n\nMEM (Methanolic Extract of Mature Leaf), EEY \n\n(Ethanolic Extract of Young leaf), EEM (Ethanolic Extract \n\nof Mature Leaf), TPC (Total Phenolic Content), TFC \n\n(Total Flavonoid Content)  \n\nAuthor\u2019s contributions \nThis research was performed in collaboration with all \n\nauthors. RM and KK have contributed to the plan of the \n\nresearch work. The first draft was written by RM, \n\nresponsible for data analysis, who conducted technical \n\nwork (Lab). SKK supervised the research project, PT \n\nrevised the manuscript. The final manuscript was read \n\nand approved by all authors. \n\nCompeting interests  \nNo competing interests. \n\nFunding  \nThe research was completed by financial support from \n\nthe University Grant Commission (Sanothimi, \n\nBhaktapur, Nepal). \n\nAcknowledgments \nThe authors extend their appreciation to the Central \n\nDepartment of Chemistry, Tribhuvan University for \n\nproviding an opportunity to conduct this research and \n\nProf. Dr. Suresh Kumar Ghimire of Central Department \n\nof Botany, Tribhuvan University for plant identification  \n\nEthical approval and consent  \nNot applicable \n\nReferences \n1.  IDF Diabetes Atlas 9th edition 2019 [Internet]. [cited 2020 Sep 2].   \n\nAvailable from:  https://www.google.com/search?client=firefox-\nb-d&q=IDF+Diabetes+Atlas+9th+edition+2019.+%5B \nOnline%5D.%28URL+https%3A%2F%2Fwww.diabetesatlas.org \n%2Fen%2F%29.+%28Accessed+27+January+2020%9 \n\n2.  WHO | Global report on diabetes [Internet]. WHO. World Health \nOrganization; [cited 2020 Sep 2]. Available from: \nhttp://www.who.int/diabetes/global-report/en/ \n\n3.  Rowley WR, Bezold C. Creating Public Awareness: State 2025 \nDiabetes Forecasts. Popul Health Manag [Internet]. [cited 2020 Oct \n30].  Available from:  https://www.liebertpub.com/doi/ \nfull/ 10.1089/pop.2011.0053 \n\n4.  Sansar N. World Diabetes Day 2018: 4% of Nepal Population \nDiabetic! [Internet]. Nepali Sansar. 2018 [cited 2020 Sep 2]. \nAvailable from: https://www.nepalisansar.com/health/ world-\ndiabetes-day-2018-4-of-nepal-population-diabetic/ \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20  Maharjan et al. \n\n\u00a9NJB, BSN 19 \n\n5. Baynes JW. Role of oxidative stress in development of \ncomplications in diabetes. Diabetes. 1991;40(4):405-12.\nhttps://doi.org/10.2337/diab.40.4.405\n\n6. Tahrani AA, Piya MK, Kennedy A, Barnett AH. Glycaemic control \nin type 2 diabetes: targets and new therapies. Pharmacol Ther. \n2010;125(2):328-61. https://doi.org/10.1016/ j.pharmthera \n.2009.11.001 \n\n7. Kumanan R, Manimaran S, Saleemulla K, Dhanabal SP, Nanjan \nMJ. Screening of bark of Cinnamomum tamala (Lauraceae) by using \n\u03b1-amylase inhibition assay for anti-diabetic activity. Int J Pharm \nBiomed Res. 2010;1(2):69-72. \n\n8. Sudha P, Zinjarde SS, Bhargava SY, Kumar AR. Potent \u03b1-amylase \ninhibitory activity of Indian Ayurvedic medicinal plants.", "start_char_idx": 21572, "end_char_idx": 24542, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6d33203b-97f1-45ac-a798-c105c89b3798": {"__data__": {"id_": "6d33203b-97f1-45ac-a798-c105c89b3798", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "31777f0f-8104-409b-a976-03c13210ca4f", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "45fa34974eef91444dab6021c360b4219fce139a6783f53c8f650ae75bd8edeb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d7dfe381-6ce6-4f2a-9f18-20b76fdde1d3", "node_type": "1", "metadata": {}, "hash": "cb1ad5306b673745b521e19818790918e90177acd991dbbd0979a0527bde1434", "class_name": "RelatedNodeInfo"}}, "text": "Potent \u03b1-amylase \ninhibitory activity of Indian Ayurvedic medicinal plants. BMC \nComplement Altern Med. 2011;11(1):5.\nhttps://doi.org/10.1186/1472-6882-11-5 \n\n9. Barni F, Berti A, Rapone C, Lago G. \u03b1-Amylase kinetic test in bodily \nsingle and mixed stains. J Forensic Sci. 2006;51(6):1389-96. \nhttp//doi.org/10.1111/j.1556-4029.2006.00259.x\n\n10. Yang J-H, Lin H-C, Mau J-L. Antioxidant properties of several \ncommercial mushrooms. Food Chem. 2002;77(2):229-35. \nhttps://doi.org/10.1016/S0308-8146(01)00342-9 \n\n11. Majumder P, Paridhavi M. An ethno-phytochemical and \npharmacological review on novel Indian medicinal plants used \nin herbal formulations. Int J Pharm Pharm Sci. 2013;5(4):74-83.\n\n12. Flora of Nepal - Online Flora [Internet]. [cited 2020 Sep 2]. \nAvailable from: http://www.floraofnepal.org /onlineflora? \nname=  rossulariaceae&pubname=FloraOfNepal\n\n13. Kapoor IPS, Singh B, Singh G. Essential oil and oleoresins of \nCinnamomum tamala (tejpat) as natural food preservatives for \npineapple fruit juice. J Food Process Preserv. 2008;32(5):719-28.\nhttps://doi.org/10.1111/j.1745-4549.2008.00200.x \n\n14. Gaur RD. Traditional dye yielding plants of Uttarakhand, India. \nIndian J. Nat Prod Resour. 2008;7:154-265. \n\n15. Malla SB, Shakya PR. Medicinal plants of Nepal. His Majesty's \nGovernment of Nepal, Ministry of Forests, Department of \nMedicinal Plants; 1970. \n\n16. Prasad KN, Yang B, Dong X, Jiang G, Zhang H, Xie H, et al. \nFlavonoid contents and antioxidant activities from \nCinnamomum species. Innov Food Sci Emerg Technol. \n2009;10(4):627-32. https://doi.org/10.1016/j.ifset.2009.05.009\n\n17. Sultana S, Ripa FA, Hamid K. Comparative antioxidant activity \nstudy of some commonly used spices in Bangladesh. Pak J Biol \nSci. 2010;13(7):340.\n\n18. Parekh J. In vitro screening of antibacterial activity of aqueous \nand alcoholic extracts of various Indian plant species against \nselected pathogens from Enterobacteriaceae. Afr J Microbiol Res. \n2007;1(6):92-9.  https://doi.org/10.5897/AJMR.9000402\n\n19. Jeyasree P, Dasarathan P. Screening of phytochemicals and \nimmuneomodulatory potential of amedicinal plant, \nCinnamomum tamala. Int J Pharma Sci Res. 2012;3:1049-52. \n\n20. Thombre RS, Shinde V, Thaiparambil E, Zende S, Mehta S. \nAntimicrobial activity and mechanism of inhibition of silver \nnanoparticles against extreme halophilic archaea. Front \nMicrobiol. 2016;7:1424. https://doi.org/10.3389 \n/fmicb.2016.01424 \n\n21. Sun P, Wang T, Chen L, Yu BW, Jia Q, Chen KX, Fan HM, Li YM, \nWang HY. Trimer procyanidin oligomers contribute to the \nprotective effects of cinnamon extracts on pancreatic \u03b2-cells in \nvitro. Acta Pharmacol Sin.", "start_char_idx": 24467, "end_char_idx": 27115, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d7dfe381-6ce6-4f2a-9f18-20b76fdde1d3": {"__data__": {"id_": "d7dfe381-6ce6-4f2a-9f18-20b76fdde1d3", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6d33203b-97f1-45ac-a798-c105c89b3798", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "dce4d0bbb7b17c5310f02343a7808e46648a0e385ee4841eada448affbf17973", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3ce2c39d-2f59-4dff-84ab-b9990d64f1aa", "node_type": "1", "metadata": {}, "hash": "f8d44cc878cd1d82c7ae053d1983875785da69acf80b917279e014b92912bfaa", "class_name": "RelatedNodeInfo"}}, "text": "Acta Pharmacol Sin. 2016;37(8):1083-90. \nhttps://doi.org/10.1038/aps.2016.29\n\n22. Udupa KN, Tripathi SN, Chandola HM. Effect of Cinnamomum \ntamala on plasma insulin vis-\u00e0-vis blood sugar in patients \nofdiabetes mellitus. J Res Ayu Siddha. 1980;1:345-57. \n\n23. Wang T, Sun P, Chen L, Huang Q, Chen K, Jia Q, Li Y, Wang H. \nCinnamtannin D-1 protects pancreatic \u03b2-cells from palmitic acid-\ninduced apoptosis by attenuating oxidative stress. J Agric Food \nChem. 2014 Jun 4;62(22):5038-45.\nhttps://doi.org/10.1021/jf500387d\n\n24. Shahwar D, Ullaha S, Khan MA, Ahmad N, Saeed A, Ullah S. \nAnticancer activity of Cinnamomum tamala leaf constituents \ntowards human ovarian cancer cells. Pak J Pharm Sci. \n2015;28(3):969-72. \n\n25. Dumbre RK, Kamble MB, Patil VR. Inhibitory effects by \nayurvedic plants on prostate enlargement induced in rats. \nPharmacogn Res. 2014;6(2):127-32.  \nhttps://dx.doi.org/10.4103%2F0974-8490.129031 \n\n26. Zhang S, Bi H, Liu C. Extraction of bio-active components from \nRhodiola sachalinensis under ultrahigh hydrostatic pressure.\nSep Purif Technol. 2007;57(2):277-82.  https://doi.org/10.1016/j. \nseppur.2007.04.022\n\n27. Sofowora A. Medicinal plants and traditional medicine in Africa. \nIbadan. Niger Spectr Books Ltd. 1993:191-289. \n\n28. Harborne AJ. Phytochemical methods a guide to modern \ntechniques of plant analysis. Springer science and business \nmedia; 1998. \n\n29. Evans WC. Trease and Evans Pharmacognosy. 15th ed. Sanders \nCo Ltd Singap. 2002. \n\n30. Ainsworth EA, Gillespie KM. Estimation of total phenolic \ncontent and other oxidation substrates in plant tissues using \nFolin\u2013Ciocalteu reagent. Nat Protoc. 2007;2(4):875-77.\nhttps://doi.org/10.1038/nprot.2007.102\n\n31. Lu X, Ross CF, Powers JR, Aston DE, Rasco BA. Determination \nof total phenolic content and antioxidant activity of Garlic \n(Allium sativum) and Elephant Garlic (Allium ampeloprasum) by \nattenuated total reflectance\u2013fourier transformed infrared \nspectroscopy. J Agric Food Chem. 2011;59(10):5215-21. \nhttps://doi.org/10.1021/jf201254f\n\n32. Sapkota BK, Khadayat K, Adhikari B, Poudel DK, \nNiraula P, Budhathoki P, Aryal B, Basnet K, Ghimire M,\nMarahatha R, Parajuli N. Phytochemical Analysis, \nAntidiabetic Potential and in-silico Evaluation of Some \nMedicinal Plants. Pharmacogn Res. 2021;13(3):140-48.  https://\ndoi.org/10.5530/pres.13.3.6 \n\n33. Chang C-C, Yang M-H, Wen H-M, Chern J-C. Estimation of total \nflavonoid content in propolis by two complementary \ncolorimetric methods. J Food Drug Anal. 2002;10(3):178-82. \nhttps://doi.org/10.38212/2224-6614.2748 \n\n34.", "start_char_idx": 27096, "end_char_idx": 29659, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3ce2c39d-2f59-4dff-84ab-b9990d64f1aa": {"__data__": {"id_": "3ce2c39d-2f59-4dff-84ab-b9990d64f1aa", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d7dfe381-6ce6-4f2a-9f18-20b76fdde1d3", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "e33f2422cda58ad534ef4a056901dc7cf293f2d4fe9c4916eee3ee3372585213", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1520fd62-d572-4f91-a92f-f51f14051a1a", "node_type": "1", "metadata": {}, "hash": "a028defb385c7f582297e50bfa11be1ecd543dfce626f6c197a3615d76996e72", "class_name": "RelatedNodeInfo"}}, "text": "Khadayat K, Marasini BP, Gautam H, Ghaju S, Parajuli N. \nEvaluation of the alpha-amylase inhibitory activity of Nepalese \nmedicinal plants used in the treatment of diabetes mellitus. Clin \nPhytoscience. 2020;6(1):1-8.\nhttps://doi.org/10.1186/s40816-020-00179-8 \n\n35. Bouguerra R, Alberti H, Salem LB, Rayana CB, Atti JE, Gaigi S, \net al. The global diabetes pandemic: the Tunisian experience. Eur \nJ Clin Nutr. 2007;61(2):160\u201365. https://doi.org/10.1038\n/sj.ejcn.1602478 \n\n36. Hogan S, Zhang L, Li J, Sun S, Canning C, Zhou K. Antioxidant \nrich grape pomace extract suppresses postprandial \nhyperglycemia in diabetic mice by specifically inhibiting \u03b1-\nglucosidase. Nutr Metab. 2010;7(1):1-9.\nhttps://doi.org/10.1186/1743-7075-7-71 \n\n37. Hamden K, Keskes H, Belhaj S, Mnafgui K, Allouche N. \nInhibitory potential of omega-3 fatty and fenugreek essential oil \non key enzymes of carbohydrate-digestion and hypertension in \ndiabetes rats. Lipids Health Dis. 2011;10(1):1-10. \nhttps://doi.org/10.1186/1476-511X-10-226 \n\n38. Mayur B, Sancheti S, Shruti S, Sung-Yum S. Antioxidant and \u03b1-\nglucosidase inhibitory properties of Carpesium abrotanoides L. \nJ Med Plants Res. 2010;4(15):1547-53. https://doi.org/ \n10.5897/JMPR.9000218 \n\n39. Stalikas CD. Extraction, separation, and detection methods for \nphenolic acids and flavonoids. J Sep Sci. 2007;30(18):3268-95.\nhttps://doi.org/10.1002/jssc.200700261\n\n40. Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, \nSoetaredjo FE, Ismadji S, et al. Effect of extraction solvent on total \nphenol content, total flavonoid content, and antioxidant activity \nof Limnophila aromatica. J Food Drug Anal. 2014;22(3):296-302. \nhttps://doi.org/10.1016/j.jfda.2013.11.001\n\n41. Duh P-D, Yeh D-B, Yen G-C. Extraction and identification of an \nantioxidative component from peanut hulls. J Am Oil Chem Soc. \n1992;69(8):814-18. https://doi.org/10.1007/BF02637703\n\n42. Gill S, Panthari P, Kharkwal H. Phytochemical investigation of \nhigh altitude medicinal plants Cinnamomum tamala (Buch-ham) \nNees and Eberm and Rhododendron arboreum smith. Am J \nPhytomed Clin Ther. 2015;3:512-28. \n\n\n\nNepal J Biotechnol. 2021 Dec.9 (2):14-20  Maharjan et al. \n\n\u00a9NJB, BSN 20 \n\n43. Apak R, G\u00fc\u00e7l\u00fc K, Demirata B, \u00d6zy\u00fcrek M, \u00c7elik SE, Bekta\u015fo\u011flu B, \net al. Comparative evaluation of various total antioxidant \ncapacity assays applied to phenolic compounds with the \nCUPRAC assay. Molecules. 2007;12(7):1496-1547. \nhttps://doi.org/10.3390/12071496 \n\n44.", "start_char_idx": 29660, "end_char_idx": 32110, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1520fd62-d572-4f91-a92f-f51f14051a1a": {"__data__": {"id_": "1520fd62-d572-4f91-a92f-f51f14051a1a", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3ce2c39d-2f59-4dff-84ab-b9990d64f1aa", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "348210b92350ba64333354283a051b080979cdb4ae0bb56a8b42740310bf2f59", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a91f038c-1145-4d36-b9a8-37508ab58091", "node_type": "1", "metadata": {}, "hash": "536b264d85091aaf44498d0adba5487046bd81230fac1ed50e9870c87f2e117f", "class_name": "RelatedNodeInfo"}}, "text": "Aryal B, Niraula P, Khadayat K, Adhikari B, Khatri Chhetri D, \nSapkota BK, Bhattarai BR, Aryal N, Parajuli N. Antidiabetic, \nAntimicrobial, and Molecular Profiling of Selected Medicinal \nPlants. Evid-Based Complementary Altern. Med. 2021 May \n7;2021. https://doi.org/10.1155/2021/5510099 \n\n45. Barone E, Calabrese V, Mancuso C. Ferulic acid and its therapeutic \npotential as hormetin for age-related diseases. Biogerontology. \n2009 Apr;10(2):97-108.  https://doi: 10.1007/s10522-008-9160-8 \n\n46. Rizvi SI, Mishra N. Anti\u2010oxidant effect of quercetin on type 2 \ndiabetic erythrocytes. J. Food Biochem. 2009 Jun;33(3):404-15.  \nhttps://doi: 10.1111/J.1745-4514.2009.00228.x \n\n47. T. Van Ngo, C. J. Scarlett, M. C. Bowyer, P. D. Ngo, and Q. Van \nVuong, \u201cImpact of different extraction solvents on bioactive \ncompounds and antioxidant capacity from the root of Salacia \nchinensis L. J. Food Qual. 2017 Jan 11: 2017.  https://doi: \n10.1155/2017/9305047 \n\n48. Bhakta D, Ganjewala D. Effect of leaf positions on total \nphenolics, flavonoids and proanthocyanidins content and \nantioxidant activities in Lantana camara (L). J Sci Res. \n2009;1(2):363-69. https://doi.org/10.3329/jsr.v1i2.1873\n\n49. Yang S, Meng Y, Yan J, Wang N, Xue Z, Zhang H, Fan Y. \nPolysaccharide-enriched fraction from Amillariella mellea \nfruiting body improves insulin resistance. Molecules. \n2019;24(1):46.  https://doi.org/10.3390/molecules24010046\n\n50. Marrelli M, Amodeo V, Statti G, Conforti F. Biological properties \nand bioactive components of Allium cepa L.: Focus on potential \nbenefits in the treatment of obesity and related comorbidities. \nMolecules. 2019;24(1):119. \n\n51. Keskes H, Mnafgui K, Hamden K, Damak M, El Feki A, Allouche \nN. In vitro anti-diabetic, anti-obesity and antioxidant proprieties \nof Juniperus phoenicea L. leaves from Tunisia. Asian Pac J  Trop \nBio. 2014;4:S649-55. https://doi.org/10.12980 \n/APJTB.4.201414B114 \n\n52. Fruit and vegetable intake and incidence of type 2 diabetes \nmellitus: systematic review and meta-analysis | The BMJ \n[Internet]. [cited 2020 Nov 3]. Available from: \nhttps://www.bmj.com/content/341/bmj.c4229\n\n53. Pochhi ML. An antioxidant activity of Cinnamonum tamala \nimproves histopathological alterations and biochemical \nparameters in alloxan-induced diabetic rats. Asian J Med Sci. \n2019;10(6):50-6.\n\n54. Lavelli V, Harsha PSS, Laureati M, Pagliarini E. Degradation \nkinetics of encapsulated grape skin phenolics and micronized \ngrape skins in various water activity environments and criteria \nto develop wide-ranging and tailor-made food applications. \nInnov Food Sci Emerg Technol. 2017;39:156-64. \nhttps://doi.org/10.1016/j.ifset.2016.12.006\n\n55.", "start_char_idx": 32111, "end_char_idx": 34784, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a91f038c-1145-4d36-b9a8-37508ab58091": {"__data__": {"id_": "a91f038c-1145-4d36-b9a8-37508ab58091", "embedding": null, "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-5", "node_type": "4", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "c7a6c38d4cf96439299c944c5051a35e77572c3fbfa5665958ff16c755442504", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1520fd62-d572-4f91-a92f-f51f14051a1a", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "3c3dfd114fd3565563fcd7cce1d182974b5faf30222d332c9786ab9a802f12ce", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "60bd7713-8456-4816-be75-b1584470998f", "node_type": "1", "metadata": {}, "hash": "71f50ea32e1151f2a2f5a7a355abfac34919ecb1d473847ed8e9c251ac58b5c3", "class_name": "RelatedNodeInfo"}}, "text": "Taslimi P, Aslan HE, Demir Y, Oztaskin N, Mara\u015f A, Gul\u00e7in \u0130, et \nal. Diarylmethanon, bromophenol and diarylmethane \ncompounds: Discovery of potent aldose reductase, \u03b1-amylase \nand \u03b1-glycosidase inhibitors as new therapeutic approach in \ndiabetes and functional hyperglycemia. Int J Biol Macromol. \n2018;119:857-63. https://doi.org/ 10.1016/j.ijbiomac. 2018 \n.08.004 \n\n56. Anderson RA, Broadhurst CL, Polansky MM, Schmidt WF, \nKhan A, Flanagan VP, et al. Isolation and characterization of \npolyphenol type-A polymers from cinnamon with insulin-like \nbiological activity. J Agric Food Chem. 2004;52(1):65-70. \nhttps://doi.org/10.1021/jf034916b\n\n57. Babu PS, Prabuseenivasan S, Ignacimuthu S. Cinnamaldehyde-a \npotential antidiabetic agent. Phytomedicine. 2007;14(1):15-22. \n https://doi.org/10.1016/ j.phymed.2006.11.005", "start_char_idx": 34785, "end_char_idx": 35603, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "60bd7713-8456-4816-be75-b1584470998f": {"__data__": {"id_": "60bd7713-8456-4816-be75-b1584470998f", "embedding": null, "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-50", "node_type": "4", "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "hash": "8847f9718bf9ece3ed153241a05e4ba609a870c6dc922f55504f09b6a019eeaa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a91f038c-1145-4d36-b9a8-37508ab58091", "node_type": "1", "metadata": {"identifier": "njb-5", "author": "Maharjan, Rasna; Thapa, Prakash; Khadayat, Karan; Kalauni, Surya Kant", "title": "Phytochemical Analysis and \u03b1-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala", "date": "2021-12-30", "file": "njb-5.pdf"}, "hash": "193fc9796caf27e982e15f8c268df855b6c587a154d2d8d4ef45037c873a1227", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3a1bc38a-4557-4b1a-a986-887a59b809db", "node_type": "1", "metadata": {}, "hash": "f3f63d628b4615328e1670d93c76cc3efc04cf0c7e7be1a31bcb1f7387605519", "class_name": "RelatedNodeInfo"}}, "text": "Microsoft Word - Editorial.docx\n\n\nTHE\t\n \u00a0FUTURE\t\n \u00a0SCIENCE\t\n \u00a0\n\n\t\n \u00a0\nThe\t\n \u00a0current\t\n \u00a0thrust\t\n \u00a0of\t\n \u00a0scientific\t\n \u00a0discourse\t\n \u00a0in\t\n \u00a0biological\t\n \u00a0science\t\n \u00a0is\t\n \u00a0centered\t\n \u00a0on\t\n \u00a0exploration\t\n \u00a0of\t\n \u00a0the\t\n \u00a0intrinsic\t\n \u00a0\nfactors\t\n \u00a0directly\t\n \u00a0involved\t\n \u00a0in\t\n \u00a0facilitating\t\n \u00a0the\t\n \u00a0life,\t\n \u00a0and\t\n \u00a0determining\t\n \u00a0other\t\n \u00a0ancillary\t\n \u00a0factors\t\n \u00a0maintaining\t\n \u00a0it\t\n \u00a0in\t\n \u00a0\nproper\t\n \u00a0order\t\n \u00a0for\t\n \u00a0conglomeration\t\n \u00a0into\t\n \u00a0the\t\n \u00a0concrete\t\n \u00a0life\t\n \u00a0system.\t\n \u00a0One\t\n \u00a0of\t\n \u00a0the\t\n \u00a0trademark\t\n \u00a0breakthroughs\t\n \u00a0has\t\n \u00a0\nbeen\t\n \u00a0 the\t\n \u00a0 Venterian\t\n \u00a0 artificial\t\n \u00a0 life,\t\n \u00a0 in\t\n \u00a0 which\t\n \u00a0 whole\t\n \u00a0 life\t\n \u00a0 was\t\n \u00a0 created\t\n \u00a0 artificially\t\n \u00a0 mediated\t\n \u00a0 by\t\n \u00a0 genetic\t\n \u00a0\nreconstitution\t\n \u00a0in\t\n \u00a0scaffold\t\n \u00a0of\t\n \u00a0cellular\t\n \u00a0system.\t\n \u00a0Similarly,\t\n \u00a0rapid\t\n \u00a0interest\t\n \u00a0and\t\n \u00a0flux\t\n \u00a0of\t\n \u00a0cutting\t\n \u00a0edge\t\n \u00a0research\t\n \u00a0is\t\n \u00a0\ncentered\t\n \u00a0 to\t\n \u00a0 understanding\t\n \u00a0 and\t\n \u00a0 thus\t\n \u00a0 devising\t\n \u00a0 mechanism\t\n \u00a0 to\t\n \u00a0 control\t\n \u00a0 the\t\n \u00a0 metabolic\t\n \u00a0 and\t\n \u00a0 physiological\t\n \u00a0\nprofile\t\n \u00a0 to\t\n \u00a0 prevent\t\n \u00a0 various\t\n \u00a0 diseases\t\n \u00a0 such\t\n \u00a0 as\t\n \u00a0 Alzheimer\u2019s\t\n \u00a0 disease,\t\n \u00a0 Parkinson\t\n \u00a0 disease,\t\n \u00a0 melanomas,\t\n \u00a0\nsarcomas\t\n \u00a0etc.\t\n \u00a0In\t\n \u00a0this\t\n \u00a0effort\t\n \u00a0whole\t\n \u00a0consortium\t\n \u00a0of\t\n \u00a0scientific\t\n \u00a0community,\t\n \u00a0academicians\t\n \u00a0and\t\n \u00a0researchers\t\n \u00a0are\t\n \u00a0\nputting\t\n \u00a0 their\t\n \u00a0 hands\t\n \u00a0 together\t\n \u00a0 for\t\n \u00a0 creation\t\n \u00a0 of\t\n \u00a0 panoramic\t\n \u00a0 globe,\t\n \u00a0 where\t\n \u00a0 there\t\n \u00a0 is\t\n \u00a0 cure\t\n \u00a0 for\t\n \u00a0 every\t\n \u00a0 kind\t\n \u00a0 of\t\n \u00a0\ndiseases\t\n \u00a0or\t\n \u00a0disorders.\t\n \u00a0\t\n \u00a0\n\nOne\t\n \u00a0of\t\n \u00a0the\t\n \u00a0most\t\n \u00a0important\t\n \u00a0aspects\t\n \u00a0associated\t\n \u00a0with\t\n \u00a0the\t\n \u00a0fast\t\n \u00a0moving\t\n \u00a0technological\t\n \u00a0intervention\t\n \u00a0has\t\n \u00a0been\t\n \u00a0\nco-\u00ad\u2010ordination,\t\n \u00a0 co-\u00ad\u2010operation\t\n \u00a0 and\t\n \u00a0 integration\t\n \u00a0 of\t\n \u00a0 scientific\t\n \u00a0 communities,\t\n \u00a0 for\t\n \u00a0 which\t\n \u00a0 the\t\n \u00a0 current\t\n \u00a0\ncommunication\t\n \u00a0technology\t\n \u00a0is\t\n \u00a0responsible.\t\n \u00a0Due\t\n \u00a0to\t\n \u00a0amalgamation\t\n \u00a0of\t\n \u00a0 ideas\t\n \u00a0and\t\n \u00a0knowledge\t\n \u00a0of\t\n \u00a0scientists\t\n \u00a0\nacross\t\n \u00a0the\t\n \u00a0cross-\u00ad\u2010country\t\n \u00a0boundaries,\t\n \u00a0the\t\n \u00a0scientific\t\n \u00a0pulse\t\n \u00a0is\t\n \u00a0rapidly\t\n \u00a0increasing.\t\n \u00a0But\t\n \u00a0still\t\n \u00a0 in\t\n \u00a0this\t\n \u00a0phase\t\n \u00a0of\t\n \u00a0\ntransition\t\n \u00a0of\t\n \u00a0world,\t\n \u00a0it\t\n \u00a0can\t\n \u00a0be\t\n \u00a0felt\t\n \u00a0that\t\n \u00a0some\t\n \u00a0patch\t\n \u00a0on\t\n \u00a0the\t\n \u00a0globe\t\n \u00a0is\t\n \u00a0still\t\n \u00a0undermined\t\n \u00a0and\t\n \u00a0to\t\n \u00a0some\t\n \u00a0extent\t\n \u00a0\nthis\t\n \u00a0part\t\n \u00a0is\t\n \u00a0unaware\t\n \u00a0of\t\n \u00a0the\t\n \u00a0technological\t\n \u00a0charisma.", "start_char_idx": 49, "end_char_idx": 2370, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3a1bc38a-4557-4b1a-a986-887a59b809db": {"__data__": {"id_": "3a1bc38a-4557-4b1a-a986-887a59b809db", "embedding": null, "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-50", "node_type": "4", "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "hash": "8847f9718bf9ece3ed153241a05e4ba609a870c6dc922f55504f09b6a019eeaa", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "60bd7713-8456-4816-be75-b1584470998f", "node_type": "1", "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "hash": "cf3e09072b1eac33acba25351c97156b0ddbc3fe72e438cd2359b7bf2a35e3cd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "141625fa-3eff-4438-99a7-1fd17db3c9e7", "node_type": "1", "metadata": {}, "hash": "daaa57263d1a68d85572af0f073379d5892487f66cc85365e04f415478cadd98", "class_name": "RelatedNodeInfo"}}, "text": "But,\t\n \u00a0this\t\n \u00a0part\t\n \u00a0too\t\n \u00a0has\t\n \u00a0some\t\n \u00a0crucial\t\n \u00a0contribution\t\n \u00a0when\t\n \u00a0\nconclusion\t\n \u00a0has\t\n \u00a0to\t\n \u00a0be\t\n \u00a0summed\t\n \u00a0up\t\n \u00a0taking\t\n \u00a0into\t\n \u00a0consideration\t\n \u00a0of\t\n \u00a0the\t\n \u00a0whole\t\n \u00a0globe\t\n \u00a0as\t\n \u00a0a\t\n \u00a0single\t\n \u00a0system.\t\n \u00a0\n\nThe\t\n \u00a0 crux\t\n \u00a0 of\t\n \u00a0 technological\t\n \u00a0 impetus\t\n \u00a0 has\t\n \u00a0 been\t\n \u00a0 the\t\n \u00a0 utilization\t\n \u00a0 of\t\n \u00a0 natural\t\n \u00a0 resources,\t\n \u00a0 so\t\n \u00a0 the\t\n \u00a0 developed\t\n \u00a0\ncommunity\t\n \u00a0is\t\n \u00a0moving\t\n \u00a0forward\t\n \u00a0for\t\n \u00a0the\t\n \u00a0world\t\n \u00a0with\t\n \u00a0flux\t\n \u00a0of\t\n \u00a0artificial\t\n \u00a0stuffs.\t\n \u00a0The\t\n \u00a0current\t\n \u00a0research\t\n \u00a0in\t\n \u00a0top\t\n \u00a0tier\t\n \u00a0\neconomic\t\n \u00a0colossus\t\n \u00a0is\t\n \u00a0pivoted\t\n \u00a0on\t\n \u00a0automation\t\n \u00a0with\t\n \u00a0robotics,\t\n \u00a0complementation\t\n \u00a0and\t\n \u00a0supplementation\t\n \u00a0of\t\n \u00a0\nrequisites\t\n \u00a0with\t\n \u00a0artificial\t\n \u00a0stuffs,\t\n \u00a0and\t\n \u00a0designing\t\n \u00a0of\t\n \u00a0efficient\t\n \u00a0strategies\t\n \u00a0to\t\n \u00a0curtail\t\n \u00a0the\t\n \u00a0natural\t\n \u00a0requirements.\t\n \u00a0\nBut,\t\n \u00a0 regarding\t\n \u00a0 the\t\n \u00a0 sustainability\t\n \u00a0 it\t\n \u00a0 is\t\n \u00a0 still\t\n \u00a0 questionable\t\n \u00a0 in\t\n \u00a0 terms\t\n \u00a0 of\t\n \u00a0 rationality.\t\n \u00a0 In\t\n \u00a0 contrast,\t\n \u00a0 on\t\n \u00a0 the\t\n \u00a0\nbackdoor\t\n \u00a0 we\t\n \u00a0 can\t\n \u00a0 realize\t\n \u00a0 that\t\n \u00a0 undermined\t\n \u00a0 patch\t\n \u00a0 of\t\n \u00a0 world\t\n \u00a0 is\t\n \u00a0 still\t\n \u00a0 furbished\t\n \u00a0 with\t\n \u00a0 enormous\t\n \u00a0 natural\t\n \u00a0\nresource\t\n \u00a0 in\t\n \u00a0 terms\t\n \u00a0 of\t\n \u00a0 flora\t\n \u00a0 and\t\n \u00a0 fauna.\t\n \u00a0 It\t\n \u00a0 is\t\n \u00a0 unquestionable\t\n \u00a0 that\t\n \u00a0 we\t\n \u00a0 can\t\n \u00a0 go\t\n \u00a0 for\t\n \u00a0 sustainable\t\n \u00a0 use\t\n \u00a0 of\t\n \u00a0 the\t\n \u00a0\nnatural\t\n \u00a0resources\t\n \u00a0rather\t\n \u00a0than\t\n \u00a0purging\t\n \u00a0all\t\n \u00a0our\t\n \u00a0effort\t\n \u00a0in\t\n \u00a0artifice\t\n \u00a0creation.\t\n \u00a0It\t\n \u00a0is\t\n \u00a0more\t\n \u00a0rational\t\n \u00a0to\t\n \u00a0devise\t\n \u00a0the\t\n \u00a0\nstrategic\t\n \u00a0approaches\t\n \u00a0to\t\n \u00a0maintain\t\n \u00a0the\t\n \u00a0sustainable\t\n \u00a0utilization\t\n \u00a0of\t\n \u00a0the\t\n \u00a0natural\t\n \u00a0resources\t\n \u00a0available.\t\n \u00a0For\t\n \u00a0that\t\n \u00a0\nthe\t\n \u00a0 idea\t\n \u00a0 from\t\n \u00a0 the\t\n \u00a0 developed\t\n \u00a0 community\t\n \u00a0 should\t\n \u00a0 be\t\n \u00a0 mingled\t\n \u00a0 with\t\n \u00a0 the\t\n \u00a0 natural\t\n \u00a0 resources\t\n \u00a0 available\t\n \u00a0 in\t\n \u00a0\nhotspots\t\n \u00a0patches\t\n \u00a0of\t\n \u00a0globe,\t\n \u00a0to\t\n \u00a0create\t\n \u00a0a\t\n \u00a0utopian\t\n \u00a0era.\t\n \u00a0Hence,\t\n \u00a0the\t\n \u00a0future\t\n \u00a0science\t\n \u00a0should\t\n \u00a0be\t\n \u00a0BE\t\n \u00a0NATURAL\t\n \u00a0\nWITH\t\n \u00a0SUSTAINABILITY!\t\n \u00a0\n\n \n\n \n\nDipesh Dhakal \nExecutive editor \nNepal Journal of Biotechnology", "start_char_idx": 2374, "end_char_idx": 4431, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "141625fa-3eff-4438-99a7-1fd17db3c9e7": {"__data__": {"id_": "141625fa-3eff-4438-99a7-1fd17db3c9e7", "embedding": null, "metadata": {"identifier": "njb-51", "author": "Dhakal, Dipesh", "title": "Race for Excellence", "date": "2022-07-11", "file": "njb-51.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-51", "node_type": "4", "metadata": {"identifier": "njb-51", "author": "Dhakal, Dipesh", "title": "Race for Excellence", "date": "2022-07-11", "file": "njb-51.pdf"}, "hash": "15558406bf6ad05ef0d6b9f1b1517f031910ad3cb5176453b8fd1fb92988f985", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3a1bc38a-4557-4b1a-a986-887a59b809db", "node_type": "1", "metadata": {"identifier": "njb-50", "author": "Dhakal, Dipesh", "title": "The Future Science", "date": "2022-07-11", "file": "njb-50.pdf"}, "hash": "974b6eb0ada6ba531ec3c522b0549663fe9776c666a616aa2907542a0bbd0168", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "02391b62-7059-4093-8abb-adc1c2b87636", "node_type": "1", "metadata": {}, "hash": "74db2dfffc58a79e289abaa54782fe1b0ec141c7ad7087ee3c8659539765c446", "class_name": "RelatedNodeInfo"}}, "text": "RACE FOR EXCELLENCE \n\nScience and technology has always been frontiers for prosperity and well-being of human life. \nThe fundamental backbone of scientific clues and technological intervention relies on course \nand discourse of the natural attributes within the nature itself; taking into consideration of the \nphysical, chemical, biological and computational prospect. Nature is no doubt the greatest \nscientist, researcher and expertise, who timely challenges us, evokes us and motivates us for \ninnovations, creations and constructive implications.  \n\nBiological sciences and allied research is on the hype in current scenario, because it is direct or \nindirect (through model animals) discourse of aspects pertaining to human at genetic, \nbiochemical or molecular level. Utilizing the science of biology, chemistry, physics, engineering, \nand information technology to develop products and services of great promise is the threshold \nof current scientific research. With different technological intervention and theological \nimplications, the scientific community has not only been able to manipulate organisms but also \nmanipulate life and materials at the atomic level for benefit of human civilization. \n\nHuman capacity has been exemplified by cutting edge innovations and implications for better \nlife. Human has been able to unravel from bottom of sea, through the surface of earth, and \nbeyond the atmosphere into the space. But, as one goes on unraveling the intricate aspects of \nnature, you face with new meshwork of mysteries. This is the charm and vigor of science and \ntechnology that makes the things just moving on forward and ahead. The scientific community: \nthe team leaders, associates, and assistants in academia and industries are enormously \ninvolved in breaking through these mysteries, modulate them and utilize them for worthy \noutputs. With this manifestation of the hypothesis to final accomplishment, the current science \nand technology is moving rapidly and efficiently. But, the story starts from here, the deeper we \ngo the wider the horizon becomes; accomplishing one landmark is starting point for a longer \nrace to compete with nature. \n\nIt is race for excellence; nature versus the scientific nurture, zeal, passion and commitment. Are \nyou ready to go is the call? If yes, get set ready and go! If not then wait and let others to go \nbefore you but you have to move on. It is something like, you make a choice or the choice will \nmake you! Be ready with the strategy! We heard the voice, culminated our energies and got on \nthe track for the journey! Before, saying \u201cHere, it is\u201d; we are thankful to all the stakeholders \nand bolstering hands on our effort. \n\n \n\n  \n\nDipesh Dhakal \nExecutive editor \nNepal Journal of Biotechnology", "start_char_idx": 52, "end_char_idx": 2821, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "02391b62-7059-4093-8abb-adc1c2b87636": {"__data__": {"id_": "02391b62-7059-4093-8abb-adc1c2b87636", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "141625fa-3eff-4438-99a7-1fd17db3c9e7", "node_type": "1", "metadata": {"identifier": "njb-51", "author": "Dhakal, Dipesh", "title": "Race for Excellence", "date": "2022-07-11", "file": "njb-51.pdf"}, "hash": "876d9fb4cb9cb64091a72c29059031b5666b02156c7a24c2249a32ed2fec6132", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d4ae27eb-5057-4677-ab5b-a7bab2b0824b", "node_type": "1", "metadata": {}, "hash": "23769c59f612f627fb9de8ea9eda239c364b6844ff60579bc3d6f725c9a09fcd", "class_name": "RelatedNodeInfo"}}, "text": "7\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a055\u201058\u00a0\n\n55\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nBRIEF\u00a0COMMUNICATION\u00a0\u00a0\n\nBiotechnology\u00a0Growth\u00a0Partnership:\u00a0A\u00a0Potential\u00a0\nFlagship\u00a0Program\u00a0in\u00a0S&T\u00a0\n\nRaju\u00a0Adhikari\u00a0and\u00a0Benu\u00a0Adhikari\u00a0\n\nNRN\u00a0Skill,\u00a0Knowledge\u00a0and\u00a0Innovation\u00a0(SKI)\u00a0Exchange\u00a0Task\u00a0Force,\u00a0Non\u00a0Resident\u00a0Nepali\u00a0Association\u00a0(NRNA),\u00a0www.nrn.org.np\u00a0\n\nCorrespondence\u00a0Author:\u00a0\u00a0\nRaju\u00a0Adhikari\u00a0\nE\u2010mail:\u00a0r_adhikari@hotmail.com\u00a0\n\nBackground\u00a0\nNepal\u00a0 is\u00a0a\u00a0 land\u00a0 locked\u00a0country\u00a0with\u00a0very\u00a0few\u00a0mineral\u00a0\n\nresources.\u00a0 However,\u00a0 she\u00a0 is\u00a0 vastly\u00a0 rich\u00a0 in\u00a0 her\u00a0 diverse,\u00a0\n\nnative,\u00a0high\u00a0altitude\u00a0flora\u00a0and\u00a0fauna\u00a0from\u00a0the\u00a0tropical\u00a0\n\nto\u00a0subalpine\u00a0region;\u00a0well\u00a0known\u00a0for\u00a0their\u00a0high\u00a0value\u00a0in\u00a0\n\nmedicinal\u00a0 application.\u00a0 These\u00a0 natural\u00a0 resources\u00a0 have\u00a0\n\nnot\u00a0 been\u00a0 exploited\u00a0 to\u00a0 their\u00a0 full\u00a0 potential\u00a0 due\u00a0 to\u00a0 the\u00a0\n\nlack\u00a0 of\u00a0 necessary\u00a0 expertise\u00a0 and\u00a0 required\u00a0 technical\u00a0\n\nfacilities\u00a0 needed\u00a0 for\u00a0 their\u00a0 full\u00a0 scientific\u00a0 investigation.\u00a0\n\nNepal\u00a0 is\u00a0 primarily\u00a0 an\u00a0 agricultural\u00a0 country\u00a0 and\u00a0 the\u00a0\n\nAgriculture\u00a0 contributes\u00a0 to\u00a0 approximately\u00a0 38%\u00a0 of\u00a0\n\nNepal\u2019s\u00a0 31\u00a0 B$\u00a0 GDP.\u00a0 \u00a0 In\u00a0 the\u00a0 past\u00a0 decade,\u00a0 there\u00a0 were\u00a0\n\nmany\u00a0 initiatives\u00a0 by\u00a0 government\u00a0 research\u00a0 agencies,\u00a0\n\nprivate\u00a0sectors\u00a0and\u00a0foreign\u00a0agencies\u00a0such\u00a0as\u00a0USAID\u00a0and\u00a0\n\nRockefeller\u00a0foundation\u00a0to\u00a0promote\u00a0biotechnology\u00a0and\u00a0\n\nnatural\u00a0product\u00a0research\u00a0activities\u00a0in\u00a0Nepal.\u00a0However,\u00a0\n\ndue\u00a0 to\u00a0 lack\u00a0 of\u00a0 policies\u00a0 and\u00a0 collaborations\u00a0 at\u00a0 an\u00a0\n\nInstitutional\u00a0level,\u00a0most\u00a0of\u00a0the\u00a0R&D\u00a0work\u00a0had\u00a0remained\u00a0\n\nfocused\u00a0 only\u00a0 in\u00a0 the\u00a0 tissue\u00a0 culturing\u00a0 of\u00a0 few\u00a0 plants\u00a0\n\n(medicinal/horticulture/ornamental)\u00a0 and\u00a0 fruits/\n\nvegetables\u00a0so\u00a0far.\u00a0\u00a0\n\nNepal\u00a0Academy\u00a0of\u00a0Science\u00a0and\u00a0Technology\u00a0(NAST)\u00a0and\u00a0\n\nthe\u00a0Ministry\u00a0of\u00a0S&T\u00a0have\u00a0strongly\u00a0emphasized\u00a0the\u00a0need\u00a0\n\nto\u00a0promote\u00a0innovation\u00a0and\u00a0collaboration\u00a0in\u00a0the\u00a0above\u00a0\n\nstated\u00a0priority\u00a0areas\u00a0whilst\u00a0working\u00a0in\u00a0partnership\u00a0with\u00a0\n\noverseas\u00a0 countries.\u00a0 The\u00a0 Nepal\u00a0 government\u00a0 has\u00a0\n\nannounced\u00a0a\u00a0new\u00a0national\u00a0plan\u00a0for\u00a0the\u00a0biotechnology\u00a0\n\nresearch\u00a0by\u00a0focusing\u00a0on\u00a0altitude\u00a0medicinal\u00a0plants\u00a0and\u00a0\n\nsetting\u00a0 up\u00a0 a\u00a0 national\u00a0 biotechnology\u00a0 research\u00a0 and\u00a0\n\ndevelopment\u00a0 centre\u00a0 to\u00a0 speed\u00a0 up\u00a0 research\u00a0 and\u00a0\n\nencourage\u00a0 private\u00a0 sector\u00a0 participation.\u00a0 The\u00a0 plan\u00a0\n\nrecommends\u00a0 biotechnology\u00a0 courses\u00a0 and\u00a0 scholarships\u00a0\n\nin\u00a0Nepal\u2019s\u00a0universities\u00a0and\u00a0provides\u00a0financial\u00a0incentives\u00a0\n\nin\u00a0tissue\u00a0culture\u00a0and\u00a0genetic\u00a0technologies\u00a0to\u00a0select\u00a0and\u00a0\n\nbreed\u00a0 improved\u00a0 crop\u00a0 varieties,\u00a0 usage\u00a0 of\u00a0 microbial\u00a0\n\ncultures\u00a0 to\u00a0 manage\u00a0 industrial\u00a0 waste,\u00a0 biosensors\u00a0 for\u00a0\n\nmonitoring\u00a0 soil\u00a0 and\u00a0 air\u00a0 pollutants,\u00a0 and\u00a0 scientific\u00a0\n\nassessment\u00a0of\u00a0the\u00a0country's\u00a0bio\u00a0diverse\u00a0floras\u00a0for\u00a0use\u00a0in\u00a0\n\npharmaceutical\u00a0and\u00a0cosmetic\u00a0industries.", "start_char_idx": 45, "end_char_idx": 2659, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d4ae27eb-5057-4677-ab5b-a7bab2b0824b": {"__data__": {"id_": "d4ae27eb-5057-4677-ab5b-a7bab2b0824b", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "02391b62-7059-4093-8abb-adc1c2b87636", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "c35edcea1bebd570f0b381bf470bf09bc1a013f5833ead1d333b0a93fd265d89", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "69657cf2-e52b-455a-a33a-9978a18cbbd7", "node_type": "1", "metadata": {}, "hash": "cad5deb29ab706b6ecc105d45f53edc86d30c9eb9713b1d845b686da2573bf1f", "class_name": "RelatedNodeInfo"}}, "text": "Biotechnology\u00a0 research\u00a0 globally\u00a0 and\u00a0 especially\u00a0 in\u00a0\n\ncountries\u00a0 like\u00a0 USA,\u00a0 UK,\u00a0 European\u00a0 Union,\u00a0 Australia,\u00a0\u00a0\n\nChina\u00a0 and\u00a0 India\u00a0 have\u00a0 a\u00a0 proven\u00a0 track\u00a0 record\u00a0 of\u00a0\n\ncommercial\u00a0 success.\u00a0 \u00a0 Australia\u00a0 has\u00a0 signed\u00a0 an\u00a0\n\ninternational\u00a0 Biotechnology\u00a0 growth\u00a0 partnership\u00a0\n\ninitiative\u00a0with\u00a0a\u00a0few\u00a0countries\u00a0and\u00a0such\u00a0collaborations\u00a0\n\nare\u00a0expected\u00a0to\u00a0induce\u00a0a\u00a0great\u00a0benefit\u00a0to\u00a0the\u00a0economy\u00a0\n\nof\u00a0both\u00a0countries\u00a0through\u00a0sharing\u00a0of\u00a0ideas,\u00a0innovation\u00a0\n\nand\u00a0 resources.\u00a0 Nepal\u00a0 Academy\u00a0 of\u00a0 Science\u00a0 and\u00a0\n\nTechnology\u00a0 (NAST)\u00a0 and\u00a0 Nepal\u00a0 Agriculture\u00a0 Research\u00a0\n\nCouncil\u00a0(NARC)\u00a0have\u00a0shown\u00a0interest\u00a0in\u00a0developing\u00a0such\u00a0\n\npartnerships\u00a0with\u00a0Australia\u00a0and\u00a0overseas\u00a0organizations\u00a0\n\nto\u00a0help\u00a0Nepal\u00a0biotechnology\u00a0Industry\u00a0in\u00a0Agriculture\u00a0and\u00a0\n\nHealth\u00a0sectors.\u00a0Recently,\u00a0NRNA\u00a0and\u00a0NAST\u00a0have\u00a0signed\u00a0\n\na\u00a0memorandum\u00a0of\u00a0understanding\u00a0(MoU)\u00a0in\u00a0S&T\u00a0field\u00a0to\u00a0\n\nwork\u00a0 jointly\u00a0 in\u00a0 national\u00a0 priority\u00a0 areas\u00a0 such\u00a0 as\u00a0\n\nBiotechnology\u00a0and\u00a0Environment.\u00a0This\u00a0will\u00a0allow\u00a0NAST,\u00a0\n\nNRNA\u00a0and\u00a0other\u00a0scientific\u00a0institutions\u00a0in\u00a0Nepal\u00a0to\u00a0work\u00a0\n\njointly\u00a0on\u00a0collaborative\u00a0projects\u00a0and\u00a0submit\u00a0proposals\u00a0\n\nRaju\u00a0 Adhikari\u00a0 is\u00a0 currently\u00a0 working\u00a0 as\u00a0 a\u00a0 Principal\u00a0\nResearch\u00a0 Scientist\u00a0 at\u00a0 CSIRO\u00a0 Material\u00a0 Science\u00a0 and\u00a0\nEngineering,\u00a0Vic,\u00a0Australia\u00a0and\u00a0is\u00a0the\u00a0chair\u00a0of\u00a0SKI\u00a0Task\u00a0\nforce.\u00a0Dr\u00a0Benu\u00a0Adhikari,\u00a0 is\u00a0a\u00a0member\u00a0SKI\u00a0task\u00a0force\u00a0\nand\u00a0 a\u00a0 Senior\u00a0 lecturer\u00a0 at\u00a0 University\u00a0 of\u00a0 Ballarat,\u00a0 Vic\u00a0\nAustralia\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a055\u201058\u00a0\n\n56\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nto\u00a0 interested\u00a0 overseas\u00a0 Biotechnology\u00a0 companies\u00a0 and\u00a0\n\nresearch\u00a0organizations\u00a0for\u00a0funding.\u00a0\n\nPotential\u00a0Project\u00a0Area\u00a0\nNepal\u2019s\u00a0high\u00a0altitude\u00a0herbal\u00a0medicinal\u00a0plants\u00a0are\u00a0native\u00a0\n\nand\u00a0unique\u00a0to\u00a0geo\u2010climatic\u00a0regions\u00a0and\u00a0have\u00a0potential\u00a0\n\nniches\u00a0 for\u00a0 the\u00a0 global\u00a0 market.\u00a0 Nepal\u00a0 currently\u00a0 trades\u00a0\n\nabout\u00a0 36\u00a0 different\u00a0 types\u00a0 of\u00a0 products\u00a0 derived\u00a0 from\u00a0\n\nherbal\u00a0plants\u00a0and\u00a0the\u00a0total\u00a0revenue\u00a0earned\u00a0is\u00a0reported\u00a0\n\nto\u00a0 be\u00a0 approximately\u00a0 12\u00a0 million\u00a0 NRs\u00a0 (US\u00a0 $240,000).\u00a0\n\nAround\u00a098%\u00a0raw\u00a0products\u00a0are\u00a0exported\u00a0to\u00a0Dabur\u00a0India,\u00a0\n\na\u00a0 leader\u00a0 in\u00a0 manufacturing/marketing\u00a0 herbal,\u00a0 nature\u2010\n\nbased\u00a0products\u00a0 in\u00a0Asia.\u00a0Due\u00a0to\u00a0the\u00a0 lack\u00a0of\u00a0any\u00a0patent\u00a0\n\nprotection\u00a0alongside\u00a0the\u00a0Governments\u00a0weak\u00a0policy\u00a0on\u00a0\n\nfarming\u00a0 and\u00a0 trade\u00a0 of\u00a0 medicinal\u00a0 plants,\u00a0 the\u00a0 revenue\u00a0\n\nsource\u00a0from\u00a0these\u00a0herbal\u00a0medicinal\u00a0plants\u00a0are\u00a0mostly\u00a0\n\nfrom\u00a0 the\u00a0 selling\u00a0 of\u00a0 raw\u00a0 products.\u00a0 In\u00a0 Nepal\u00a0 Ayurvedic\u00a0\n\nIndustries\u00a0 are\u00a0 also\u00a0 selling\u00a0 mostly\u00a0 the\u00a0 raw\u00a0 materials\u00a0\n\nwithout\u00a0 any\u00a0 value\u00a0 added\u00a0 research\u00a0 input\u00a0 and\u00a0 thus\u00a0\n\ncontributing\u00a0very\u00a0little\u00a0to\u00a0the\u00a0country\u2019s\u00a0economy\u00a0(NTFP\u00a0\n\nTrade\u00a0Bulletin,\u00a02005)\u00a0\n\nBiotechnology\u00a0Growth\u00a0Partnership\u00a0could\u00a0be\u00a0initiated\u00a0as\u00a0\n\na\u00a0 long\u00a0 term\u00a0 collaborative\u00a0 project\u00a0 in\u00a0 the\u00a0 above\u00a0\n\nmentioned\u00a0 areas.", "start_char_idx": 2662, "end_char_idx": 5365, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "69657cf2-e52b-455a-a33a-9978a18cbbd7": {"__data__": {"id_": "69657cf2-e52b-455a-a33a-9978a18cbbd7", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d4ae27eb-5057-4677-ab5b-a7bab2b0824b", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "ac92d82a418b7352c8ae060f5d9ca88af60b87b00486894da69f85d1d15570c9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9d5bcbd2-119b-4f9c-b4d4-7f0bcfb2c4af", "node_type": "1", "metadata": {}, "hash": "ada4998c2d1935175b19d5ade5790a2bd3ec40acac6d9637aed1d6319933f467", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 project\u00a0 could\u00a0 aim\u00a0 to\u00a0\n\ncommercially\u00a0exploit\u00a0high\u00a0altitude\u00a0flora\u00a0such\u00a0as\u00a0Yarsha\u00a0\n\nGumba\u00a0(Cordyceps\u00a0sinensis)\u00a0(Figure\u00a01)\u00a0by\u00a0carrying\u00a0out\u00a0\n\nresearch\u00a0tailored\u00a0to\u00a0value\u00a0add\u00a0by\u00a0conducting\u00a0rapid\u00a0high\u00a0\n\nthroughput\u00a0bioassay\u00a0screenings\u00a0against\u00a0certain\u00a0specific\u00a0\n\ntarget\u00a0 diseases\u00a0 and\u00a0 developing\u00a0 appropriate\u00a0\n\nformulations\u00a0 for\u00a0 its\u00a0 use\u00a0 as\u00a0 a\u00a0 common\u00a0 medicines\u00a0 and\u00a0\n\nhealth\u00a0 products\u00a0 [1\u20109].\u00a0 A\u00a0 medicinal\u00a0 herb\u00a0 of\u00a0 illustrious\u00a0\n\nhistory\u00a0 known\u00a0 as\u00a0 Himalayan\u00a0 Viagra\u00a0 has\u00a0 been\u00a0 well\u00a0\n\ndocumented\u00a0 in\u00a0 the\u00a0 literature\u00a0 for\u00a0 its\u00a0 use\u00a0 as\u00a0 a\u00a0 drug\u00a0 to\u00a0\n\nboost\u00a0 immune\u00a0 system,\u00a0 anti\u2010ageing\u00a0 and\u00a0 aphrodisiac\u00a0\n\nproperties.\u00a0Only\u00a03\u00a0species\u00a0of\u00a0the\u00a0caterpillar\u00a0fungus\u00a0are\u00a0\n\nreportedly\u00a0found\u00a0in\u00a0Nepal\u00a0at\u00a0a\u00a0reserve\u00a0~\u00a04,900\u00a0m\u00a0with\u00a0\n\n50%\u00a0 collection\u00a0 coming\u00a0 from\u00a0 Dolpa\u00a0 region\u00a0 alone.\u00a0 The\u00a0\n\nraw\u00a0materials\u00a0collected\u00a0in\u00a0this\u00a0manner\u00a0are\u00a0exported\u00a0to\u00a0\n\nTibet,\u00a0 China\u00a0 and\u00a0 other\u00a0 countries.\u00a0 Government\u00a0 had\u00a0\n\nlegalized\u00a0 the\u00a0 trade\u00a0 of\u00a0 raw\u00a0 material\u00a0 in\u00a0 2001\u00a0 and\u00a0\n\nimposed\u00a0 a\u00a0 royalty\u00a0 of\u00a0 $280/kg\u00a0 whereas\u00a0 the\u00a0 global\u00a0\n\nMarket\u00a0price\u00a0currently\u00a0stands\u00a0between\u00a0US$\u00a03,\u00a0000/kg\u00a0\n\nfor\u00a0 the\u00a0 lowest\u00a0 quality\u00a0 to\u00a0 over\u00a0 US$18,\u00a0 000/Kg\u00a0 for\u00a0 the\u00a0\n\nhighest\u00a0 quality\u00a0 larvae.\u00a0 The\u00a0 project\u00a0 will\u00a0 also\u00a0 aim\u00a0 to\u00a0\n\nestablish\u00a0 a\u00a0 National\u00a0 Germplasm\u00a0 bank\u00a0 of\u00a0 flora\u00a0 of\u00a0 high\u00a0\n\nmedicinal\u00a0values\u00a0to\u00a0protect\u00a0their\u00a0genetic\u00a0diversity.\u00a0\u00a0\n\nSimilarly\u00a0 in\u00a0 Food\u00a0 biotechnology,\u00a0 Yak\u00a0 cheese\u00a0 could\u00a0 be\u00a0\n\nused\u00a0as\u00a0a\u00a0valuable\u00a0resource\u00a0due\u00a0to\u00a0 its\u00a0 low\u00a0fat\u00a0(26%),\u00a0\n\nhigh\u00a0 protein\u00a0 (26%\u00a0 )\u00a0 and\u00a0 non\u00a0 conjugated\u00a0 linoleic\u00a0 acid\u00a0\n\n(CLAs)\u00a0content\u00a0compared\u00a0to\u00a0cow\u00a0cheddar\u00a0cheese\u00a0(see\u00a0\n\nTable\u00a0 1).\u00a0 The\u00a0 above\u00a0 fatty\u00a0 acid\u00a0 is\u00a0 reported\u00a0 to\u00a0 posses\u00a0\n\nanticancer\u00a0 and\u00a0 anti\u00a0 Type\u00a0 T2\u00a0 diabetes\u00a0 properties\u00a0 [10\u2010\n\n15).\u00a0 A\u00a0 project\u00a0 proposal\u00a0 to\u00a0 address\u00a0 its\u00a0 large\u00a0 scale\u00a0\n\nproduction,\u00a0 assess\u00a0 scientific\u00a0 effectiveness\u00a0 and\u00a0 quality\u00a0\n\ncontrol\u00a0could\u00a0help\u00a0diary\u00a0food\u00a0industries\u00a0and\u00a0bring\u00a0huge\u00a0\n\neconomic\u00a0benefit\u00a0to\u00a0the\u00a0farming\u00a0community.\u00a0\u00a0\n\nFurthermore,\u00a0 in\u00a0 a\u00a0 country\u00a0 like\u00a0 Nepal,\u00a0 where\u00a0 large\u00a0\n\nquantity\u00a0 of\u00a0 fruits\u00a0 are\u00a0 wasted\u00a0 due\u00a0 to\u00a0 lack\u00a0 of\u00a0\n\ninfrastructure\u00a0 (processing,\u00a0 storage,\u00a0 transportation),\u00a0 a\u00a0\n\nproject\u00a0focused\u00a0to\u00a0converting\u00a0these\u00a0fruits\u00a0into\u00a0powders\u00a0\n\nwithout\u00a0 compromising\u00a0 their\u00a0 nutritional\u00a0 value\u00a0 could\u00a0\n\nhelp\u00a0food\u00a0industry\u00a0in\u00a0a\u00a0big\u00a0innovative\u00a0way.\u00a0A\u00a0research\u00a0\n\nteam\u00a0 led\u00a0 by\u00a0 a\u00a0 NRN\u00a0 STF\u00a0 team\u00a0 member\u00a0 Dr.\u00a0 Benu\u00a0\n\nAdhikari\u00a0(University\u00a0of\u00a0Ballarat,\u00a0Victoria,\u00a0Australia)\u00a0has\u00a0\n\ndeveloped\u00a0 some\u00a0 breakthrough\u00a0 technologies\u00a0 in\u00a0 this\u00a0\n\narea.\u00a0\u00a0\n\nThere\u00a0 are\u00a0 many\u00a0 other\u00a0 potential\u00a0 areas\u00a0 in\u00a0 agriculture\u00a0\n\nwhere\u00a0such\u00a0collaboration\u00a0could\u00a0be\u00a0 initiated.", "start_char_idx": 5367, "end_char_idx": 7909, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9d5bcbd2-119b-4f9c-b4d4-7f0bcfb2c4af": {"__data__": {"id_": "9d5bcbd2-119b-4f9c-b4d4-7f0bcfb2c4af", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "69657cf2-e52b-455a-a33a-9978a18cbbd7", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "4173a9d8a06af390229633a4b412f4e532a34015de2a2cf914fc80f61082a95a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "e05ccffd-a2e4-44b2-8e52-9e98c7991506", "node_type": "1", "metadata": {}, "hash": "e3e9860dcc6296744c138b27f0b96e3a928dbfdb670cd2edd97108930d930336", "class_name": "RelatedNodeInfo"}}, "text": "SKI\u00a0takes\u00a0\n\nthis\u00a0 opportunity\u00a0 to\u00a0 invite\u00a0 our\u00a0 NRN\u00a0 professionals\u00a0 who\u00a0\n\nare\u00a0working\u00a0in\u00a0the\u00a0Biotechnology\u00a0field\u00a0to\u00a0come\u00a0forward\u00a0\n\nand\u00a0 contribute\u00a0 in\u00a0 this\u00a0 new\u00a0 venture\u00a0 initiative\u00a0 of\u00a0 S&T\u00a0\n\nstream.\u00a0\n\nPath\u00a0to\u00a0Market\u00a0\nThe\u00a0collaboration\u00a0initially\u00a0could\u00a0focus\u00a0on\u00a0high\u00a0altitude\u00a0\n\nnative\u00a0 medicinal\u00a0 plants\u00a0 in\u00a0 order\u00a0 to\u00a0 develop\u00a0 common\u00a0\n\ndrugs\u00a0 and\u00a0 cosmetic\u00a0 products\u00a0 for\u00a0 pharmaceutical\u00a0\n\nindustries.\u00a0These\u00a0types\u00a0of\u00a0products\u00a0serve\u00a0an\u00a0advantage\u00a0\n\nas\u00a0they\u00a0do\u00a0not\u00a0require\u00a0stringent\u00a0regulatory\u00a0criteria\u00a0and\u00a0\n\nare\u00a0an\u00a0easy\u00a0path\u00a0to\u00a0the\u00a0market.\u00a0The\u00a0products\u00a0based\u00a0on\u00a0\n\ndeveloped\u00a0formulation\u00a0from\u00a0the\u00a0above\u00a0project\u00a0can\u00a0be\u00a0\n\nmanufactured\u00a0 and\u00a0 marketed\u00a0 as\u00a0 a\u00a0 common\u00a0 drug\u00a0 and\u00a0\n\ncan\u00a0be\u00a0sold\u00a0under\u00a0the\u00a0patent/trade\u00a0rights\u00a0to\u00a0guarantee\u00a0\n\nits\u00a0claim.\u00a0Nepal\u2019s\u00a0commitment\u00a0will\u00a0be\u00a0to\u00a0provide\u00a0raw\u00a0\n\nmaterials\u00a0 and\u00a0 manpower\u00a0 and\u00a0 conduct\u00a0 preliminary\u00a0\n\nscreening\u00a0 research\u00a0 while\u00a0 advanced\u00a0 testing\u00a0 and\u00a0\n\nformulation\u00a0 matrix\u00a0 could\u00a0 be\u00a0 carried\u00a0 out\u00a0 in\u00a0 Australia.\u00a0\n\nThe\u00a0clinical\u00a0trials\u00a0and\u00a0manufacturing\u00a0of\u00a0products\u00a0could\u00a0\n\nbe\u00a0 carried\u00a0 out\u00a0 in\u00a0 Nepal\u00a0 which\u00a0 will\u00a0 help\u00a0 develop\u00a0\n\nnecessary\u00a0 infrastructure\u00a0 for\u00a0 the\u00a0 production\u00a0 of\u00a0 such\u00a0\n\nvalue\u00a0added\u00a0products\u00a0and\u00a0clinical\u00a0trial\u00a0facilities\u00a0 in\u00a0the\u00a0\n\nlong\u00a0 run\u00a0 on\u00a0 low\u00a0 cost.\u00a0 \u00a0 The\u00a0 patent\u00a0 royalties\u00a0 will\u00a0 be\u00a0\n\nshared\u00a0and\u00a010%\u00a0of\u00a0profit\u00a0from\u00a0the\u00a0sale\u00a0of\u00a0such\u00a0product\u00a0\n\ncan\u00a0be\u00a0reinvested\u00a0in\u00a0R&D\u00a0to\u00a0develop\u00a0further\u00a0advanced\u00a0\n\ntechnologies\u00a0 or\u00a0 to\u00a0 extend\u00a0 research\u00a0 in\u00a0other\u00a0 potential\u00a0\n\nareas.\u00a0The\u00a0 life\u00a0of\u00a0the\u00a0above\u00a0flagship\u00a0project\u00a0could\u00a0be", "start_char_idx": 7910, "end_char_idx": 9397, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "e05ccffd-a2e4-44b2-8e52-9e98c7991506": {"__data__": {"id_": "e05ccffd-a2e4-44b2-8e52-9e98c7991506", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9d5bcbd2-119b-4f9c-b4d4-7f0bcfb2c4af", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "fe934e4825bc4a89ad7cd8db7409943f957a3e3e1ccf356a27c9a57849205695", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4f2a5607-bef5-40f7-9ddf-b53573890055", "node_type": "1", "metadata": {}, "hash": "b90082224733636867322ce0f192338fb14d5514efd7ae8face7ee2b2d51b0d5", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 life\u00a0of\u00a0the\u00a0above\u00a0flagship\u00a0project\u00a0could\u00a0be\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a055\u201058\u00a0\n\n57\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\napproximately\u00a03\u20105\u00a0years\u00a0to\u00a0allow\u00a0patent\u00a0protection\u00a0and\u00a0\n\ndevelopment\u00a0work.\u00a0\n\nApproach\u00a0\nNRN\u00a0SKI\u00a0team\u00a0 is\u00a0working\u00a0closely\u00a0with\u00a0Nepal\u00a0Academy\u00a0\n\nof\u00a0Science\u00a0and\u00a0Technology\u00a0in\u00a0Nepal\u00a0to\u00a0develop\u00a0a\u00a0joint\u00a0\n\ncollaborative\u00a0 project\u00a0 proposal\u00a0 to\u00a0 explore\u00a0 fund\u00a0 from\u00a0\n\npotential\u00a0investors\u00a0or\u00a0overseas\u00a0S&T\u00a0organisations.\u00a0The\u00a0\n\nS&T\u00a0organisations\u00a0in\u00a0Nepal\u00a0will\u00a0provide\u00a0commitment\u00a0to\u00a0\n\nraw\u00a0 materials,\u00a0 manpower\u00a0 and\u00a0 facilities\u00a0 to\u00a0 conduct\u00a0\n\npreliminary\u00a0 research\u00a0 work\u00a0 and\u00a0 the\u00a0 advanced\u00a0 testing\u00a0\n\nand\u00a0 formulation\u00a0 development\u00a0 work\u00a0 could\u00a0 be\u00a0 carried\u00a0\n\nout\u00a0 overseas.\u00a0 Final\u00a0 formulation\u00a0 trials\u00a0 and\u00a0\n\nmanufacturing\u00a0 of\u00a0 such\u00a0 value\u00a0 added\u00a0 products\u00a0 will\u00a0 be\u00a0\n\ncarried\u00a0out\u00a0in\u00a0Nepal\u00a0(cheap\u00a0resources)\u00a0to\u00a0help\u00a0develop\u00a0\n\nresearch\u00a0 infrastructure.\u00a0 The\u00a0 patent\u00a0 will\u00a0 be\u00a0 shared\u00a0\n\ndepending\u00a0 upon\u00a0 the\u00a0 nature\u00a0 of\u00a0 the\u00a0 agreement\u00a0 and\u00a0\n\nNepal\u00a0 will\u00a0 get\u00a0 the\u00a0 dollar\u00a0 value\u00a0 from\u00a0 the\u00a0 royalty\u00a0\n\nobtained\u00a0 from\u00a0 the\u00a0 sale\u00a0 of\u00a0 such\u00a0 products.\u00a0 \u00a0 NRNA\u00a0 will\u00a0\n\nlobby\u00a0Nepal\u00a0government\u00a0to\u00a0provide\u00a0tax\u00a0incentives\u00a0and\u00a0\n\nother\u00a0 flexibility\u00a0 for\u00a0 establishment\u00a0 of\u00a0 any\u00a0 spin\u2010off\u00a0\n\ncompany\u00a0 arising\u00a0 from\u00a0 such\u00a0 an\u00a0 investment.\u00a0 A\u00a0 general\u00a0\n\nscheme\u00a0of\u00a0research\u00a0approach\u00a0is\u00a0given\u00a0in\u00a0Figure\u00a02.\u00a0\u00a0\n\nFigure\u00a01.\u00a0(a)\u00a0Dolpa\u00a0community\u00a0collecting\u00a0Yarsha\u00a0\n\nGumba,\u00a0\u00a0\n\nFigure\u00a01.\u00a0(b)\u00a0a\u00a0raw\u00a0specimen\u00a0of\u00a0Yarsha\u00a0Gumba \u00a0\n\nFatty\u00a0acid\u00a0 Yak\u00a0cheese\u00a0 Cow\u2010cheddar\u00a0cheese\u00a0\ncis\u20109,\u00a0trans\u201011\u00a0CLA\u00a0 2.01\u00b10.07\u00a0 0.48\u00b10.07\u00a0\ntrans\u20109,\u00a0cis\u201011\u00a0CLA\u00a0 0.13\u00b10.01\u00a0 0.03\u00b10.01\u00a0\ntrans\u201010,\u00a0cis\u201012\u00a0CLA\u00a0 0.038\u00b10.001\u00a0 0.009\u00b10.001\u00a0\ntrans\u201011,\u00a0trans\u201013\u00a0CLA\u00a0 0.039\u00b10.001\u00a0 0.018\u00b10.001\u00a0\ntrans\u20109,\u00a0trans\u201011\u00a0CLA\u00a0+\u00a0trans\u201010,\u00a0trans\u201012\u00a0CLA\u00a0 0.057\u00b10.003\u00a0 0.031\u00b10.003\u00a0\n\nTable\u00a01.\u00a0A\u00a0comparison\u00a0of\u00a0cheese\u00a0composition\u00a0of\u00a0Yak\u00a0and\u00a0Cow\u2019s\u00a0milk\u00a0\n\nFigure\u00a02.\u00a0A\u00a0general\u00a0\nscheme\u00a0of\u00a0approach\u00a0in\u00a0\nFlagship\u00a0projects\u00a0\n\n5\u00a0yrs\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a055\u201058\u00a0\n\n58\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nExpected\u00a0Outcome\u00a0\nThe\u00a0 above\u00a0 conceived\u00a0 research\u00a0 programs\u00a0 will\u00a0 bring\u00a0\n\ninnovation\u00a0 and\u00a0 expertise\u00a0 to\u00a0 Nepal\u00a0 and\u00a0 help\u00a0 in\u00a0\n\nconducting\u00a0 applied\u00a0 research\u00a0 activities\u00a0 to\u00a0 asses\u2019\u00a0 the\u00a0\n\nscientific\u00a0 effectiveness\u00a0 of\u00a0 the\u00a0 above\u00a0 mentioned\u00a0\n\nresources\u00a0 and\u00a0 value\u00a0 add\u00a0 the\u00a0 product\u00a0 for\u00a0 their\u00a0\n\ncommercial\u00a0 exploitation.\u00a0 \u00a0 The\u00a0 above\u00a0 mentioned\u00a0\n\nresearch\u00a0 streams\u00a0 will\u00a0 generate\u00a0 new\u00a0 patents\u00a0 and\u00a0\n\npublications.", "start_char_idx": 9349, "end_char_idx": 11818, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4f2a5607-bef5-40f7-9ddf-b53573890055": {"__data__": {"id_": "4f2a5607-bef5-40f7-9ddf-b53573890055", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "e05ccffd-a2e4-44b2-8e52-9e98c7991506", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "251cd5b153d0e58d4f1e877512eabc2213ce08e17ee5c9ea2ae96584a6afa991", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "83a7d2ef-245f-467b-bc3f-369c44eeb5c0", "node_type": "1", "metadata": {}, "hash": "56fcd6b29fa9b75aeadc781ee1613be9f3dceac7dbb9ebbd4ee6d54ab0c35fa0", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 revenue\u00a0 will\u00a0 be\u00a0 generated\u00a0 by\u00a0\n\nlicensing\u00a0the\u00a0technology\u00a0or\u00a0through\u00a0spin\u00a0off\u00a0companies\u00a0\n\nwith\u00a0 the\u00a0 royalties\u00a0 that\u00a0 can\u00a0 be\u00a0 shared\u00a0 mutually.\u00a0 Such\u00a0\n\ncollaborative\u00a0 research\u00a0 programs\u00a0 will\u00a0 build\u00a0 strong,\u00a0\n\nlonger\u00a0term\u00a0partnership\u00a0between\u00a0Nepal\u00a0and\u00a0overseas\u00a0\n\ninstitutions\u00a0 in\u00a0 the\u00a0 field\u00a0 S&T\u00a0 and\u00a0 extend\u00a0 the\u00a0\n\ncollaborations\u00a0 further\u00a0 in\u00a0 other\u00a0 areas\u00a0 of\u00a0 commercial\u00a0\n\npotentiality.\u00a0Nepal\u00a0as\u00a0well\u00a0as\u00a0the\u00a0collaborating\u00a0partner\u00a0\n\ncountry\u00a0stands\u00a0to\u00a0win\u00a0from\u00a0these\u00a0ventures.\u00a0 \u00a0The\u00a0S&T\u00a0\n\nstream\u00a0will\u00a0also\u00a0explore\u00a0and\u00a0help\u00a0conduct\u00a0a\u00a0feasibility\u00a0\n\nstudy\u00a0to\u00a0establish\u00a0a\u00a0National\u00a0Biotechnology\u00a0Centre\u00a0of\u00a0\n\nExcellence,\u00a0lobby\u00a0with\u00a0the\u00a0Government\u00a0to\u00a0formulate\u00a0a\u00a0\n\nBiotechnology\u00a0 policy\u00a0 and\u00a0 help\u00a0 establish\u00a0 a\u00a0 separate\u00a0\n\nBiotechnology\u00a0department\u00a0under\u00a0Ministry\u00a0of\u00a0S&T.\u00a0\n\nRisk\u00a0Factor\u00a0\nThe\u00a0 current\u00a0 political\u00a0 instability,\u00a0 lack\u00a0 of\u00a0 collaborative\u00a0\n\nagreements\u00a0 and\u00a0 the\u00a0 absence\u00a0 of\u00a0 commercial\u00a0 focus,\u00a0\n\npoor\u00a0 governance\u00a0 and\u00a0 funding\u00a0 of\u00a0 S&T\u00a0 projects,\u00a0 long\u00a0\n\nterm\u00a0 commitment,\u00a0 poor\u00a0 private\u00a0 sector\u00a0 participation\u00a0\n\nand\u00a0 the\u00a0 absence\u00a0 of\u00a0 clear\u00a0 S&T\u00a0 plan\u00a0 are\u00a0 some\u00a0 of\u00a0 the\u00a0\n\nfactors\u00a0 that\u00a0 may\u00a0 undermine\u00a0 confidence\u00a0 of\u00a0 stake\u2010\n\nholders.\u00a0 Because\u00a0 of\u00a0 these\u00a0 reasons\u00a0 the\u00a0 government\u00a0\n\nneeds\u00a0 to\u00a0 provide\u00a0 better\u00a0 initiatives\u00a0 and\u00a0 assurance\u00a0 to\u00a0\n\nprotect\u00a0such\u00a0investment\u00a0by\u00a0brining\u00a0a\u00a0special\u00a0legislation\u00a0\n\nin\u00a0S&T\u00a0investment.\u00a0The\u00a0S&T\u00a0organisations\u00a0in\u00a0Nepal\u00a0are\u00a0\n\ninterested\u00a0 to\u00a0 enter\u00a0 into\u00a0 such\u00a0 a\u00a0 partnership\u00a0 program\u00a0\n\nand\u00a0 by\u00a0 signing\u00a0 MoU\u00a0 with\u00a0 NAST;\u00a0 NRNA\u00a0 has\u00a0 shown\u00a0 its\u00a0\n\ncommitment\u00a0 to\u00a0 provide\u00a0 a\u00a0 helping\u00a0 hand\u00a0 and\u00a0 work\u00a0 in\u00a0\n\npartnership\u00a0to\u00a0achieve\u00a0the\u00a0above\u00a0goals.\u00a0\u00a0\n\nReferences\u00a0\nAdhikari,\u00a0 M.\u00a0 K.,\u00a0 2008.\u00a0 The\u00a0 medicinal\u00a0 fungi\u00a0 from\u00a0 Nepal.\u00a0 Pp.\u00a0\n\n107\u2010118.\u00a0In:\u00a0Jha,\u00a0P.\u00a0K.,\u00a0Karmacharya,\u00a0S.\u00a0B.,\u00a0Chhetri,\u00a0M.\u00a0K.,\u00a0\n\nThapa,\u00a0 C.\u00a0 B.,\u00a0 Shrestha,\u00a0 B.\u00a0 B.,\u00a0 Eds,\u00a0 \u00a0 Medicinal\u00a0 Plants\u00a0 in\u00a0\n\nNepal:\u00a0An\u00a0Anthology\u00a0of\u00a0Contemporary\u00a0Research,\u00a0Ecological\u00a0\n\nSociety,\u00a0Kathmandu,\u00a0Nepal.\u00a0\n\nBok,\u00a0J.\u00a0W.\u00a0Lermer,\u00a0L.,\u00a0Chilton,\u00a0J.,\u00a01999.\u00a0Antitumor\u00a0sterols\u00a0from\u00a0\n\nthe\u00a0mycelia\u00a0of\u00a0Cordyceps\u00a0sinensis.\u00a0Phytochemistry,\u00a051,\u00a0891\u2010\n\n898.\u00a0\n\nChen,\u00a0 Y.\u00a0 Q.,\u00a0 Hu,\u00a0 B.,\u00a0 Xu,\u00a0 F.,\u00a0 Zhang,\u00a0 W.,\u00a0 Zhou,\u00a0 H.,\u00a0 Qu,\u00a0 H.\u00a0 L.,\u00a0\n\n2004.\u00a0\u00a0Genetic\u00a0variation\u00a0of\u00a0Cordyceps\u00a0sinensis,\u00a0a\u00a0fruitbody\u00a0\n\nproducing\u00a0 entomopathogenic\u00a0 species\u00a0 from\u00a0 different\u00a0\n\ngeographical\u00a0 region\u00a0 in\u00a0 China.\u00a0 FEMS\u00a0 Microbioliological\u00a0\n\nLetttter,\u00a0230,\u00a0153\u2010158.\u00a0\n\nDong,\u00a0C.\u2010H.,\u00a0Yao,\u00a0Y.\u2010J.2008.\u00a0In\u00a0vitro\u00a0evaluation\u00a0of\u00a0antioxidant\u00a0\n\nactivities\u00a0 of\u00a0 aqueous\u00a0 extracts\u00a0 from\u00a0 natural\u00a0 and\u00a0 cultured\u00a0\n\nmycelia\u00a0of\u00a0Cordyceps\u00a0sinensis.\u00a0LWT,\u00a041,\u00a0669\u2010677.", "start_char_idx": 11820, "end_char_idx": 14320, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "83a7d2ef-245f-467b-bc3f-369c44eeb5c0": {"__data__": {"id_": "83a7d2ef-245f-467b-bc3f-369c44eeb5c0", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4f2a5607-bef5-40f7-9ddf-b53573890055", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "73d997e93a3ccd7e2451b89599bf48b79ad1bb0bde7300cda66f4bc7845c1ddd", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f3af971a-f512-4ebd-b33a-1f355012a0b8", "node_type": "1", "metadata": {}, "hash": "edfc5f9df5515a89e81fc4a932dcd3d7c9cd714f0df05e5593e6330cae64298f", "class_name": "RelatedNodeInfo"}}, "text": "Koh,\u00a0 J.\u00a0 H.,\u00a0 Kim,\u00a0 J.\u00a0 M.,\u00a0 Chang,\u00a0 U.\u00a0 J.,\u00a0 \u00a0 Sub,\u00a0 H.\u00a0 J.,\u00a0 2003.\u00a0\n\nHypocholesterolemic\u00a0 effect\u00a0 of\u00a0 hot\u00a0 water\u00a0 extract\u00a0 from\u00a0\n\nmycelia\u00a0of\u00a0Cordyceps\u00a0sinensis.\u00a0Biol.\u00a0Pharm.\u00a0Bull.\u00a026,\u00a084\u201087.\u00a0\n\nKoh,\u00a0J.\u00a0H.,\u00a0Yu,\u00a0K.W.,\u00a0Shu,\u00a0H.\u00a0J.,\u00a0Choi,\u00a0Y.\u00a0M.,\u00a0Ahn,\u00a0T.S.,\u00a02002.\u00a0\n\nActivation\u00a0 of\u00a0 macrophages\u00a0 and\u00a0 the\u00a0 intestinal\u00a0 immune\u00a0\n\nsystem\u00a0by\u00a0an\u00a0orally\u00a0administrated\u00a0decoction\u00a0from\u00a0culture\u00a0\n\nmycelia\u00a0 of\u00a0 Cordyceps\u00a0 sinensis.\u00a0 Bioscience,\u00a0 Biotechnology\u00a0\n\nand\u00a0Biochemistry,\u00a066\u00a0(2),\u00a0407\u2010411.\u00a0\n\nLi,\u00a0 S.\u00a0 P.,\u00a0 Li,\u00a0 P.,\u00a0 Dong,\u00a0 T.\u00a0 T.\u00a0 X.,\u00a0 Tsim,\u00a0 K.\u00a0 W.\u00a0 K.,\u00a0 2001.\u00a0 Anti\u2010\n\noxidation\u00a0 activity\u00a0 of\u00a0 different\u00a0 types\u00a0 of\u00a0 natural\u00a0 Cordyceps\u00a0\n\nsinensis\u00a0and\u00a0cultured\u00a0Cordyceps\u00a0mycelia.\u00a0Phytomedicine,\u00a08,\u00a0\n\n207\u2010212.\u00a0\n\nShrestha,\u00a0 B.,\u00a0 Lee,\u00a0 W.\u2010H.,\u00a0 Han,\u00a0 S.\u2010K.,\u00a0 Sung,\u00a0 J.\u2010M.,\u00a0 2006.\u00a0\n\nObservations\u00a0 on\u00a0 some\u00a0 of\u00a0 the\u00a0 mycelia\u00a0 growth\u00a0 and\u00a0\n\npigmentation\u00a0characteristics\u00a0of\u00a0Cordyceps\u00a0militaris\u00a0isolates.\u00a0\n\nMycobiology,\u00a034,\u00a083\u201091.\u00a0\n\nZhu,\u00a0 J.\u00a0 S.,\u00a0 Halpern,\u00a0 G.\u00a0 M.,\u00a0 Jones,\u00a0 K.,\u00a0 1998.\u00a0 The\u00a0 scientific\u00a0\n\nrediscovery\u00a0 of\u00a0 an\u00a0 ancient\u00a0 Chinese\u00a0 herbal\u00a0 medicine:\u00a0\n\nCordyceps\u00a0 sinensis:\u00a0 Part\u00a0 I.\u00a0 J.\u00a0 Alt.\u00a0 Comp.\u00a0 Med.\u00a0 4\u00a0 (3),\u00a0 289\u2010\n\n303.\u00a0\n\nFatty\u00a0 Acid\u00a0 Composition\u00a0 of\u00a0 Yak\u00a0 (Bos\u00a0 grunniens)\u00a0 Cheese\u00a0\n\nIncluding\u00a0 Conjugated\u00a0 Linoleic\u00a0 Acid\u00a0 and\u00a0 trans\u201018:1\u00a0 Fatty\u00a0\n\nAcids;\u00a0Mamun\u00a0M.\u00a0Or\u2010Rashid,\u00a0Nicholas\u00a0E.\u00a0Odongo,\u00a0Bhishma\u00a0\n\nSubedi,\u00a0Pralhad\u00a0Karki\u00a0and\u00a0Brian\u00a0W.\u00a0McBride,\u00a0Department\u00a0of\u00a0\n\nAnimal\u00a0and\u00a0Poultry\u00a0Science,\u00a0University\u00a0of\u00a0Guelph,\u00a0Guelph,\u00a0\n\nOntario,\u00a0Canada\u00a0N1G\u00a02W1;\u00a0Asia,\u00a0\u00a0Agric.\u00a0Food\u00a0Chem.,\u00a02008,\u00a0\n\n56\u00a0(5),\u00a0pp\u00a01654\u20131660.\u00a0\n\nThe\u00a0big\u00a0cheese\u00a0\"Nepal\u00a0is\u00a0perfect\u00a0for\u00a0cheese\u00a0production.\u00a0You\u00a0\n\ncouldn't\u00a0ask\u00a0for\u00a0more,\u00a0Nepali\u00a0Times,\u00a0\"\u00a0FROM\u00a0 ISSUE\u00a0#427\u00a0\n\n(28\u00a0NOV\u00a02008\u00a0\u2010\u00a004\u00a0DEC\u00a02008).\u00a0\u00a0\n\nHigh\u00a0 Hill\u00a0 Yak\u00a0 Cheese\u00a0 Production\u00a0 in\u00a0 Nepal:\u00a0 An\u00a0 Analysis\u00a0 of\u00a0\n\nPrivatization\u00a0 Policy\u00a0 Incorporating\u00a0 the\u00a0 Impacts\u00a0 of\u00a0 Market\u00a0\n\nFailures\u00a0 or\u00a0 Agro\u2010Industries\u00a0 in\u00a0 Developing\u00a0 Countries,\u00a0 by\u00a0\n\nLuke\u00a0 A.\u00a0 Colavito,\u00a0 Virginia\u00a0 Polytechnic\u00a0 Institute\u00a0 and\u00a0 State\u00a0\n\nUniversity,\u00a0July\u00a08,\u00a01997.\u00a0\n\nYak\u00a0Cheese:\u00a0Exotic,\u00a0but\u00a0Healthy?", "start_char_idx": 14323, "end_char_idx": 16307, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f3af971a-f512-4ebd-b33a-1f355012a0b8": {"__data__": {"id_": "f3af971a-f512-4ebd-b33a-1f355012a0b8", "embedding": null, "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-52", "node_type": "4", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "a161d139b6d2bd2483800fd1caa0fde22733135373e3b4b5aa891670feb2052c", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "83a7d2ef-245f-467b-bc3f-369c44eeb5c0", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "24e6b29acc92240b08469d3aa318862f21b66b57e1fa5327097681a0051ac2a0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d6831088-7e2f-4a33-9824-4a97d0aca3fe", "node_type": "1", "metadata": {}, "hash": "1d459dc3f40997f6fb2580b424d173e325e8a8eaedd2a76b468689a6be547303", "class_name": "RelatedNodeInfo"}}, "text": "Yak\u00a0Cheese:\u00a0Exotic,\u00a0but\u00a0Healthy?\u00a0Cheese\u00a0From\u00a0Grass\u2010Fed\u00a0Yaks\u00a0\n\nMay\u00a0 Have\u00a0 Better\u00a0 Fatty\u00a0 Acid\u00a0 Profile\u00a0 Than\u00a0 Cheese\u00a0 From\u00a0\n\nGrain\u2010Fed\u00a0 Cows,\u00a0 Study\u00a0 Shows,\u00a0By\u00a0 Miranda\u00a0 Hitti,\u00a0 WebMD\u00a0\n\nHealth\u00a0News,\u00a0WEBMED,\u00a0March\u00a014,2008.\u00a0\n\nYak\u00a0Cheese\u00a0Is\u00a0a\u00a0Miracle;\u00a0http://news.softpedia.com/news/\n\nYak\u2010Cheese\u2010is\u2010Better\u2010for\u2010the\u2010Heart81087.shtml", "start_char_idx": 16275, "end_char_idx": 16601, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d6831088-7e2f-4a33-9824-4a97d0aca3fe": {"__data__": {"id_": "d6831088-7e2f-4a33-9824-4a97d0aca3fe", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f3af971a-f512-4ebd-b33a-1f355012a0b8", "node_type": "1", "metadata": {"identifier": "njb-52", "author": "Adhikari, Raju; Adhikari, Benu", "title": "Biotechnology Growth Partnership: A Potential Flagship Program in S&T", "date": "2022-07-11", "file": "njb-52.pdf"}, "hash": "fe7c42af581ecda0b65bdbf40a4792385ce87040b444376e2a0ec45ecdb2a44c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4e14f28c-702f-4208-bcaf-f43ab9c35d2e", "node_type": "1", "metadata": {}, "hash": "3cdbbd4e5c7e8dd999eae14959941f1cec49cbf72681038602c3a2b8b9cbbb5d", "class_name": "RelatedNodeInfo"}}, "text": "6\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n49\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nBRIEF\u00a0COMMUNICATION\u00a0\u00a0\n\nIn\u00a0vitro\u00a0cultivation\u00a0and\u00a0regeneration\u00a0of\u00a0Solanum\u00a0\nmelongena(L.)\u00a0using\u00a0stem,\u00a0root\u00a0and\u00a0leaf\u00a0explants.\u00a0\n\nBishnu\u00a0Pada\u00a0Ray,\u00a0Lutful\u00a0Hassan\u00a0and\u00a0Smreeti\u00a0Kana\u00a0Sarker\u00a0\n\nBiotechnology\u00a0Laboratory,\u00a0Department\u00a0of\u00a0Biotechnology,\u00a0Bangladesh\u00a0Agricultural\u00a0University\u00a0(BAU),\u00a0Mymensingh.\u00a0\n\nAbstract\u00a0\n\nThe\u00a0treatment\u00a0combinations\u00a0was\u00a0BAP\u00a0(0,\u00a02.0,\u00a03.0\u00a0and\u00a04.0\u00a0mg/L)\u00a0and\u00a0NAA\u00a0(0,\u00a00.1,\u00a00.5,\u00a0and\u00a01.0\u00a0mg/L).\u00a0The\u00a0rate\u00a0of\u00a0\n\ncallus\u00a0formation\u00a0varied\u00a0 in\u00a0different\u00a0treatments.\u00a0The\u00a0highest\u00a0amount\u00a0of\u00a0callus\u00a0(48.66%)\u00a0 was\u00a0produced\u00a0on\u00a0MS\u00a0\n\nmedium\u00a0containing\u00a02.0\u00a0mg/l\u00a0BAP\u00a0and\u00a00.5\u00a0mg/l\u00a0NAA\u00a0from\u00a0stem\u00a0and\u00a08.2\u00a0days\u00a0required\u00a0for\u00a0callus\u00a0induction.\u00a0The\u00a0\n\nnumber\u00a0 of\u00a0 shoot\u00a0 regenerated\u00a0 through\u00a0 callus\u00a0 from\u00a0 stem\u00a0 containing\u00a0 2.0\u00a0 mg/l\u00a0 BAP\u00a0 and\u00a0 0.5\u00a0 mg/l\u00a0 NAA\u00a0 was\u00a0 3.4\u00a0\n\n(23.287%)\u00a0and\u00a0days\u00a0required\u00a0for\u00a038.8\u00a0days.\u00a0\u00a0\n\n\u00a0\nKey\u00a0words:\u00a0Regeneration,\u00a0BAP,\u00a0NAA.\u00a0\n\n\u00a0\nCorrespondence\u00a0Author:\u00a0\nE\u2010mail:\u00a0bpray2003@yahoo.com\u00a0\n\nIntroduction\u00a0\nBrinjal\u00a0(Solanum\u00a0melongena\u00a0L.),\u00a0belongs\u00a0to\u00a0the\u00a0family\u00a0\n\nSolanaceae,\u00a0is\u00a0one\u00a0of\u00a0the\u00a0most\u00a0popular,\u00a0palatable\u00a0and\u00a0\n\nnutritious\u00a0 vegetable\u00a0 crop\u00a0 in\u00a0 Bangladesh.\u00a0 It\u00a0 is\u00a0 thought\u00a0\n\nto\u00a0 be\u00a0 originated\u00a0 in\u00a0 Indian\u00a0 subcontinent\u00a0 with\u00a0 the\u00a0\n\nsecondary\u00a0 centre\u00a0 of\u00a0 origin\u00a0 in\u00a0 China\u00a0 (Zeven\u00a0 and\u00a0\n\nZhukovsky,\u00a01975).\u00a0Brinjal\u00a0 is\u00a0cultivated\u00a0throughout\u00a0the\u00a0\n\nentire\u00a0 tropics\u00a0 and\u00a0 sub\u2010tropics.\u00a0 It\u00a0 has\u00a0 higher\u00a0 calorie,\u00a0\n\niron,\u00a0phosphorus\u00a0and\u00a0riboflavin\u00a0than\u00a0tomato.\u00a0Brinjal\u00a0is\u00a0\n\nthe\u00a0second\u00a0most\u00a0important\u00a0vegetable\u00a0crop\u00a0after\u00a0potato\u00a0\n\nin\u00a0respect\u00a0of\u00a0total\u00a0acreage\u00a0(million\u00a0ha)\u00a0and\u00a0production\u00a0\n\n(370,000\u00a0mt)\u00a0in\u00a0Bangladesh\u00a0(BBS,\u00a02003).\u00a0It\u00a0also\u00a0plays\u00a0a\u00a0\n\nvital\u00a0 role\u00a0 in\u00a0 the\u00a0 national\u00a0 economy\u00a0 as\u00a0 a\u00a0 cash\u00a0 crop.\u00a0\n\nBrinjal\u00a0 is\u00a0 highly\u00a0 susceptible\u00a0 to\u00a0 different\u00a0 insects,\u00a0 pests\u00a0\n\nand\u00a0 diseases\u00a0 that\u00a0 exert\u00a0 a\u00a0 deleterious\u00a0 effect\u00a0 on\u00a0 yield,\u00a0\n\nmarket\u00a0quality,\u00a0storability\u00a0and\u00a0international\u00a0germplasm\u00a0\n\ndistribution.\u00a0 The\u00a0 seed\u2010borne\u00a0 pathogens\u00a0 of\u00a0 previous\u00a0\n\nyears\u00a0 can\u00a0 be\u00a0 perpetuated\u00a0 over\u00a0 the\u00a0 generations\u00a0 with\u00a0\n\nsymptoms\u00a0expressed.\u00a0To\u00a0overcome\u00a0this\u00a0situation,\u00a0plant\u00a0\n\ntissue\u00a0culture\u00a0offers\u00a0an\u00a0efficient\u00a0method\u00a0for\u00a0pathogen\u00a0\n\nfree\u00a0 materials\u00a0 and\u00a0 germplasm\u00a0 preservation\u00a0 of\u00a0 plants.", "start_char_idx": 45, "end_char_idx": 2275, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4e14f28c-702f-4208-bcaf-f43ab9c35d2e": {"__data__": {"id_": "4e14f28c-702f-4208-bcaf-f43ab9c35d2e", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d6831088-7e2f-4a33-9824-4a97d0aca3fe", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ca74a05d40d99900818df1f64ab71b11bba165a2f26374b971dcdc4c651f2a66", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8cf1c87d-14a8-4523-a86c-feda5e0da668", "node_type": "1", "metadata": {}, "hash": "81b8f3283385e06b2df2f1fa71c62763cb7f5c61760f61ff4aff9b7faeba292c", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0potential\u00a0value\u00a0of\u00a0tissue\u00a0culture\u00a0in\u00a0plant\u00a0breeding\u00a0\n\nhas\u00a0been\u00a0widely\u00a0recognized,\u00a0and\u00a0it\u00a0is\u00a0generally\u00a0used\u00a0as\u00a0\n\nuseful\u00a0 tool\u00a0 for\u00a0 crop\u00a0 improvement.\u00a0 Regeneration\u00a0 of\u00a0\n\nvaluable\u00a0economic\u00a0plants\u00a0through\u00a0tissue\u00a0culture\u00a0based\u00a0\n\non\u00a0the\u00a0principle\u00a0of\u00a0totipotency,\u00a0 individual\u00a0plant\u00a0cell\u00a0 is\u00a0\n\ncapable\u00a0 of\u00a0 regenerating\u00a0 new\u00a0 plantlets.\u00a0 Anwar\u00a0 et\u00a0 al.\u00a0\n\n(2002)\u00a0 cultured\u00a0 the\u00a0 aborigine\u00a0 leaf\u00a0 explants\u00a0 on\u00a0 MS\u00a0\n\nmedia\u00a0containing\u00a0IAA,\u00a0BA\u00a0(benzyl\u00a0adenine),\u00a0IBA,\u00a0NAA\u00a0or\u00a0\n\n2,4\u2010D\u00a0at\u00a02\u00a0mg/l.\u00a0NAA\u00a0produced\u00a0greenish,\u00a0fast\u2010growing\u00a0\n\ncallus.\u00a02,\u00a04\u2010D\u00a0induced\u00a0early\u00a0callus\u00a0production\u00a0from\u00a0the\u00a0\n\npetiole,\u00a0while\u00a0BA\u00a0induced\u00a0green\u00a0callus\u00a0production\u00a0from\u00a0\n\nthe\u00a0upper\u00a0surface\u00a0of\u00a0the\u00a0 lamina.\u00a0The\u00a0addition\u00a0of\u00a0NAA\u00a0\n\nor\u00a0 IBA\u00a0 at\u00a0 0.5\u00a0 mg/l\u00a0 in\u00a0 BA\u00a0 supplemented\u00a0 medium\u00a0\n\nincreased\u00a0 the\u00a0 mass\u00a0 production\u00a0 of\u00a0 callus\u00a0 and\u00a0 shoot\u00a0\n\nregeneration.\u00a0The\u00a0regeneration\u00a0efficiency\u00a0of\u00a0the\u00a0plant\u00a0\n\ndecreased\u00a0in\u00a0MS\u00a0medium\u00a0supplied\u00a0with\u00a0kinetin\u00a0(2\u00a0mg/l)\u00a0\n\nand\u00a0NAA\u00a0(0.5\u00a0mg/l).\u00a0\u00a0 \u00a0\n\nThe\u00a0 seeds\u00a0 of\u00a0 brinjal\u00a0 cv.\u00a0 Jhumki\u00a0 were\u00a0 collected\u00a0 from\u00a0\n\nBangladesh\u00a0 Agricultural\u00a0 Research\u00a0 Institute\u00a0 (BARI),\u00a0\n\nJoydebpur,\u00a0Gazipur\u00a0and\u00a0stem,\u00a0root\u00a0and\u00a0leaf\u00a0were\u00a0used\u00a0\n\nfor\u00a0 establishment\u00a0 of\u00a0 culture.\u00a0 Healthy\u00a0 seeds\u00a0 of\u00a0 brinjal\u00a0\n\ncv.\u00a0 Jhumki\u00a0 were\u00a0 collected\u00a0 from\u00a0 the\u00a0 BARI.\u00a0 The\u00a0 seeds\u00a0\n\nwere\u00a0 then\u00a0 washed\u00a0 thoroughly\u00a0 in\u00a0 running\u00a0 tap\u00a0 water.\u00a0\n\nThe\u00a0 instruments\u00a0 like\u00a0 scalpels,\u00a0 forceps,\u00a0 needles\u00a0 etc.\u00a0\n\nwere\u00a0 sterilized\u00a0 inside\u00a0 the\u00a0 Laminar\u00a0 Air\u00a0 Flow\u00a0 Cabinet.\u00a0\n\nOther\u00a0requirements\u00a0like\u00a0petridishes,\u00a0distilled\u00a0water\u00a0and\u00a0\n\nglassware\u00a0were\u00a0sterilized\u00a0by\u00a0an\u00a0autoclave.\u00a0The\u00a0surface\u00a0\n\nsterilization\u00a0of\u00a0these\u00a0seeds\u00a0was\u00a0carried\u00a0out\u00a0by\u00a0dipping\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n50\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nand\u00a0flaming\u00a0method\u00a0under\u00a0a\u00a0Laminar\u00a0Air\u00a0Flow\u00a0Cabinet\u00a0\n\nand\u00a0 others\u00a0 were\u00a0 rinsed\u00a0 in\u00a0 70%\u00a0 ethyl\u00a0 alcohol\u00a0 for\u00a0 one\u00a0\n\nminute,\u00a0 and\u00a0 then\u00a0 thoroughly\u00a0 washed\u00a0 with\u00a0 sterilized\u00a0\n\ndistilled\u00a0 water.\u00a0 The\u00a0 alcohol\u00a0 treated\u00a0 seeds\u00a0 were\u00a0\n\nsterilized\u00a0 with\u00a0 0.1%\u00a0 HgCl2\u00a0 solution\u00a0 for\u00a0 8\u201010\u00a0 minutes,\u00a0\n\nfew\u00a0drops\u00a0Tween\u201020\u00a0per\u00a0100\u00a0ml\u00a0was\u00a0also\u00a0added\u00a0at\u00a0that\u00a0\n\ntime.\u00a0 The\u00a0 seeds\u00a0 were\u00a0 then\u00a0 washed\u00a0 5\u20106\u00a0 times\u00a0 with\u00a0\n\nsterilized\u00a0distilled\u00a0water.\u00a0The\u00a0seeds\u00a0were\u00a0then\u00a0ready\u00a0for\u00a0\n\nplacement\u00a0into\u00a0the\u00a0media.\u00a0Sterilized\u00a0seeds\u00a0were\u00a0placed\u00a0\n\ninto\u00a0 seed\u00a0 germination\u00a0 medium\u00a0 in\u00a0 Petridish.\u00a0 Six\u00a0 seeds\u00a0\n\nwere\u00a0 placed\u00a0 in\u00a0 each\u00a0 Petridish.", "start_char_idx": 2278, "end_char_idx": 4635, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8cf1c87d-14a8-4523-a86c-feda5e0da668": {"__data__": {"id_": "8cf1c87d-14a8-4523-a86c-feda5e0da668", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4e14f28c-702f-4208-bcaf-f43ab9c35d2e", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "cf6dc67ace7a73b7fab749d5a8219a713e4924db3507c52f3f31730b7864dda8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a1064fa6-f707-4f66-bfef-8f2a22a3490f", "node_type": "1", "metadata": {}, "hash": "04ef479b7c49eef4d4e97b3f21ad3850deb0dc400378041ef5ff4cb13aa95e55", "class_name": "RelatedNodeInfo"}}, "text": "Six\u00a0 seeds\u00a0\n\nwere\u00a0 placed\u00a0 in\u00a0 each\u00a0 Petridish.\u00a0 The\u00a0 culture\u00a0 was\u00a0 then\u00a0\n\nincubated\u00a0 in\u00a0dark\u00a0till\u00a0the\u00a0germination\u00a0of\u00a0seeds.\u00a0These\u00a0\n\nwere\u00a0 then\u00a0 transferred\u00a0 to\u00a0 16\u00a0 hours\u00a0 light\u00a0 for\u00a0 normal\u00a0\n\nseedling\u00a0growth.\u00a0MS\u00a0(Murashige\u00a0&\u00a0Skoog,\u00a01962)\u00a0basal\u00a0\n\nmedium\u00a0 with\u00a0 different\u00a0 concentrations\u00a0 and\u00a0\n\ncombinations\u00a0of\u00a0BAP\u00a0(0,\u00a02.0,\u00a03.0\u00a0and\u00a04.0\u00a0mg/l)\u00a0and\u00a0NAA\u00a0\n\n(0,\u00a0 0.1,\u00a0 0.5\u00a0 and\u00a0 1.0\u00a0 mg/l)\u00a0 were\u00a0 used.\u00a0 Six\u00a0 pieces\u00a0 (2\u20103\u00a0\n\nmm)\u00a0of\u00a0stem\u00a0segments\u00a0were\u00a0arranged\u00a0horizontally\u00a0on\u00a0\n\neach\u00a0petridish\u00a0 and\u00a0 gently\u00a0pressed\u00a0 into\u00a0 the\u00a0 surface\u00a0 of\u00a0\n\nthe\u00a0 sterilized\u00a0 culture\u00a0 medium\u00a0 with\u00a0 various\u00a0\n\nconcentrations\u00a0 and\u00a0 combinations\u00a0 of\u00a0 hormones\u00a0 like\u00a0\n\nNAA\u00a0 and\u00a0 BAP.\u00a0 The\u00a0 petridish\u00a0 was\u00a0 covered\u00a0 and\u00a0 sealed\u00a0\n\nwith\u00a0 Para\u00a0 film.\u00a0 Leaf\u00a0 segment\u00a0 from\u00a0 each\u00a0 germinated\u00a0\n\nseedling\u00a0 were\u00a0 cut\u00a0 into\u00a0 small\u00a0 pieces\u00a0 using\u00a0 sterilized\u00a0\n\nscalpel\u00a0under\u00a0a\u00a0Laminar\u00a0Air\u00a0Flow\u00a0Cabinet.\u00a0Six\u00a0pieces\u00a0of\u00a0\n\nleaf\u00a0 segments\u00a0 were\u00a0 arranged\u00a0 on\u00a0 each\u00a0 petridish\u00a0 and\u00a0\n\ngently\u00a0pressed\u00a0into\u00a0the\u00a0surface\u00a0of\u00a0the\u00a0sterilized\u00a0culture\u00a0\n\nmedium.\u00a0 The\u00a0 Petri\u00a0 dishes\u00a0 were\u00a0 covered\u00a0 and\u00a0 sealed\u00a0\n\nwith\u00a0Para\u00a0film.\u00a0Root\u00a0tip\u00a0segments\u00a0(0.5mm)\u00a0were\u00a0placed\u00a0\n\non\u00a0 a\u00a0 sterilized\u00a0 petridish\u00a0 under\u00a0 a\u00a0 Laminar\u00a0 Air\u00a0 Flow\u00a0\n\nCabinet.\u00a0 The\u00a0 petridish\u00a0 was\u00a0 covered\u00a0 and\u00a0 sealed\u00a0 with\u00a0\n\nPara\u00a0film.\u00a0\n\nPlant\u00a0regeneration\u00a0from\u00a0induced\u00a0calli\u00a0of\u00a0brinjal\u00a0through\u00a0\n\nMS\u00a0 medium\u00a0 supplemented\u00a0 with\u00a0 different\u00a0\n\ncombinations\u00a0of\u00a0hormones\u00a0was\u00a0used.\u00a0Stem,\u00a0 leaf,\u00a0root\u00a0\n\nsegments\u00a0 were\u00a0 used\u00a0 as\u00a0 explants\u00a0 to\u00a0 observe\u00a0 their\u00a0\n\ncallusing\u00a0response.\u00a0Thirty\u00a0explants\u00a0were\u00a0 inoculated\u00a0 in\u00a0\n\neach\u00a0 treatment.\u00a0 Among\u00a0 the\u00a0 explants\u00a0 used,\u00a0 stem\u00a0 was\u00a0\n\ncomparatively\u00a0 more\u00a0 responsive\u00a0 for\u00a0 callus\u00a0 induction\u00a0\n\nthan\u00a0other\u00a0explants.\u00a0The\u00a0combined\u00a0effect\u00a0of\u00a0explants\u00a0\n\nand\u00a0different\u00a0combinations\u00a0of\u00a0BAP\u00a0and\u00a0NAA\u00a0on\u00a0callus\u00a0\n\ninduction\u00a0has\u00a0been\u00a0presented\u00a0in\u00a0Table\u00a01.Stem\u00a0showed\u00a0\n\nthe\u00a0 highest\u00a0 callusing\u00a0 mean\u00a0 (8.363)\u00a0 whereas\u00a0 leaf\u00a0\n\nsegments\u00a0 gave\u00a0 callusing\u00a0 mean\u00a0 (6.950)\u00a0 and\u00a0 root\u00a0\n\nsegments\u00a0had\u00a0the\u00a0 lowest\u00a0callusing\u00a0mean\u00a0(6.688).\u00a0The\u00a0\n\nhighest\u00a0callusing\u00a0was\u00a0obtained\u00a0in\u00a02.0\u00a0mg/l\u00a0BAP\u00a0(8.567)\u00a0\n\nand\u00a00.5\u00a0mg/l\u00a0NAA\u00a0(9.333).\u00a0Also\u00a0minimum\u00a0days\u00a0(9.725)\u00a0\n\nwere\u00a0 required\u00a0 for\u00a0 callus\u00a0 induction\u00a0 from\u00a0 stem.\u00a0 Days\u00a0\n\nrequired\u00a0for\u00a0callus\u00a0 induction\u00a0from\u00a02.0\u00a0mg/l\u00a0BAP\u00a0were\u00a0\n\n10.350\u00a0and\u00a0days\u00a0required\u00a0for\u00a0callus\u00a0induction\u00a0from\u00a00.5\u00a0\n\nmg/l\u00a0were\u00a010.367.", "start_char_idx": 4588, "end_char_idx": 6853, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a1064fa6-f707-4f66-bfef-8f2a22a3490f": {"__data__": {"id_": "a1064fa6-f707-4f66-bfef-8f2a22a3490f", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8cf1c87d-14a8-4523-a86c-feda5e0da668", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "e091869d2aaefb8cb5c69d0b12e1bf511d50eceb626f5aebbe9374fd56c9ac79", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "74c07ec0-9b90-4b67-8bdd-7cd0c5046c78", "node_type": "1", "metadata": {}, "hash": "1c96d7900154fcd240e81f9e2ab59e6ba5cefce73574588aa936b113177acf0d", "class_name": "RelatedNodeInfo"}}, "text": "In\u00a0case\u00a0of\u00a0stem,\u00a0among\u00a0the\u00a0different\u00a0\n\ncombination\u00a0 of\u00a0 MS\u00a0 media\u00a0 containing\u00a0 2.0\u00a0 mg/l\u00a0 BAP\u00a0 +\u00a0\n\n0.5\u00a0mg/l\u00a0NAA\u00a0and\u00a04.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0showed\u00a0\n\nbetter\u00a0 callus\u00a0 induction\u00a0 i.e.\u00a0 14.600\u00a0 and\u00a0 11.600\u00a0\n\nrespectively\u00a0 out\u00a0 of\u00a0 30\u00a0 cultured\u00a0 explants\u00a0 in\u00a0 Fig.\u00a0 2\u00a0 and\u00a0\n\nFig.\u00a0 3.\u00a0 On\u00a0 the\u00a0 other\u00a0 hand,\u00a0 in\u00a0 case\u00a0 of\u00a0 leaf\u00a0 the\u00a0\n\ncombination\u00a0of\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0showed\u00a0\n\nbetter\u00a0callus\u00a0induction\u00a0i.e.\u00a013.4.\u00a0The\u00a0explants\u00a0cultured\u00a0\n\non\u00a0MS\u00a0medium\u00a0without\u00a0hormones\u00a0did\u00a0not\u00a0produce\u00a0any\u00a0\n\ncallus.\u00a0 It\u00a0 was\u00a0 also\u00a0 found\u00a0 that\u00a0 calli\u00a0 were\u00a0 induced\u00a0 in\u00a0\n\nmedium\u00a0supplemented\u00a0with\u00a0BAP\u00a0and\u00a0NAA\u00a0which\u00a0 is\u00a0 in\u00a0\n\nsupport\u00a0 of\u00a0 the\u00a0 results\u00a0 obtained\u00a0 by\u00a0 Jayasree\u00a0 et\u00a0 al.\u00a0\n\n(2001).\u00a0The\u00a0percentage\u00a0of\u00a0callus\u00a0induction\u00a0was\u00a0highest\u00a0\n\nin\u00a0MS\u00a0media\u00a0containing\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0\n\nfrom\u00a0stem\u00a0i.e.\u00a048.666%\u00a0followed\u00a0by\u00a0callus\u00a0induction\u00a0in\u00a0\n\nFigure\u00a01:\u00a0Seed\u00a0germination\u00a0from\u00a0brinjal\u00a0cv.\u00a0Jhumki\u00a0on\u00a0\nMS\u00a0media\u00a0without\u00a0\u00a0hormones\u00a0at\u00a07\u00a0days \u00a0\n\nFigure\u00a02.\u00a0Callus\u00a0induction\u00a0from\u00a0brinjal\u00a0cv.\u00a0Jhumki\u00a0on\u00a0MS\u00a0\nmedia\u00a0with\u00a0hormones\u00a0(BAP\u00a0and\u00a0NAA)\u00a0at\u00a022\u00a0days \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n51\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nExplants\u00a0 were\u00a0 cultured\u00a0 on\u00a0 MS\u00a0 media\u00a0 supplemented\u00a0\n\nwith\u00a0different\u00a0combinations\u00a0and\u00a0concentrations\u00a0of\u00a0BAP\u00a0\n\n(0,\u00a02.0,\u00a03.0\u00a0and\u00a04.0\u00a0mg/l)\u00a0and\u00a0NAA\u00a0(0,\u00a00.1,\u00a00.5,\u00a0and\u00a01.0\u00a0\n\nmg/l).\u00a0 The\u00a0 highest\u00a0 amount\u00a0 of\u00a0 callus\u00a0 (48.66%)\u00a0 was\u00a0\n\nproduced\u00a0on\u00a0MS\u00a0medium\u00a0containing\u00a02.0\u00a0mg/l\u00a0BAP\u00a0and\u00a0\n\n0.5\u00a0 mg/l\u00a0 NAA\u00a0 from\u00a0 stem\u00a0 and\u00a0 8.2\u00a0 days\u00a0 required\u00a0 for\u00a0\n\ncallus\u00a0formation.\u00a0The\u00a0growth\u00a0of\u00a0callus\u00a0was\u00a0faster\u00a0on\u00a0MS\u00a0\n\nmedia\u00a0supplemented\u00a0with\u00a02.0\u00a0mg/l\u00a0BAP\u00a0and\u00a00.5\u00a0mg/l\u00a0\n\nNAA\u00a0 from\u00a0 the\u00a0 stem.\u00a0 \u00a0 Maximum\u00a0 number\u00a0 of\u00a0 plant\u00a0\n\nregeneration\u00a0through\u00a0callus\u00a0from\u00a0stem\u00a0containing\u00a02.0\u00a0\n\nmg/l\u00a0 BAP\u00a0 and\u00a0 0.5\u00a0 mg/l\u00a0 NAA\u00a0 were\u00a0 3.4\u00a0 (23.287%)\u00a0 and\u00a0\n\nfrom\u00a0 leaf\u00a0 containing\u00a0 2.0\u00a0 mg/l\u00a0 BAP\u00a0 and\u00a0 0.5\u00a0 mg/l\u00a0 NAA\u00a0\n\nwere\u00a01.6\u00a0(11.94%).", "start_char_idx": 6854, "end_char_idx": 8730, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "74c07ec0-9b90-4b67-8bdd-7cd0c5046c78": {"__data__": {"id_": "74c07ec0-9b90-4b67-8bdd-7cd0c5046c78", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a1064fa6-f707-4f66-bfef-8f2a22a3490f", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "b113693dc4ebfbfc56a6bde6f30922aadd93ca12230dd9aeaf54a25cc3c4a916", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cd4dd7e3-8ae0-4629-9fa5-7b3bc65cb50f", "node_type": "1", "metadata": {}, "hash": "c2c811deb801c321373be45766974fa2d17df7f0b4a045457e4f797beee4e67f", "class_name": "RelatedNodeInfo"}}, "text": "Acknowledgement\u00a0\nAll\u00a0 praises\u00a0 are\u00a0 due\u00a0 to\u00a0 Almighty\u00a0 God\u00a0 ,for\u00a0 bestowing\u00a0\n\nmercy\u00a0upon\u00a0me\u00a0and\u00a0for\u00a0imbibing\u00a0confidence\u00a0on\u00a0me\u00a0to\u00a0\n\nmaterialize\u00a0 the\u00a0 research\u00a0 work\u00a0 .The\u00a0 author\u00a0 deem\u00a0 it\u00a0 a\u00a0\n\nproud\u00a0 privilege\u00a0 to\u00a0 express\u00a0 his\u00a0 deep\u00a0 gratitude,\u00a0 over\u00a0\n\nindebtedness\u00a0 sincere\u00a0 appreciation\u00a0 to\u00a0 his\u00a0 reverend\u00a0\n\nteacher\u00a0 and\u00a0 supervisor\u00a0 Foreign\u00a0 professor\u00a0 Dr.\u00a0 Lutful\u00a0\n\nHassan,\u00a0 Dept.\u00a0 of\u00a0 Genetics\u00a0 &\u00a0 Plant\u00a0\n\nBreeding\u00a0 ,Bangladesh\u00a0 Agricultural\u00a0 University\u00a0 ,\u00a0\n\nMymensingh\u00a0 for\u00a0 his\u00a0 constant\u00a0 supervision,\u00a0 untiring\u00a0\n\nassistance\u00a0 ,\u00a0 scholastic\u00a0 ,continuous\u00a0 impression\u00a0 and\u00a0\n\nconstructive\u00a0comments.\u00a0The\u00a0authors\u00a0also\u00a0wish\u00a0to\u00a0thank\u00a0\n\nUSDA\u00a0 project\u00a0 for\u00a0 providing\u00a0 fund.\u00a0 This\u00a0 paper\u00a0 is\u00a0\n\nsupported\u00a0 by\u00a0 Bangladesh\u00a0 Association\u00a0 for\u00a0\n\nBiotechnology\u00a0(BABT). \u00a0\n\nleaf.\u00a0The\u00a0combination\u00a0of\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0\n\nrequired\u00a0 8.2\u00a0 days\u00a0 for\u00a0 callus\u00a0 induction\u00a0 from\u00a0 stem\u00a0\n\nexplants.\u00a0 On\u00a0 the\u00a0 otherhand,\u00a0 the\u00a0 combination\u00a0 of\u00a0 2.0\u00a0\n\nmg/l\u00a0BAP\u00a0+\u00a00.1\u00a0mg/l\u00a0NAA\u00a0needed\u00a010.8\u00a0days\u00a0for\u00a0callus\u00a0\n\nfrom\u00a0 root\u00a0 explants.\u00a0 So,\u00a0 callus\u00a0 induction\u00a0 from\u00a0 stem\u00a0\n\nrequired\u00a0minimum\u00a0days.\u00a0Among\u00a0the\u00a0supplements,\u00a0the\u00a0\n\nhighest\u00a0 regeneration\u00a0 potentiality\u00a0 observed\u00a0 from\u00a0 2.0\u00a0\n\nmg/l\u00a0BAP\u00a0(0.717)\u00a0and\u00a00.5\u00a0mg/l\u00a0NAA\u00a0(0.667).\u00a0But\u00a0there\u00a0\n\nwas\u00a0 no\u00a0 regeneration\u00a0 ability\u00a0 without\u00a0 hormones.\u00a0 The\u00a0\n\ncombined\u00a0effect\u00a0of\u00a0different\u00a0combinations\u00a0of\u00a0BAP\u00a0and\u00a0\n\nNAA\u00a0in\u00a0MS\u00a0medium\u00a0on\u00a0plant\u00a0regeneration\u00a0from\u00a0stem,\u00a0\n\nleaf\u00a0and\u00a0root\u00a0of\u00a0brinjal\u00a0cv.\u00a0Jhumki\u00a0have\u00a0been\u00a0presented\u00a0\n\nin\u00a0 Table\u00a0 2.\u00a0 Various\u00a0 combinations\u00a0 of\u00a0 supplements\u00a0\n\nshowed\u00a0 significant\u00a0 variation\u00a0 in\u00a0 regeneration\u00a0 ability.\u00a0\n\nAmong\u00a0the\u00a0used\u00a0combinations,\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0\n\nNAA\u00a0showed\u00a0the\u00a0highest\u00a0regeneration\u00a0of\u00a0plantlets\u00a0from\u00a0\n\nstem\u00a0 (3.400).\u00a0 The\u00a0 regeneration\u00a0 of\u00a0 plantlets\u00a0 was\u00a0 (1.6)\u00a0\n\nfrom\u00a0 leaf\u00a0 in\u00a0 2.0\u00a0 mg/l\u00a0 BAP\u00a0 and\u00a0 0.5\u00a0 mg/l\u00a0 NAA\u00a0\n\ncombinations.\u00a0 Root\u00a0 showed\u00a0 lowest\u00a0 regeneration.\u00a0 The\u00a0\n\npercentage(i.e.\u00a023.28%)\u00a0of\u00a0regeneration\u00a0was\u00a0recorded\u00a0\n\nthe\u00a0highest\u00a0in\u00a0MS\u00a0media\u00a0containing\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0\n\nmg/l\u00a0 NAA\u00a0 from\u00a0 stem\u00a0 and\u00a0 days\u00a0 required\u00a0 for\u00a0\n\nregeneration\u00a0 was\u00a0 minimum\u00a0 (38.8\u00a0 days).\u00a0 The\u00a0\n\npercentage\u00a0 (11.94%)\u00a0 of\u00a0 regeneration\u00a0 was\u00a0 the\u00a0 highest\u00a0\n\nin\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0from\u00a0leaf.\u00a0i.e.\u00a01.6\u00a0and\u00a0\n\npercentage\u00a0 of\u00a0 regeneration\u00a0 from\u00a0 root\u00a0 is\u00a0 the\u00a0 lowest.\u00a0\n\nPlant\u00a0regeneration\u00a0from\u00a0leaf\u00a0in\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0\n\nNAA\u00a0combination\u00a0required\u00a0minimum\u00a0days\u00a0(46.2\u00a0days).", "start_char_idx": 8733, "end_char_idx": 11071, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cd4dd7e3-8ae0-4629-9fa5-7b3bc65cb50f": {"__data__": {"id_": "cd4dd7e3-8ae0-4629-9fa5-7b3bc65cb50f", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "74c07ec0-9b90-4b67-8bdd-7cd0c5046c78", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "d1174927547bd90aa28dcca214a61c0da3fe72cd8717f8bdedca1a6966aef6a8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c986a51f-6eba-4fbd-b1b3-7718ab15c9b5", "node_type": "1", "metadata": {}, "hash": "373006e76188e8fc8cbf2a87cfd1e4c9df72d118ac8b778d4542208c54f6b073", "class_name": "RelatedNodeInfo"}}, "text": "From\u00a0 the\u00a0 above\u00a0 discussion,\u00a0 we\u00a0 found\u00a0 that\u00a0 the\u00a0 best\u00a0\n\nshoot\u00a0 regeneration\u00a0 was\u00a0 recorded\u00a0 from\u00a0 media\u00a0\n\nsupplemented\u00a0with\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0NAA\u00a0in\u00a0Fig.\u00a0\n\n4.\u00a0\n\nFigure\u00a03.\u00a0Callus\u00a0induction\u00a0from\u00a0brinjal\u00a0cv.\u00a0Jhumki\u00a0on\u00a0MS\u00a0\nmedia\u00a0with\u00a0hormones\u00a0(BAP\u00a0and\u00a0NAA)\u00a0at\u00a022\u00a0days. \u00a0\n\nFigure\u00a04.\u00a0Direct\u00a0regeneration\u00a0from\u00a0brinjal\u00a0cv.\u00a0Jhumki\u00a0on\u00a0\nMS\u00a0medium\u00a0supplemented\u00a0with\u00a02.0\u00a0mg/l\u00a0BAP\u00a0+\u00a00.5\u00a0mg/l\u00a0\nNAA \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n52\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nTreatment\u00a0combinations\u00a0\nNo.\u00a0of\u00a0explant\u00a0showing\u00a0\n\ncallus\u00a0induction\u00a0\n%\u00a0of\u00a0callus\u00a0induction\u00a0\n\nDays\u00a0required\u00a0for\u00a0callus\u00a0\ninduction\u00a0Explants\u00a0\n\nTreatments\u00a0\nBAP\u00a0(mg/l)\u00a0 NAA\u00a0(mg/l)\u00a0\n\n\u00a0\u00a0\n\nStem\u00a0\n\n0\u00a0\n\n0\u00a0 0.000\u00a0R\u00a0 0.000\u00a0R\u00a0 0.000I\u00a0\n0.1\u00a0 7.000\u00a0JKLMN\u00a0 23.333\u00a0JKLMN\u00a0 10.400\u00a0ABCDEF\u00a0\n0.5\u00a0 6.600\u00a0KLMNO\u00a0 22.000\u00a0KLMNO\u00a0 10.44\u00a0ABCDEF\u00a0\n1.0\u00a0 7.400\u00a0HIJKLM\u00a0 \u00a0 24.066\u00a0HIJKIM\u00a0 10.600\u00a0ABCDEF\u00a0\n\n2.0\u00a0\n\n0\u00a0 6.200\u00a0LMNO\u00a0 20.660\u00a0LMNO\u00a0 10.400\u00a0ABCDEF\u00a0\n0.1\u00a0 8.600\u00a0FGHI\u00a0 28.666\u00a0FGHI\u00a0 9.600\u00a0EBCDEFG\u00a0\n0.5\u00a0 14.600\u00a0A\u00a0 48.666\u00a0A\u00a0 8.200\u00a0GH\u00a0\n1.0\u00a0 9.600\u00a0DEFG\u00a0 32.000\u00a0DEFG\u00a0 9.600\u00a0EFG\u00a0\n\n3.0\u00a0\n\n0\u00a0 6.800\u00a0KLMNO\u00a0 22.660\u00a0KLMNO\u00a0 11.200\u00a0ABCDE\u00a0\n0.1\u00a0 9.400\u00a0DEFG\u00a0 31.330\u00a0DEFG\u00a0 10.800\u00a0ABCDE\u00a0\n0.5\u00a0 10.200\u00a0DE\u00a0 34.000\u00a0DE\u00a0 9.800\u00a0DEFG\u00a0\n1.0\u00a0 9.200\u00a0DEFG\u00a0 30.666\u00a0DEFG\u00a0 10.600\u00a0ABCDEF\u00a0\n\n4.0\u00a0\n\n0\u00a0 6.600\u00a0KLMNO\u00a0 22.000\u00a0KLMNO\u00a0 11.400\u00a0ABCDE\u00a0\n0.1\u00a0 10.000\u00a0DEF\u00a0 33.333\u00a0DEF\u00a0 11.200\u00a0ABCDE\u00a0\n0.5\u00a0 11.600\u00a0C\u00a0 38.666\u00a0C\u00a0 10.000\u00a0CDEF\\G\u00a0\n1.0\u00a0 10.000\u00a0DEF\u00a0 33.333\u00a0DEF\u00a0 11.400\u00a0ABCDE\u00a0\n\n\u00a0\u00a0\n\nLeaf\u00a0\n\n0\u00a0\n\n0\u00a0 0.000\u00a0R\u00a0 0.000\u00a0R\u00a0 0.000\u00a0ABCDE\u00a0\n0.1\u00a0 5.800\u00a0NOP\u00a0 19.333\u00a0NOP\u00a0 11.000\u00a0ABCDE\u00a0\n0.5\u00a0 6.000\u00a0MNO\u00a0 20.000\u00a0MNO\u00a0 10.800\u00a0ABCDE\u00a0\n1.0\u00a0 9.200\u00a0DEFG\u00a0 30.666\u00a0DEFG\u00a0 10.600\u00a0ABCDF\u00a0\n\n2.0\u00a0\n\n0\u00a0 4.600\u00a0PQ\u00a0 15.330\u00a0PQ\u00a0 11.000\u00a0ABCDE\u00a0\n0.1\u00a0 7.400\u00a0IJKLM\u00a0 24.660\u00a0IJKLM\u00a0 10.200\u00a0BCDEF\u00a0\n0.", "start_char_idx": 11074, "end_char_idx": 12765, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c986a51f-6eba-4fbd-b1b3-7718ab15c9b5": {"__data__": {"id_": "c986a51f-6eba-4fbd-b1b3-7718ab15c9b5", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cd4dd7e3-8ae0-4629-9fa5-7b3bc65cb50f", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "4d72243979f34edbbeeae37fa235e0abad751459083578c935fc66baba36ee34", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "18ad941e-4b95-41b6-89ce-288075f2a1b4", "node_type": "1", "metadata": {}, "hash": "c7c0498973e9eb11eee7755f69a781cbba769c006e52307998516ea571820236", "class_name": "RelatedNodeInfo"}}, "text": "660\u00a0IJKLM\u00a0 10.200\u00a0BCDEF\u00a0\n0.5\u00a0 13.400\u00a0B\u00a0 44.660\u00a0B\u00a0 8.600\u00a0FH\u00a0\n1.0\u00a0 9.800\u00a0DEF\u00a0 32.666\u00a0DEF\u00a0 10.800\u00a0ABCDE\u00a0\n\n3.0\u00a0\n\n0\u00a0 4.400\u00a0Q\u00a0 14.666\u00a0Q\u00a0 10.800\u00a0ABCDE\u00a0\n0.1\u00a0 6.400\u00a0KLMNO\u00a0 21.333\u00a0KLMNO\u00a0 10.800\u00a0ABCDE\u00a0\n0.5\u00a0 6.200\u00a0LMNO\u00a0 20.660\u00a0LMNO\u00a0 11.200\u00a0ABCDE\u00a0\n1.0\u00a0 10.000\u00a0DEF\u00a0 33.333\u00a0DEF\u00a0 10.800\u00a0ABCDE\u00a0\n\n4.0\u00a0\n\n0\u00a0 4.400\u00a0Q\u00a0 14.666\u00a0Q\u00a0 11.400\u00a0ABCDE\u00a0\n0.1\u00a0 7.600\u00a0HIJK\u00a0 25.333\u00a0HIJK\u00a0 11.600\u00a0ABCDE\u00a0\n0.5\u00a0 7.400\u00a0IJKLM\u00a0 24.661\u00a0IJKLM\u00a0 11.600\u00a0ABCDE\u00a0\n1.0\u00a0 8.600\u00a0FGHI\u00a0 28.660\u00a0FGHI\u00a0 11.200\u00a0ABCDE\u00a0\n\n\u00a0\u00a0\n\nRoot\u00a0\n\n0\u00a0\n\n0\u00a0 0.000\u00a0R\u00a0 0.000\u00a0R\u00a0 0.000\u00a0I\u00a0\n0.1\u00a0 4.400\u00a0Q\u00a0 14.666\u00a0Q\u00a0 12.200\u00a0AB\u00a0\n0.5\u00a0 6.400\u00a0KLMNO\u00a0 21.333\u00a0KLMNO\u00a0 12.400\u00a0A\u00a0\n1.0\u00a0 6.600\u00a0KLMNO\u00a0 22.000\u00a0KLMNO\u00a0 11.800\u00a0ABCD\u00a0\n\n2.0\u00a0\n\n0\u00a0 4.200\u00a0Q\u00a0 14.000\u00a0Q\u00a0 12.000\u00a0ABC\u00a0\n0.1\u00a0 6.600\u00a0KLMNO\u00a0 22.000\u00a0KLMNO\u00a0 11.400\u00a0ABCDE\u00a0\n0.5\u00a0 9.600\u00a0DEFG\u00a0 32.000\u00a0DEFG\u00a0 10.800\u00a0ABCDE\u00a0\n1.0\u00a0 8.200\u00a0GHIJ\u00a0 27.330\u00a0GHIJ\u00a0 11.600\u00a0ABCDE\u00a0\n\n3.0\u00a0\n\n0\u00a0 5.400\u00a0OPQ\u00a0 18.000\u00a0PQ\u00a0 11.400\u00a0ABCDE\u00a0\n0.1\u00a0 7.800\u00a0HIJK\u00a0 26.000\u00a0HIJK\u00a0 11.000\u00a0ABCDE\u00a0\n0.5\u00a0 10.600\u00a0CD\u00a0 35.330\u00a0CD\u00a0 10.200\u00a0BCDEF\u00a0\n1.0\u00a0 8.800\u00a0EFGH\u00a0 29.330\u00a0EFGH\u00a0 11.600\u00a0ABCDE\u00a0\n\n4.0\u00a0\n\n0\u00a0 4.400\u00a0Q\u00a0 14.666\u00a0Q\u00a0 7.600\u00a0H\u00a0\n0.1\u00a0 6.800\u00a0JKLMNO\u00a0 22.660\u00a0JKLMNO\u00a0 11.800\u00a0ABCD\u00a0\n0.5\u00a0 9.400\u00a0DEFG\u00a0 31.330\u00a0DEFG\u00a0 10.400\u00a0ABCDEF\u00a0\n1.0\u00a0 7.800\u00a0HIJK\u00a0 26.00\u00a0HIJK\u00a0 11.400\u00a0ABCDE\u00a0\n\nTable\u00a01.\u00a0The\u00a0effect\u00a0of\u00a0BAP\u00a0and\u00a0NAA\u00a0in\u00a0MS\u00a0medium\u00a0on\u00a0callus\u00a0induction\u00a0from\u00a0different\u00a0explants.\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n53\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nTable\u00a02.\u00a0The\u00a0effect\u00a0of\u00a0BAP\u00a0and\u00a0NAA\u00a0in\u00a0MS\u00a0medium\u00a0on\u00a0plant\u00a0regeneration\u00a0from\u00a0different\u00a0explants. \u00a0\n\nTreatment\u00a0combinations\u00a0 No.", "start_char_idx": 12738, "end_char_idx": 14239, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "18ad941e-4b95-41b6-89ce-288075f2a1b4": {"__data__": {"id_": "18ad941e-4b95-41b6-89ce-288075f2a1b4", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c986a51f-6eba-4fbd-b1b3-7718ab15c9b5", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "2962128cf80db533643a3c766c20b4920ed84d09b34c950bf828e4a83759a920", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "672eb55d-3060-4e56-a724-ddc085e31be5", "node_type": "1", "metadata": {}, "hash": "c98a43669d2946088dd0f36211c0588387c68838c907da8a8bc92f078fd8ebf0", "class_name": "RelatedNodeInfo"}}, "text": "Treatment\u00a0combinations\u00a0 No.\u00a0of\u00a0plants\u00a0regenerated\u00a0through\u00a0\ncallus\u00a0\n\n%\u00a0of\u00a0regeneration\u00a0 Days\u00a0required\u00a0for\u00a0regeneration\u00a0\nExplants\u00a0 BAP\u00a0(mg/l)\u00a0 NAA\u00a0(mg/l)\u00a0\n\nStem\u00a0\n\n0\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.5\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n\n2\u00a0\n\n0\u00a0 0.200\u00a0CD\u00a0 3.222\u00a0CD\u00a0 39.200\u00a0G\u00a0\n0.1\u00a0 0.600\u00a0CD\u00a0 6.976\u00a0CD\u00a0 39.800\u00a0G\u00a0\n0.5\u00a0 3.400\u00a0A\u00a0 23.287\u00a0A\u00a0 38.800\u00a0G\u00a0\n1\u00a0 0.600\u00a0CD\u00a0 6.25\u00a0CD\u00a0 39.000\u00a0G\u00a0\n\n3\u00a0\n\n0\u00a0 0.200\u00a0CD\u00a0 2.94\u00a0CD\u00a0 39.400\u00a0G\u00a0\n0.1\u00a0 0.800\u00a0C\u00a0 8.510\u00a0C\u00a0 39.800\u00a0G\u00a0\n0.5\u00a0 0.800\u00a0C\u00a0 7.843\u00a0C\u00a0 39.800\u00a0G\u00a0\n1\u00a0 0.600\u00a0CD\u00a0 6.521\u00a0CD\u00a0 39.600\u00a0G\u00a0\n\n4\u00a0\n\n0\u00a0 0.400\u00a0CD\u00a0 6.060\u00a0CD\u00a0 40.000\u00a0G\u00a0\n0.1\u00a0 0.600\u00a0CD\u00a0 6.000\u00a0CD\u00a0 40.000\u00a0G\u00a0\n0.5\u00a0 0.400\u00a0CD\u00a0 3.448\u00a0CD\u00a0 39.800\u00a0G\u00a0\n1\u00a0 0.400\u00a0CD\u00a0 4.00\u00a0CD\u00a0 39.600\u00a0G\u00a0\n\nLeaf\u00a0\n\n0\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.5\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n\n2\u00a0\n\n0\u00a0 0.400\u00a0CD\u00a0 8.695\u00a0CD\u00a0 48.800\u00a0CD\u00a0\n0.1\u00a0 0.600\u00a0CD\u00a0 8.108\u00a0CD\u00a0 48.000\u00a0DE\u00a0\n0.5\u00a0 1.600\u00a0B\u00a0 11.940\u00a0B\u00a0 46.200\u00a0F\u00a0\n1\u00a0 0.600\u00a0CD\u00a0 6.122\u00a0CD\u00a0 49.000\u00a0CD\u00a0\n\n3\u00a0\n\n0\u00a0 0.400\u00a0CD\u00a0 9.090\u00a0CD\u00a0 48.800\u00a0CD\u00a0\n0.1\u00a0 0.400\u00a0CD\u00a0 6.25\u00a0CD\u00a0 48.400\u00a0CDE\u00a0\n0.5\u00a0 0.600\u00a0CD\u00a0 9.677\u00a0CD\u00a0 47.400\u00a0E\u00a0\n1\u00a0 0.400\u00a0CD\u00a0 4.00\u00a0CD\u00a0 48.200CDE\u00a0\n\n4\u00a0\n\n0\u00a0 0.200\u00a0CD\u00a0 4.545\u00a0CD\u00a0 49.000\u00a0CD\u00a0\n0.1\u00a0 0.400\u00a0CD\u00a0 5.361\u00a0CD\u00a0 49.000\u00a0BC\u00a0\n0.5\u00a0 0.400\u00a0CD\u00a0 5.405\u00a0CD\u00a0 50.200\u00a0C\u00a0\n1\u00a0 0.400\u00a0CD\u00a0 4.651\u00a0CD\u00a0 49.200\u00a0BC\u00a0\n\nRoot\u00a0\n\n0\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.5\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n\n2\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.5\u00a0 0.200\u00a0CD\u00a0 2.083\u00a0CD\u00a0 60.200\u00a0A\u00a0\n1\u00a0 0.200\u00a0CD\u00a0 2.439\u00a0CD\u00a0 60.000\u00a0A\u00a0\n\n3\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 0.200\u00a0CD\u00a0 2.564\u00a0CD\u00a0 59.600\u00a0A\u00a0\n0.5\u00a0 0.", "start_char_idx": 14212, "end_char_idx": 15560, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "672eb55d-3060-4e56-a724-ddc085e31be5": {"__data__": {"id_": "672eb55d-3060-4e56-a724-ddc085e31be5", "embedding": null, "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-53", "node_type": "4", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "ec085e654cab11dfc7fbfa0769190149aee68c6977e0c14a4ac4bf7ac3daca16", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "18ad941e-4b95-41b6-89ce-288075f2a1b4", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "e1705e67af64ba8e6eae6b7d551275797b26e030e8c35c529ab11d700d73ac9d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b7d0676f-5de3-4539-9f5b-b711ee59629a", "node_type": "1", "metadata": {}, "hash": "682618240b1d19f40858355548e612da1e1175181fb65e5aa86bb5c9ae7ba4a0", "class_name": "RelatedNodeInfo"}}, "text": "564\u00a0CD\u00a0 59.600\u00a0A\u00a0\n0.5\u00a0 0.400\u00a0CD\u00a0 5.128\u00a0CD\u00a0 59.800\u00a0A\u00a0\n1\u00a0 0.200\u00a0CD\u00a0 2.272\u00a0CD\u00a0 59.600\u00a0A\u00a0\n\n4\u00a0\n\n0\u00a0 \u2010\u00a0 \u2010\u00a0 \u2010\u00a0\n0.1\u00a0 0.200\u00a0CD\u00a0 2.947\u00a0CD\u00a0 60.400\u00a0A\u00a0\n0.5\u00a0 0.200\u00a0CD\u00a0 2.127\u00a0CD\u00a0 59.400\u00a0A\u00a0\n1\u00a0 0.400\u00a0CD\u00a0 5.128\u00a0CD\u00a0 60.000\u00a0A\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a049\u201054\u00a0\n\n54\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nReferences\u00a0\nAnwar,\u00a0S;\u00a0D.\u00a0Sabana;\u00a0S.\u00a0A.\u00a0Siddiqui;\u00a0A.\u00a0Shahzad\u00a0and\u00a0S.\u00a0\nDin.\u00a0(2002).\u00a0Clonal\u00a0\u00a0\u00a0\u00a0\u00a0Propagation\u00a0of\u00a0brinjal,\u00a0Solanum\u00a0\n\nmelongena,\u00a0 through\u00a0 young\u00a0 petiolated\u00a0 leaf\u00a0 culture.\u00a0\n\nBionotesx,\u00a04(3):\u00a061.\u00a0\n\nBBS.2003.\u00a0 Statistical\u00a0 Yearbook\u00a0 of\u00a0 Bangladesh.\u00a0\n\nBangladesh\u00a0Bureau\u00a0of\u00a0Statistics.\u00a0Ministry\u00a0of\u00a0Planning,\u00a0\n\nGovernment\u00a0of\u00a0the\u00a0people\u2019s\u00a0Republic\u00a0of\u00a0Bangladesh.\u00a0\u00a0\u00a0\n\nDhaka,\u00a0Bangladesh.\u00a0p.\u00a0150.\u00a0\n\nJayasree,\u00a0 T.;\u00a0 V.\u00a0 Paban\u00a0 ;\u00a0 M.\u00a0 Ramesh;\u00a0 A.V.\u00a0 Rao\u00a0 and\u00a0 K.\u00a0\n\nJ.M.\u00a0Reddy.\u00a0(2001).\u00a0Somatic\u00a0Embryogenesis\u00a0from\u00a0leaf\u00a0\n\ncultures\u00a0of\u00a0potato.\u00a0Plant\u00a0Cell\u00a0Tissue\u00a0Organ\u00a0Cult.,\u00a064\n\n(1):\u00a0\u00a013\u201017.\u00a0\n\nMurashige,\u00a0T.\u00a0and\u00a0F.\u00a0Skoog.\u00a0(1962).\u00a0A\u00a0revised\u00a0medium\u00a0\n\nfor\u00a0rapid\u00a0growth\u00a0and\u00a0Bioassays\u00a0with\u00a0tobacco\u00a0\u00a0tissue\u00a0\n\ncultures.\u00a0Physiol.\u00a0Plant.,\u00a015:\u00a0473\u2010497.\u00a0\n\nZeven,\u00a0A.\u00a0C.\u00a0and\u00a0P.\u00a0M.\u00a0Zhukovsky.(1975).\u00a0Dictionary\u00a0of\u00a0\ncultivated\u00a0 plants\u00a0 and\u00a0 their\u00a0 Centre\u00a0 of\u00a0 Diversity.\u00a0\nWageningen,\u00a0Netherlands.p.\u00a0219", "start_char_idx": 15535, "end_char_idx": 16756, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b7d0676f-5de3-4539-9f5b-b711ee59629a": {"__data__": {"id_": "b7d0676f-5de3-4539-9f5b-b711ee59629a", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "672eb55d-3060-4e56-a724-ddc085e31be5", "node_type": "1", "metadata": {"identifier": "njb-53", "author": "Ray, Bishnu Pada; Hassan, Lutful; Sarker, Smreeti Kana", "title": "In vitro cultivation and regeneration of Solanum melongena (L.) using stem, root and leaf explants", "date": "2022-07-11", "file": "njb-53.pdf"}, "hash": "18d5f79054432a51d588fa1de66a5b89d021de1030f35b8c63afe96ba923099b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "637a0d07-ebc7-45a4-b37d-579b34a339b8", "node_type": "1", "metadata": {}, "hash": "185ad2c4dbc049effa5de01409c1befd42e2e19a8465a2db39167c2fcc85fdc9", "class_name": "RelatedNodeInfo"}}, "text": "5\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n31\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nREVIEW\u00a0ARTICLE\u00a0\n\nMolecular\u00a0differences\u00a0between\u00a0GM\u2010\u00a0and\u00a0non\u2010GM\u00a0\ncrops\u00a0over\u2010estimated?\u00a0\n\nKlaus\u00a0Ammann\u00a0\u00a0\n\nAF\u201010\u00a020101228\u00a0opensource,\u00a0New\u00a0update\u00a0from\u00a028.\u00a0December\u00a02010\u00a0\n\nCorrespondence\u00a0Author:\u00a0\u00a0\nklaus.ammann@ips.unibe.ch\u00a0\u00a0\u00a0\n\n\u00a0\nPeer\u00a0reviewed\u00a0contribution\u00a0available\u00a0on\u00a0the\u00a0following\u00a0websites:\u00a0\n\nPublic\u00a0Research\u00a0Initiative:\u00a0www.pubresreg.org\u00a0\u00a0\nEuropean\u00a0Federation\u00a0of\u00a0Biotechnology:\u00a0http://www.efb\u2010central.org/\u00a0\n\nS.\u00a0No.\u00a0 Title\u00a0 Page\u00a0No.\u00a0\n\n1.0.\u00a0 Issue\u00a0 31\u00a0\n\n2.0.\u00a0 Summary\u00a0 31\u00a0\n\n3.0.\u00a0 Differences\u00a0between\u00a0GM\u2010\u00a0and\u00a0non\u2010GM\u2010crops\u00a0overestimated\u00a0 32\u00a0\n\n3.1.\u00a0 Early\u00a0phase\u00a0of\u00a0risk\u00a0assessment:\u00a0Discovery\u00a0of\u00a0the\u00a0dynamics\u00a0of\u00a0DNA\u00a0processes.\u00a0 32\u00a0\n\n3.2.\u00a0 Molecular\u00a0processes\u00a0similar\u00a0in\u00a0natural\u00a0mutation\u00a0and\u00a0transgenesis\u00a0 32\u00a0\n\n3.3.\u00a0 More\u00a0recent\u00a0publications\u00a0about\u00a0genomic\u00a0\u00a0comparisons\u00a0of\u00a0GM\u2010\u00a0and\u00a0non\u2010GM\u00a0crops\u00a0 33\u00a0\n\n4.0.\u00a0 Natural\u00a0Genetically\u00a0Modified\u00a0Plants,\u00a0DNA\u00a0as\u00a0a\u00a0highly\u00a0dynamic\u00a0system\u00a0 36\u00a0\n\n4.1.\u00a0\nDramatic\u00a0rearrangement\u00a0of\u00a0R\u00a0gene\u00a0loci:\u00a0This\u00a0class\u00a0of\u00a0genes\u00a0diversifies\u00a0more\u00a0rapidly\u00a0than\u00a0\nother\u00a0genes\u00a0in\u00a0the\u00a0crops\u00a0studies\u00a0\n\n36\u00a0\n\n4.2.\u00a0\nJumping\u00a0Genes:\u00a0Their\u00a0dynamics\u00a0falsify\u00a0the\u00a0erroneous\u00a0picture\u00a0of\u00a0regulators\u00a0that\u00a0DNA\u00a0is\u00a0a\u00a0\nstable\u00a0string\u00a0of\u00a0genes\u00a0\n\n38\u00a0\n\n4.3.\u00a0 Helitrons\u00a0contribute\u00a0to\u00a0the\u00a0lack\u00a0of\u00a0gene\u00a0colinearity\u00a0observed\u00a0in\u00a0modern\u00a0maize\u00a0inbreds\u00a0 39\u00a0\n\n4.4.\u00a0 Polyploids,\u00a0Alloploids\u00a0in\u00a0Flowering\u00a0Plants\u00a0 39\u00a0\n\n4.5.\u00a0 Horizontal\u00a0Geneflow\u00a0between\u00a0Pro\u2010Caryotes\u00a0and\u00a0Eu\u2010Caryotes\u00a0 40\u00a0\n\n5.0.\u00a0 Some\u00a0conventional\u00a0breeding\u00a0causes\u00a0lots\u00a0of\u00a0genomic\u00a0alteration\u00a0 40\u00a0\n\n6.0.\u00a0 Regulatory\u00a0dissent\u00a0over\u00a0molecular\u00a0differences\u00a0causes\u00a0transatlantic\u00a0divide\u00a0 41\u00a0\n\n6.1.\u00a0 Perspectives\u00a0for\u00a0a\u00a0dissolution\u00a0of\u00a0this\u00a0divide\u00a0 41\u00a0\n\n8.0.\u00a0 Cited\u00a0literature\u00a0 42\u00a0\n\n7.0.\u00a0 Conclusion\u00a0 42\u00a0\n\n1.0.\u00a0Issue\u00a0\nThe\u00a0 difference\u00a0 between\u00a0 GM\u2010\u00a0 and\u00a0 non\u2010GM\u2010crops\u00a0 has\u00a0\nbeen\u00a0 overestimated,\u00a0 as\u00a0 soon\u00a0 as\u00a0 genetic\u00a0 engineering\u00a0\nhas\u00a0 been\u00a0 applied\u00a0 to\u00a0 crop\u00a0 breeding.\u00a0 The\u00a0 uncontested\u00a0\nunderstanding\u00a0among\u00a0scientists\u00a0and\u00a0in\u00a0particular\u00a0in\u00a0risk\u00a0\nassessment\u00a0community\u00a0was\u00a0that\u00a0GM\u00a0crops\u00a0pose\u00a0some\u00a0\nnovel\u00a0 risks,\u00a0 unprecedented\u00a0 in\u00a0 conventionally\u00a0 bred\u00a0\ncrops.\u00a0This\u00a0has\u00a0then\u00a0condensed\u00a0 in\u00a0the\u00a0United\u00a0Nations\u00a0\nCartagena\u00a0 Protocol\u00a0 on\u00a0 Biosafety1,\u00a0 which\u00a0 needs\u00a0 to\u00a0 be\u00a0\nquestioned\u00a0in\u00a0certain\u00a0basic\u00a0aspects.\u00a0\n\n2.0.", "start_char_idx": 45, "end_char_idx": 2308, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "637a0d07-ebc7-45a4-b37d-579b34a339b8": {"__data__": {"id_": "637a0d07-ebc7-45a4-b37d-579b34a339b8", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b7d0676f-5de3-4539-9f5b-b711ee59629a", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "a7edb6e5e61957399ee4e166bc81c7393ee6b3c4437b813cdf8fb90d3bf2f8de", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70065749-a12e-40e7-a42b-36e469923f79", "node_type": "1", "metadata": {}, "hash": "aaafab20284a3300e82d2a8c7f3c81d237517565331ca19caa72347827aea2c4", "class_name": "RelatedNodeInfo"}}, "text": "2.0.\u00a0Summary\u00a0\nAfter\u00a0an\u00a0early\u00a0phase\u00a0of\u00a0risk\u00a0assessment,\u00a0 including\u00a0the\u00a0\nresults\u00a0 of\u00a0 the\u00a0 Asilomar\u00a0 Conference\u00a0 on\u00a0 biosafety,\u00a0 an\u00a0\nearly\u00a0 divide\u00a0 in\u00a0 risk\u00a0 assessment\u00a0 basic\u00a0 concepts\u00a0\ndeveloped\u00a0 between\u00a0 Canada,\u00a0 the\u00a0 USA\u00a0 and\u00a0 Europe\u00a0\nincluding\u00a0 a\u00a0 majority\u00a0 of\u00a0 UN\u00a0 signatory\u00a0 countries.\u00a0\nResearchers\u00a0 like\u00a0 Werner\u00a0 Arber,\u00a0 based\u00a0 on\u00a0 earlier\u00a0\nmolecular\u00a0 insights\u00a0 and\u00a0 on\u00a0 his\u00a0 own\u00a0 experience\u00a0 in\u00a0\ngenetic\u00a0 engineering\u00a0 claim\u00a0 that\u00a0 related\u00a0 to\u00a0 molecular\u00a0\nprocesses\u00a0 there\u00a0 is\u00a0 no\u00a0 difference\u00a0 between\u00a0 genetically\u00a0\nengineering\u00a0 and\u00a0 natural\u00a0 mutation.\u00a0 This\u00a0 transatlantic\u00a0\n\n1\nCartagena\u00a0Protocol\u00a0on\u00a0Biosafety:\u00a0http://www.cbd.int/biosafety/\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n32\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\ndivide\u00a0 can\u00a0 be\u00a0 solved\u00a0 with\u00a0 some\u00a0 more\u00a0 innovative\u00a0\nregulatory\u00a0proceedings.\u00a0\u00a0\n\n3.0.\u00a0Differences\u00a0between\u00a0GM\u2010\u00a0and\u00a0\nnon\u2010GM\u2010crops\u00a0overestimated\u00a0\n3.1.\u00a0Early\u00a0phase\u00a0of\u00a0risk\u00a0assessment:\u00a0\nDiscovery\u00a0of\u00a0the\u00a0dynamics\u00a0of\u00a0DNA\u00a0processes.\u00a0\n\nIn\u00a0 the\u00a0 wake\u00a0 of\u00a0 molecular\u00a0 breeding,\u00a0 in\u00a0 particular\u00a0 with\u00a0\nthe\u00a0 first\u00a0 successes\u00a0 of\u00a0 \u201cgene\u00a0 splicing\u201d,\u00a0 the\u00a0 safety\u00a0\ndebates\u00a0 started\u00a0 soon\u00a0 after\u00a0 the\u00a0 discovery\u00a0 of\u00a0 the\u00a0 DNA\u00a0\nstructure\u00a0by\u00a0Watson\u00a0&\u00a0Crick\u00a0(Watson\u00a0&\u00a0Crick,\u00a01953a,\u00a0b;\u00a0\nWilkins\u00a0 et\u00a0 al.,\u00a0 1953),\u00a0 followed\u00a0 by\u00a0 the\u00a0 Asilomar\u00a0\nConference\u00a0 (Berg\u00a0 et\u00a0 al.,\u00a0 1975;\u00a0 Berg\u00a0 &\u00a0 Singer,\u00a0 1995)\u00a0 \u2010\u00a0\nsee\u00a0 also\u00a0 some\u00a0 historical\u00a0 accounts\u00a0 (Chassy,\u00a0 2007;\u00a0\nFriedberg,\u00a02007;\u00a0Klug,\u00a02004).\u00a0The\u00a0fascination\u00a0about\u00a0the\u00a0\nnovelty\u00a0 of\u00a0 transgenesis\u00a0 was\u00a0 justified,\u00a0 but\u00a0 also\u00a0\noverwhelming,\u00a0 and\u00a0 the\u00a0 many\u00a0 unforeseen\u00a0 scientific\u00a0\nbreakthroughs\u00a0 following\u00a0 were\u00a0 unprecedented\u00a0 in\u00a0 the\u00a0\nhistory\u00a0 of\u00a0 molecular\u00a0 biology.\u00a0 Unfortunately,\u00a0 the\u00a0\nenthusiasm\u00a0 also\u00a0 lashed\u00a0 back\u00a0 in\u00a0 an\u00a0 overacting\u00a0 in\u00a0 risk\u00a0\nassessment,\u00a0 when\u00a0 the\u00a0 first\u00a0 GM\u00a0 crops\u00a0 went\u00a0 into\u00a0\nproduction.\u00a0 The\u00a0 debate\u00a0 on\u00a0 how\u00a0 GM\u00a0 crops\u00a0 should\u00a0 be\u00a0\nregulated,\u00a0started\u00a0very\u00a0early\u00a0with\u00a0an\u00a0emerging\u00a0divide\u00a0\nbetween\u00a0 regulation\u00a0 in\u00a0 the\u00a0 US\u00a0 and\u00a0 Great\u00a0 Britain,\u00a0\nincluding\u00a0 later\u00a0 the\u00a0 whole\u00a0 of\u00a0 Europe\u00a0 (Bennett\u00a0 et\u00a0 al.,\u00a0\n1986;\u00a0National\u2010Research\u2010Council,\u00a01989).\u00a0\n\nThe\u00a0seemingly\u00a0absolute\u00a0novelty\u00a0of\u00a0genetic\u00a0engineering\u00a0\non\u00a0the\u00a0molecular\u00a0 level\u00a0has\u00a0been\u00a0contested\u00a0already\u00a0 in\u00a0\nthe\u00a0 early\u00a0 days\u00a0 of\u00a0 molecular\u00a0 biology\u00a0 in\u00a0 the\u00a0 1930s\u00a0 and\u00a0\n1950s\u00a0 with\u00a0 the\u00a0 discovery\u00a0 of\u00a0 cellular\u00a0 systems\u00a0 for\u00a0\ngenome\u00a0 restructuring\u00a0 discovered\u00a0 with\u00a0 the\u00a0 classic\u00a0\npapers\u00a0 of\u00a0 McClintock\u00a0 (McClintock,\u00a0 1930,\u00a0 1953),\u00a0 more\u00a0\ndetails\u00a0about\u00a0jumping\u00a0genes\u00a0see\u00a0chapter\u00a04.2.\u00a0\n\n3.2.", "start_char_idx": 2304, "end_char_idx": 4794, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70065749-a12e-40e7-a42b-36e469923f79": {"__data__": {"id_": "70065749-a12e-40e7-a42b-36e469923f79", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "637a0d07-ebc7-45a4-b37d-579b34a339b8", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "1f542b5d87e21879f694dec32730df44a56eed7f542c3612a3125ccb141bdb50", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f938a973-5b35-41fe-a0d0-1581ffd85ff3", "node_type": "1", "metadata": {}, "hash": "60852d796586f3197a49bff3588692cd06e242c3dcd5770107fbe14481b6cfa1", "class_name": "RelatedNodeInfo"}}, "text": "3.2.\u00a0 Molecular\u00a0 processes\u00a0 similar\u00a0 in\u00a0 natural\u00a0\nmutation\u00a0and\u00a0transgenesis\u00a0\n\nThe\u00a0 concept\u00a0 of\u00a0 violated\u00a0 intrinsic\u00a0 naturalness\u00a0 of\u00a0 the\u00a0\ngenomes\u00a0is\u00a0still\u00a0erroneously\u00a0maintained\u00a0by\u00a0proponents\u00a0\nof\u00a0 organic\u00a0 farmers\u00a0 (van\u00a0 Bueren\u00a0 et\u00a0 al.,\u00a0 2008;\u00a0 Van\u00a0\nBueren\u00a0&\u00a0Struik,\u00a02004,\u00a02005;\u00a0Van\u00a0Bueren\u00a0et\u00a0al.,\u00a02003).\u00a0\nThis\u00a0 concept\u00a0of\u00a0 singling\u00a0 out\u00a0 transgenity\u00a0 is\u00a0 falsified\u00a0by\u00a0\nthe\u00a0publications\u00a0of\u00a0Arber\u00a0(Nobel\u00a0Laureate\u00a01978).\u00a0\u00a0\n\nGenetic\u00a0 engineering\u00a0 has\u00a0 been\u00a0 brought\u00a0 into\u00a0\nevolutionary\u00a0 perspective\u00a0 of\u00a0 natural\u00a0 mutation\u00a0 by\u00a0\nauthorities\u00a0 such\u00a0 as\u00a0 Werner\u00a0 Arber:\u00a0 his\u00a0 view\u00a0 remains\u00a0\nscientifically\u00a0 uncontested\u00a0 that\u00a0 molecular\u00a0 processes\u00a0 in\u00a0\n\ntransgenesis\u00a0and\u00a0natural\u00a0mutation\u00a0are\u00a0basically\u00a0similar\u00a0\n(Arber,\u00a0 1994,\u00a0 2000,\u00a0 2002,\u00a0 2003,\u00a0 2004;\u00a0 Arber,\u00a0 2010).\u00a0\nThe\u00a0same\u00a0claim\u00a0is\u00a0made\u00a0with\u00a0a\u00a0more\u00a0organismal\u00a0view\u00a0\nby\u00a0Hackett\u00a0(Hackett,\u00a02002).\u00a0\n\nArber\u00a0 compared\u00a0 designed\u00a0 genetic\u00a0 alterations\u00a0\n(including\u00a0 genetic\u00a0 engineering)\u00a0 with\u00a0 the\u00a0 spontaneous\u00a0\ngenetic\u00a0 variation\u00a0 known\u00a0 to\u00a0 form\u00a0 the\u00a0 substrate\u00a0 for\u00a0\nbiological\u00a0evolution\u00a0(Arber,\u00a02002):\u00a0\n\n\u201cSite\u2010directed\u00a0 mutagenesis\u00a0 usually\u00a0 affects\u00a0 only\u00a0 a\u00a0 few\u00a0\n\nnucleotides.\u00a0 Still\u00a0 another\u00a0 genetic\u00a0 variation\u00a0 sometimes\u00a0\n\nproduced\u00a0by\u00a0genetic\u00a0engineering\u00a0is\u00a0the\u00a0reshuffling\u00a0of\u00a0genomic\u00a0\n\nsequences,\u00a0 e.g.\u00a0 if\u00a0 a\u00a0 given\u00a0 open\u00a0 reading\u00a0 frame\u00a0 is\u00a0 brought\u00a0\n\nunder\u00a0a\u00a0different\u00a0signal\u00a0for\u00a0expression\u00a0control\u00a0or\u00a0if\u00a0a\u00a0gene\u00a0is\u00a0\n\nknocked\u00a0out.\u00a0All\u00a0such\u00a0changes\u00a0have\u00a0little\u00a0chance\u00a0to\u00a0change\u00a0in\u00a0\n\nfundamental\u00a0 ways,\u00a0 the\u00a0 properties\u00a0 of\u00a0 the\u00a0 organism.\u00a0 In\u00a0\n\naddition,\u00a0 it\u00a0 should\u00a0 be\u00a0 remembered\u00a0 that\u00a0 the\u00a0 methods\u00a0 of\u00a0\n\nmolecular\u00a0 genetics\u00a0 themselves\u00a0 enable\u00a0 the\u00a0 researchers\u00a0\n\nanytime\u00a0 to\u00a0 verify\u00a0 whether\u00a0 the\u00a0effective\u00a0 genomic\u00a0 alterations\u00a0\n\ncorrespond\u00a0to\u00a0their\u00a0intentions,\u00a0and\u00a0to\u00a0explore\u00a0the\u00a0phenotypic\u00a0\n\nchanges\u00a0 due\u00a0 to\u00a0 the\u00a0 alterations.\u00a0 This\u00a0 forms\u00a0 part\u00a0 of\u00a0 the\u00a0\n\nexperimental\u00a0 procedures\u00a0 of\u00a0 any\u00a0 research\u00a0 seriously\u00a0 carried\u00a0\n\nout.\u00a0Interestingly,\u00a0naturally\u00a0occurring\u00a0molecular\u00a0evolution,\u00a0i.e.\u00a0\n\nthe\u00a0 spontaneous\u00a0 generation\u00a0 of\u00a0 genetic\u00a0 variants\u00a0 has\u00a0 been\u00a0\n\nseen\u00a0to\u00a0follow\u00a0exactly\u00a0the\u00a0same\u00a0three\u00a0strategies\u00a0as\u00a0those\u00a0used\u00a0\n\nin\u00a0genetic\u00a0engineering.\u00a0These\u00a0three\u00a0strategies\u00a0are:\u00a0\u00a0\n\n(a)\u00a0 small\u00a0local\u00a0changes\u00a0in\u00a0the\u00a0nucleotide\u00a0sequences,\u00a0\u00a0\n\n(b)\u00a0 internal\u00a0reshuffling\u00a0of\u00a0genomic\u00a0DNA\u00a0segments,\u00a0and\u00a0\n\n(c)\u00a0 acquisition\u00a0of\u00a0usually\u00a0rather\u00a0small\u00a0segments\u00a0of\u00a0DNA\u00a0from\u00a0\n\nanother\u00a0type\u00a0of\u00a0organism\u00a0by\u00a0horizontal\u00a0gene\u00a0transfer.\u00a0\n\nHowever,\u00a0 there\u00a0 is\u00a0 a\u00a0 principal\u00a0 difference\u00a0 between\u00a0 the\u00a0\n\nprocedures\u00a0of\u00a0genetic\u00a0engineering\u00a0and\u00a0those\u00a0serving\u00a0in\u00a0nature\u00a0\n\nfor\u00a0 biological\u00a0 evolution.\u00a0 While\u00a0 the\u00a0 genetic\u00a0 engineer\u00a0 pre\u2010\n\nreflects\u00a0his\u00a0alteration\u00a0and\u00a0verifies\u00a0its\u00a0results,\u00a0nature\u00a0places\u00a0its\u00a0\n\ngenetic\u00a0variations\u00a0more\u00a0randomly\u00a0and\u00a0largely\u00a0independent\u00a0of\u00a0\n\nan\u00a0identified\u00a0goal.", "start_char_idx": 4790, "end_char_idx": 7506, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f938a973-5b35-41fe-a0d0-1581ffd85ff3": {"__data__": {"id_": "f938a973-5b35-41fe-a0d0-1581ffd85ff3", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70065749-a12e-40e7-a42b-36e469923f79", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "94b87b2f045d706f5e7867d669f0ca1cae802ce8f8652597b12fafefa5c7759f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "80b918e9-78e5-4b96-ad94-0b3bad3f7b31", "node_type": "1", "metadata": {}, "hash": "cff2b1dcf9948945c669a0589669bbebb6224613908ca5a5837bd49201e64c00", "class_name": "RelatedNodeInfo"}}, "text": "Under\u00a0natural\u00a0conditions,\u00a0it\u00a0is\u00a0the\u00a0pressure\u00a0\n\nof\u00a0 natural\u00a0 selection\u00a0 which\u00a0 eventually\u00a0 determines,\u00a0 together\u00a0\n\nwith\u00a0the\u00a0available\u00a0diversity\u00a0of\u00a0genetic\u00a0variants,\u00a0the\u00a0direction\u00a0\n\ntaken\u00a0 by\u00a0 evolution.\u00a0 \u00a0 It\u00a0 is\u00a0 interesting\u00a0 to\u00a0 note\u00a0 that\u00a0 natural\u00a0\n\nselection\u00a0 also\u00a0 plays\u00a0 its\u00a0 decisive\u00a0 role\u00a0 in\u00a0 genetic\u00a0 engineering,\u00a0\n\nsince\u00a0 indeed\u00a0 not\u00a0 all\u00a0 pre\u2010reflected\u00a0 sequence\u00a0 alterations\u00a0\n\nwithstand\u00a0the\u00a0power\u00a0of\u00a0natural\u00a0selection.\u00a0Many\u00a0investigators\u00a0\n\nhave\u00a0experienced\u00a0the\u00a0effect\u00a0of\u00a0this\u00a0natural\u00a0force\u00a0which\u00a0does\u00a0\n\nnot\u00a0allow\u00a0functional\u00a0disharmony\u00a0in\u00a0a\u00a0mutated\u00a0organism.\u201d\u00a0\n\nArbers\u00a0 numerous\u00a0 writings\u00a0 (Arber,\u00a0 2000,\u00a0 2003,\u00a0 2004)\u00a0\n\nconfirm\u00a0 this\u00a0 important\u00a0 comparison\u00a0 on\u00a0 the\u00a0 genomic\u00a0\n\nlevel\u00a0 of\u00a0 evolutionary\u00a0 and\u00a0 modern\u00a0 plant\u00a0 breeding\u00a0\n\nprocesses.\u00a0 But\u00a0 there\u00a0 is\u00a0 of\u00a0 course,\u00a0 despite\u00a0 all\u00a0 the\u00a0\n\nsimilarities,\u00a0 one\u00a0 major\u00a0 difference:\u00a0 whereas\u00a0 natural\u00a0\n\nmutation\u00a0acts\u00a0completely\u00a0in\u00a0a\u00a0natural\u00a0time\u00a0scale,\u00a0that\u00a0\n\nis,\u00a0 the\u00a0 mutants\u00a0 will\u00a0 need\u00a0 hundreds\u00a0 to\u00a0 hundred\u00a0 of\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n33\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nthousands\u00a0of\u00a0years\u00a0to\u00a0overcome\u00a0selective\u00a0processes\u00a0in\u00a0\n\nnature\u00a0until\u00a0they\u00a0really\u00a0succeed\u00a0and\u00a0take\u00a0over\u00a0against\u00a0\n\ntheir\u00a0natural\u00a0competitors,\u00a0this\u00a0 is\u00a0totally\u00a0different\u00a0with\u00a0\n\nthe\u00a0transgenic\u00a0crop\u00a0products:\u00a0they\u00a0run\u00a0through\u00a0a\u00a0R&D\u00a0\n\nphase,\u00a0and\u00a0a\u00a0regulatory\u00a0process\u00a0of\u00a0an\u00a0average\u00a0of\u00a015\u00a0to\u00a0\n\n20\u00a0 years\u00a0 until\u00a0 being\u00a0 completely\u00a0 deregulated.\u00a0 But\u00a0\n\nsomewhere\u00a0along\u00a0this\u00a0process\u00a0they\u00a0will\u00a0be\u00a0propagated\u00a0\n\nto\u00a0the\u00a0millions\u00a0 in\u00a0the\u00a0field,\u00a0covering\u00a0 in\u00a0a\u00a0evolutionary\u00a0\n\nextremely\u00a0short\u00a0time\u00a0span\u00a0millions\u00a0of\u00a0hectares.\u00a0\n\nIn\u00a0 a\u00a0 recent\u00a0 paper,\u00a0 (Coll\u00a0 et\u00a0 al.,\u00a0 2009)\u00a0 come\u00a0 to\u00a0 the\u00a0\n\nconclusion,\u00a0that\u00a0Gene\u00a0expression\u00a0profiles\u00a0of\u00a0MON810\u00a0\n\nand\u00a0 comparable\u00a0 non\u2010GM\u00a0 maize\u00a0 varieties\u00a0 cultured\u00a0 in\u00a0\n\nthe\u00a0 field\u00a0 are\u00a0 more\u00a0 similar\u00a0 than\u00a0 are\u00a0 those\u00a0 of\u00a0\n\nconventional\u00a0 lines.\u00a0 Their\u00a0 bibliography\u00a0 supports\u00a0 this\u00a0\n\nview\u00a0with\u00a0numerous\u00a0peer\u00a0reviewed\u00a0publications.\u00a0 \u00a0\n\nIt\u00a0 is\u00a0 therefore\u00a0 no\u00a0 surprise\u00a0 that\u00a0 a\u00a0 natural\u00a0 transgene\u00a0\nspecies\u00a0 has\u00a0 been\u00a0 discovered\u00a0 in\u00a0 a\u00a0 widespread\u00a0 grass\u00a0\ngenus\u00a0(Ghatnekar\u00a0et\u00a0al.,\u00a02006).\u00a0\n\n(Miller\u00a0 &\u00a0 Conko,\u00a0 2004)\u00a0 provide\u00a0 important\u00a0 arguments\u00a0\nsupporting\u00a0this\u00a0view:\u00a0\u00a0\n\nThe\u00a0authors\u00a0raise\u00a0also\u00a0in\u00a0a\u00a0justified\u00a0way\u00a0doubts\u00a0about\u00a0\nthe\u00a0 commonly\u00a0 used\u00a0 concept\u00a0 of\u00a0 transgenesis.\u00a0 In\u00a0 the\u00a0\nlight\u00a0of\u00a0pre\u2010recombinant\u00a0DNA\u00a0produced\u00a0in\u00a0great\u00a0variety\u00a0\nby\u00a0 conventional\u00a0 breeding\u00a0 with\u00a0 thousands\u00a0 of\u00a0 foreign\u00a0\ngenes.\u00a0\n\n\u201cIn\u00a0 these\u00a0 examples\u00a0 of\u00a0 prerecombinant\u2010DNA\u00a0 genetic\u00a0\nimprovement,\u00a0 breeders\u00a0 and\u00a0 food\u00a0 producers\u00a0 possess\u00a0 little\u00a0\nknowledge\u00a0 of\u00a0 the\u00a0 exact\u00a0 genetic\u00a0 changes\u00a0 that\u00a0 produced\u00a0 the\u00a0\nuseful\u00a0 trait,\u00a0 information\u00a0 about\u00a0 what\u00a0 other\u00a0 changes\u00a0 have\u00a0\noccurred\u00a0concomitantly\u00a0in\u00a0the\u00a0plant\u00a0or\u00a0data\u00a0on\u00a0the\u00a0transfer\u00a0of\u00a0\nnewly\u00a0 incorporated\u00a0 genes\u00a0 into\u00a0 animals,\u00a0 humans\u00a0 or\u00a0\nmicroorganisms.", "start_char_idx": 7507, "end_char_idx": 10329, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "80b918e9-78e5-4b96-ad94-0b3bad3f7b31": {"__data__": {"id_": "80b918e9-78e5-4b96-ad94-0b3bad3f7b31", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f938a973-5b35-41fe-a0d0-1581ffd85ff3", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "1b6295f2b401bb057cda8fd1d2e170c3ebe4b224f47e2a4e6ac7a0509d991868", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91d384b3-1c8f-447a-ad42-46149d168aa5", "node_type": "1", "metadata": {}, "hash": "1f6ff9849f25929656c73fa7ff438691be377bb5a9660f13c18c962817b84c8a", "class_name": "RelatedNodeInfo"}}, "text": "Consider,\u00a0 for\u00a0 example,\u00a0 the\u00a0 relatively\u00a0 new\u00a0\nman\u2010made\u00a0wheat\u00a0 'species'\u00a0Triticum\u00a0agropyrotriticum,\u00a0which\u00a0\nresulted\u00a0from\u00a0the\u00a0wide\u2010cross\u00a0combination\u00a0of\u00a0the\u00a0genomes\u00a0of\u00a0\nbread\u00a0wheat\u00a0and\u00a0a\u00a0wild\u00a0grass\u00a0sometimes\u00a0called\u00a0quackgrass\u00a0or\u00a0\ncouchgrass\u00a0 \u00a0 (Banks\u00a0 et\u00a0 al.,\u00a0 1993;\u00a0 Sinigovets,\u00a0 1987)\u00a0 T.\u00a0\nagropyrotriticum,\u00a0 which\u00a0 possesses\u00a0 all\u00a0 the\u00a0 chromosomes\u00a0 of\u00a0\nwheat\u00a0as\u00a0well\u00a0as\u00a0the\u00a0entire\u00a0genome\u00a0of\u00a0the\u00a0quackgrass,\u00a0was\u00a0\nindependently\u00a0 produced\u00a0 for\u00a0 both\u00a0 animal\u00a0 feed\u00a0 and\u00a0 human\u00a0\nfood\u00a0 in\u00a0the\u00a0former\u00a0Soviet\u00a0Union,\u00a0Canada,\u00a0the\u00a0United\u00a0States,\u00a0\nFrance,\u00a0 Germany\u00a0 and\u00a0 China.\u201d\u00a0 See\u00a0 also\u00a0 the\u00a0 ASK\u2010FORCE\u00a0\ncontributions\u00a0on\u00a0the\u00a0web\u00a0by\u00a0K.\u00a0Ammann\u00a020092\u00a0on\u00a0the\u00a0same\u00a0\nsubject.\u00a0\n\u00a0\n3.3.\u00a0More\u00a0recent\u00a0publications\u00a0about\u00a0genomic\u00a0\u00a0\ncomparisons\u00a0of\u00a0GM\u00ad\u00a0and\u00a0non\u00adGM\u00a0crops\u00a0\n\nRecent\u00a0 publications\u00a0 demonstrate,\u00a0 that\u00a0 transgenesis\u00a0\n\ne.g.\u00a0has\u00a0less\u00a0impact\u00a0on\u00a0the\u00a0transcriptome\u00a0of\u00a0the\u00a0wheat\u00a0\n\ngrain\u00a0 than\u00a0 traditional\u00a0 breeding\u00a0 (Batista\u00a0 et\u00a0 al.,\u00a0 2008;\u00a0\n\nBaudo\u00a0et\u00a0al.,\u00a02006;\u00a0Shewry\u00a0et\u00a0al.,\u00a02007),\u00a0(more\u00a0details:\u00a0\n\n(Ammann,\u00a02008,\u00a02009)).\u00a0\n\nTwo\u00a0 figures\u00a0 may\u00a0 to\u00a0 visualize\u00a0 the\u00a0 lower\u00a0 impact\u00a0 on\u00a0\ntranscriptome\u00a0 expression\u00a0 of\u00a0 transgenic\u00a0 crops\u00a0\ncompared\u00a0to\u00a0conventional\u00a0ones:\u00a0\n\nVolcano\u00a0plots\u00a0from\u00a0(Batista\u00a0et\u00a0al.,\u00a02008):\u00a0In\u00a0all\u00a0observed\u00a0\ncases\u00a0of\u00a0the\u00a0comparison\u00a0between\u00a0transgenic\u00a0and\u00a0non\u2010\ntransgenic\u00a0 crops\u00a0 the\u00a0 observed\u00a0 alteration\u00a0 was\u00a0 more\u00a0\nextensive\u00a0 in\u00a0 the\u00a0 mutagenized\u00a0 than\u00a0 in\u00a0 the\u00a0 transgenic\u00a0\nplants:\u00a0\n\n\u201cControversy\u00a0regarding\u00a0genetically\u00a0modified\u00a0(GM)\u00a0plants\u00a0and\u00a0\n\ntheir\u00a0 potential\u00a0 impact\u00a0 on\u00a0 human\u00a0 health\u00a0 contrasts\u00a0 with\u00a0 the\u00a0\n\ntacit\u00a0acceptance\u00a0of\u00a0other\u00a0plants\u00a0that\u00a0were\u00a0also\u00a0modified,\u00a0but\u00a0\n\nnot\u00a0considered\u00a0as\u00a0GM\u00a0products\u00a0(e.g.,\u00a0varieties\u00a0raised\u00a0through\u00a0\n\nconventional\u00a0breeding\u00a0such\u00a0as\u00a0mutagenesis).\u00a0What\u00a0is\u00a0beyond\u00a0\n\nthe\u00a0phenotype\u00a0of\u00a0these\u00a0improved\u00a0plants?\u00a0Should\u00a0mutagenized\u00a0\n\nplants\u00a0 be\u00a0 treated\u00a0 differently\u00a0 from\u00a0 transgenics?\u00a0 We\u00a0 have\u00a0\n\nevaluated\u00a0the\u00a0extent\u00a0of\u00a0transcriptome\u00a0modification\u00a0occurring\u00a0\n\nduring\u00a0 rice\u00a0 improvement\u00a0 through\u00a0 transgenesis\u00a0 versus\u00a0\n\nmutation\u00a0 breeding.\u00a0 We\u00a0 used\u00a0 oligonucleotide\u00a0 microarrays\u00a0 to\u00a0\n\nanalyze\u00a0gene\u00a0expression\u00a0in\u00a0four\u00a0different\u00a0pools\u00a0of\u00a0four\u00a0types\u00a0\n\nof\u00a0rice\u00a0plants\u00a0and\u00a0respective\u00a0controls:\u00a0(i)\u00a0a\u00a0gamma\u2010irradiated\u00a0\n\nstable\u00a0 mutant,\u00a0 (ii)\u00a0 the\u00a0 M1\u00a0 generation\u00a0 of\u00a0 a\u00a0 100\u2010Gy\u00a0 gamma\u2010\n\nirradiated\u00a0 plant,\u00a0 (iii)\u00a0 a\u00a0 stable\u00a0 transgenic\u00a0 plant\u00a0 obtained\u00a0 for\u00a0\n\nproduction\u00a0 of\u00a0 an\u00a0 anticancer\u00a0 antibody,\u00a0 and\u00a0 (iv)\u00a0 the\u00a0 T1\u00a0\n\ngeneration\u00a0of\u00a0a\u00a0transgenic\u00a0plant\u00a0produced\u00a0aiming\u00a0for\u00a0abiotic\u00a0\n\nstress\u00a0 improvement,\u00a0 and\u00a0 all\u00a0 of\u00a0 the\u00a0 unmodified\u00a0 original\u00a0\n\ngenotypes\u00a0as\u00a0controls.", "start_char_idx": 10331, "end_char_idx": 12839, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91d384b3-1c8f-447a-ad42-46149d168aa5": {"__data__": {"id_": "91d384b3-1c8f-447a-ad42-46149d168aa5", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "80b918e9-78e5-4b96-ad94-0b3bad3f7b31", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "ce3d0ba5b46c0a8a5a36d30c3d7471a6634563e8d58700a1708feea98fd036c0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "33f7555c-b329-4f5a-9dff-de7518326a92", "node_type": "1", "metadata": {}, "hash": "f88f615de33a14ed9a3ca4806c7fa70e394eb559140da9c51a8e95e0b8358501", "class_name": "RelatedNodeInfo"}}, "text": "We\u00a0found\u00a0that\u00a0the\u00a0improvement\u00a0of\u00a0a\u00a0\n\nplant\u00a0variety\u00a0through\u00a0the\u00a0acquisition\u00a0of\u00a0a\u00a0new\u00a0desired\u00a0trait,\u00a0\n\nusing\u00a0 either\u00a0 mutagenesis\u00a0 or\u00a0 transgenesis,\u00a0 may\u00a0 cause\u00a0 stress\u00a0\n\nand\u00a0thus\u00a0lead\u00a0to\u00a0an\u00a0altered\u00a0expression\u00a0of\u00a0untargeted\u00a0genes.\u00a0\n\nIn\u00a0all\u00a0of\u00a0the\u00a0cases\u00a0studied,\u00a0the\u00a0observed\u00a0alteration\u00a0was\u00a0more\u00a0\n\nextensive\u00a0 in\u00a0 mutagenized\u00a0 than\u00a0 in\u00a0 transgenic\u00a0 plants.\u00a0 We\u00a0\n\npropose\u00a0 that\u00a0 the\u00a0 safety\u00a0 assessment\u00a0 of\u00a0 improved\u00a0 plant\u00a0\n\nvarieties\u00a0 should\u00a0 be\u00a0 carried\u00a0 out\u00a0 on\u00a0 a\u00a0 case\u2010by\u2010case\u00a0 basis\u00a0 and\u00a0\n\nnot\u00a0 simply\u00a0 restricted\u00a0 to\u00a0 foods\u00a0 obtained\u00a0 through\u00a0 genetic\u00a0\n\nengineering.\u201d\u00a0(Batista\u00a0et\u00a0al.,\u00a02008)\u00a0\n\nPlots\u00a0 from\u00a0 (Baudo\u00a0 et\u00a0 al.,\u00a0 2006)\u00a0 are\u00a0 also\u00a0 clearly\u00a0\n\ndemonstrating,\u00a0 that\u00a0 transcriptome\u00a0 comparisons\u00a0\n\nbetween\u00a0 transgenic\u00a0 and\u00a0 non\u2010transgenic\u00a0 comparable\u00a0\n\ntraits\u00a0show\u00a0substantial\u00a0equivalence.\u00a0\n\n\u201cDetailed\u00a0global\u00a0gene\u00a0expression\u00a0profiles\u00a0have\u00a0been\u00a0obtained\u00a0\n\nfor\u00a0a\u00a0series\u00a0of\u00a0transgenic\u00a0and\u00a0conventionally\u00a0bred\u00a0wheat\u00a0lines\u00a0\n\nexpressing\u00a0additional\u00a0genes\u00a0encoding\u00a0HMW\u00a0(high\u00a0molecular\u00a0\n\nweight)\u00a0subunits\u00a0of\u00a0glutenin,\u00a0a\u00a0 group\u00a0of\u00a0endosperm\u2010specific\u00a0\n\nseed\u00a0 storage\u00a0 proteins\u00a0 known\u00a0 to\u00a0 determine\u00a0 dough\u00a0 strength\u00a0\n\nand\u00a0 therefore\u00a0 bread\u2010making\u00a0 quality.\u00a0 \u00a0 Differences\u00a0 in\u00a0\n\nendosperm\u00a0 and\u00a0 leaf\u00a0 transcriptome\u00a0 profiles\u00a0 between\u00a0\n2ASK\u2010FORCE\u00a0contribution\u00a0K.\u00a0Ammann\u00a02009:\u00a0Regulation:\u00a0Misconcepts\u00a0\n\ncause\u00a0high\u00a0costs\u00a0and\u00a0huge\u00a0delays\u00a0in\u00a0regulation\u00a0of\u00a0GM\u00a0crops:\u00a0http://\n\nwww.efb\u2010central.org/index.php/forums/viewthread/59/\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n34\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nuntransformed\u00a0and\u00a0derived\u00a0transgenic\u00a0lines\u00a0were\u00a0consistently\u00a0\n\nextremely\u00a0 small,\u00a0 when\u00a0 analysing\u00a0 plants\u00a0 containing\u00a0 either\u00a0\n\ntransgenes\u00a0only,\u00a0or\u00a0also\u00a0marker\u00a0genes.\u00a0Differences\u00a0observed\u00a0\n\nin\u00a0gene\u00a0expression\u00a0in\u00a0the\u00a0endosperm\u00a0between\u00a0conventionally\u00a0\n\nbred\u00a0material\u00a0were\u00a0much\u00a0larger\u00a0in\u00a0comparison\u00a0to\u00a0differences\u00a0\n\nbetween\u00a0 transgenic\u00a0 and\u00a0 untransformed\u00a0 lines\u00a0 exhibiting\u00a0 the\u00a0\n\nsame\u00a0complements\u00a0of\u00a0gluten\u00a0subunits.\u00a0These\u00a0results\u00a0suggest\u00a0\n\nthat\u00a0the\u00a0presence\u00a0of\u00a0the\u00a0transgenes\u00a0did\u00a0not\u00a0significantly\u00a0alter\u00a0\n\ngene\u00a0 expression\u00a0 and\u00a0 that,\u00a0 at\u00a0 this\u00a0 level\u00a0 of\u00a0 investigation,\u00a0\n\ntransgenic\u00a0 plants\u00a0 could\u00a0 be\u00a0 considered\u00a0 substantially\u00a0\n\nequivalent\u00a0 to\u00a0 untransformed\u00a0 parental\u00a0 lines.\u201d\u00a0 (Baudo\u00a0 et\u00a0 al.,\u00a0\n\n2006)\u00a0\n\nIn\u00a0another\u00a0recent\u00a0paper\u00a0on\u00a0transcriptomic\u00a0comparison,\u00a0\n\n(Kogel\u00a0 et\u00a0 al.,\u00a0 2010)\u00a0 come\u00a0 to\u00a0 the\u00a0 following\u00a0 similar\u00a0\n\nconclusions\u00a0(see\u00a0also\u00a0the\u00a0figures):\u00a0\n\n\u201cIn\u00a0 summary,\u00a0 our\u00a0 results\u00a0 substantially\u00a0 extend\u00a0 observations\u00a0\n\nthat\u00a0 cultivar\u2010specific\u00a0 differences\u00a0 in\u00a0 transcriptome\u00a0 and\u00a0\nmetabolome\u00a0 greatly\u00a0 exceed\u00a0 effects\u00a0 caused\u00a0 by\u00a0 transgene\u00a0\n\nexpression.", "start_char_idx": 12840, "end_char_idx": 15462, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "33f7555c-b329-4f5a-9dff-de7518326a92": {"__data__": {"id_": "33f7555c-b329-4f5a-9dff-de7518326a92", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91d384b3-1c8f-447a-ad42-46149d168aa5", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "84762b6883276793a8924f50b7d56e7f49a2ee8ac10f9a92eacd13d22fa71e75", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cb191bd5-69c2-49a4-b447-5f7f3e59201a", "node_type": "1", "metadata": {}, "hash": "4f64669d21970afdaaed9db2c539373da2f699cf0a479c0f4394f32ee213b62f", "class_name": "RelatedNodeInfo"}}, "text": "Furthermore,\u00a0 we\u00a0 provide\u00a0 evidence\u00a0 that,\u00a0 (i)\u00a0 the\u00a0\nimpact\u00a0of\u00a0a\u00a0low\u00a0number\u00a0of\u00a0alleles\u00a0on\u00a0the\u00a0global\u00a0transcript\u00a0and\u00a0\n\nmetabolite\u00a0profile\u00a0is\u00a0stronger\u00a0than\u00a0transgene\u00a0expression\u00a0and\u00a0\nthat,\u00a0more\u00a0specifically,\u00a0(ii)\u00a0breeding\u00a0for\u00a0better\u00a0adaptation\u00a0and\u00a0\nhigher\u00a0 yields\u00a0 has\u00a0 coordinately\u00a0 selected\u00a0 for\u00a0 improved\u00a0\n\nresistance\u00a0to\u00a0background\u00a0levels\u00a0of\u00a0root\u00a0and\u00a0leaf\u00a0diseases,\u00a0and\u00a0\n\nthis\u00a0 selection\u00a0 appears\u00a0 to\u00a0 have\u00a0 an\u00a0 extensive\u00a0 effect\u00a0 on\u00a0\n\nsubstantial\u00a0 equivalence\u00a0 in\u00a0 the\u00a0 field\u00a0 during\u00a0 latent\u00a0 pathogen\u00a0\n\nchallenge.\u201d\u00a0(Kogel\u00a0et\u00a0al.,\u00a02010)\u00a0\n\nIn\u00a0 another\u00a0 recent\u00a0 paper,\u00a0 dealing\u00a0 with\u00a0 biosynthetic\u00a0\n\ncomparison\u00a0between\u00a0tubers\u00a0and\u00a0leaves\u00a0of\u00a0potato\u00a0traits\u00a0\n\n(Ferreira\u00a0 et\u00a0 al.,\u00a0 2010),\u00a0 the\u00a0 authors\u00a0 come\u00a0 again\u00a0 to\u00a0\n\nsimilar\u00a0 conclusions,\u00a0 as\u00a0 expressed\u00a0 in\u00a0 an\u00a0 interview\u00a0 of\u00a0\n\nGMO\u00a0safety\u00a0of\u00a0the\u00a0senior\u00a0author3\u00a0\u00a0\u00a0in\u00a0http://www.gmo\u2010\n\nsafety.eu/en/news/741.docu.html\u00a0 :\u00a0 \u201cThe\u00a0 impact\u00a0 of\u00a0\n\ntransgenes\u00a0 is\u00a0 basically\u00a0 limited\u00a0 to\u00a0 their\u00a0 immediate\u00a0\n\nfunction\u201d\u00a0.\u00a0And\u00a0further\u00a0on:\u00a0\u00a0\n\nimpact\u00a0 of\u00a0 transgenes\u00a0 is\u00a0 basically\u00a0 limited\u00a0 to\u00a0 their\u00a0\n\nimmediate\u00a0function\u201d\u00a0.\u00a0And\u00a0further\u00a0on:\u00a0\u00a0\n\n\u201cGMO\u00a0 Safety:\u00a0 The\u00a0 following\u00a0 statement\u00a0 was\u00a0 deduced\u00a0 from\u00a0\n\nyour\u00a0findings:\u00a0Conventional\u00a0breeding\u00a0causes\u00a0more\u00a0changes\u00a0in\u00a0\n\nplants\u00a0than\u00a0the\u00a0 introduction\u00a0of\u00a0a\u00a0single\u00a0transgene.\u00a0Can\u00a0you\u00a0\n\nmake\u00a0 such\u00a0 a\u00a0 generalization?\u00a0 After\u00a0 all,\u00a0 you\u00a0 only\u00a0 looked\u00a0 at\u00a0\n\nbarley.\u00a0 Have\u00a0 comparable\u00a0 studies\u00a0 been\u00a0 carried\u00a0 out\u00a0 on\u00a0 other\u00a0\n\ngenetically\u00a0modified\u00a0crops?\u00a0\u00a0\n\nUwe\u00a0 Sonnewald:\u00a0 As\u00a0 far\u00a0 as\u00a0 I\u00a0 know,\u00a0 this\u00a0 was\u00a0 the\u00a0 first\u00a0 time\u00a0 that\u00a0 both\u00a0\n\nmethods\u00a0had\u00a0been\u00a0used\u00a0in\u00a0a\u00a0simultaneous\u00a0investigation.\u00a0Researchers\u00a0have\u00a0\n\nstudied\u00a0either\u00a0gene\u00a0expression\u00a0or\u00a0plant\u00a0substances\u00a0in\u00a0wheat,\u00a0potatoes\u00a0and\u00a0\n\nmaize\u00a0and\u00a0have\u00a0come\u00a0to\u00a0very\u00a0similar\u00a0conclusions.\u00a0The\u00a0impact\u00a0of\u00a0transgenes\u00a0\n\nis\u00a0basically\u00a0 limited\u00a0to\u00a0their\u00a0 immediate\u00a0function.\u00a0For\u00a0example,\u00a0 if\u00a0 I\u00a0 insert\u00a0a\u00a0\n\ngene\u00a0for\u00a0fructan\u00a0biosynthesis\u00a0in\u00a0potatoes,\u00a0it\u00a0is\u00a0hardly\u00a0surprising\u00a0that\u00a0these\u00a0\n\npotatoes\u00a0then\u00a0produce\u00a0fructan\u00a0and\u00a0so\u00a0differ\u00a0in\u00a0this\u00a0way\u00a0from\u00a0their\u00a0parent\u00a0\n\nFig.\u00a01\u00a0\u00a0Volcano\u00a0plots\u00a0for\u00a0differentially\u00a0expressed\u00a0genes.\u00a0Differentially\u00a0expressed\u00a0genes\u00a0appear\u00a0above\u00a0the\u00a0thick\u00a0horizontal\u00a0lines.\u00a0Genes\u00a0induced\u00a0_2\u2010\nfold\u00a0are\u00a0on\u00a0the\u00a0right\u00a0of\u00a0the\u00a0right\u00a0vertical\u00a0lines,\u00a0and\u00a0the\u00a0ones\u00a0repressed\u00a0_2\u2010fold\u00a0are\u00a0on\u00a0the\u00a0left\u00a0of\u00a0the\u00a0left\u00a0vertical\u00a0line.\u00a0The\u00a0numbers\u00a0corresponding\u00a0\nto\u00a0the\u00a0differentially\u00a0expressed\u00a0genes\u00a0induced\u00a0_2\u2010fold\u00a0for\u00a0each\u00a0experiment\u00a0(red\u2010shadowed\u00a0area)\u00a0are\u00a0red,\u00a0and\u00a0those\u00a0corresponding\u00a0to\u00a0the\u00a0genes\u00a0\nrepressed\u00a0_2\u2010fold\u00a0(blue\u2010shadowed\u00a0area)\u00a0are\u00a0blue.\u00a0The\u00a0green\u2010shadowed\u00a0area\u00a0corresponds\u00a0to\u00a0differentially\u00a0expressed\u00a0genes\u00a0that\u00a0were\u00a0up\u2010\u00a0or\u00a0down\u2010\nregulated\u00a0_2\u2010fold\u00a0(green\u2010colored\u00a0numbers).", "start_char_idx": 15464, "end_char_idx": 18073, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cb191bd5-69c2-49a4-b447-5f7f3e59201a": {"__data__": {"id_": "cb191bd5-69c2-49a4-b447-5f7f3e59201a", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "33f7555c-b329-4f5a-9dff-de7518326a92", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "2f1660f7562ee7ebb39109e65c2ddf247e40d44994ad88dd65d910c8d0117c6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "74d93e8c-2511-410b-b188-93582d067d8e", "node_type": "1", "metadata": {}, "hash": "ed3ae31e5b02a2ae719c932a3b2044c446719aec645ec37f634e0fd652b5185d", "class_name": "RelatedNodeInfo"}}, "text": "Blue\u2010colored\u00a0genes\u00a0are\u00a0those\u00a0with\u00a0P\u00a0between\u00a00\u00a0and\u00a00.5,\u00a0and\u00a0red\u2010colored\u00a0genes\u00a0are\u00a0those\u00a0with\u00a0P\u00a0\nbetween\u00a00.5\u00a0and\u00a01.\u00a0From\u00a0(Batista\u00a0et\u00a0al.,\u00a02008)\u00a0\n\n3See\u00a0http://www.gmo\u2010safety.eu/en/news/741.docu.html\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n35\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nlines.\u00a0But\u00a0only\u00a0negligible\u00a0additional\u00a0differences\u00a0were\u00a0found.\u00a0 I\u00a0know\u00a0of\u00a0no\u00a0\n\ninstance\u00a0 where\u00a0 a\u00a0 more\u00a0 significant\u00a0 change\u00a0 in\u00a0 gene\u00a0 expression\u00a0 has\u00a0 been\u00a0\n\ncaused\u00a0 by\u00a0 a\u00a0 single\u00a0 transgene.\u00a0 However,\u00a0 great\u00a0 variability\u00a0 exists\u00a0 between\u00a0\n\nindividual\u00a0varieties\u00a0of\u00a0all\u00a0the\u00a0crops\u00a0mentioned\u00a0and\u00a0the\u00a0obvious\u00a0explanation\u00a0\n\nfor\u00a0 this\u00a0 is\u00a0 that\u00a0 often\u00a0 the\u00a0 breeding\u00a0 objective\u00a0 is\u00a0 to\u00a0 create\u00a0 resistance\u00a0 to\u00a0\n\nexternal\u00a0stress\u00a0factors,\u00a0and\u00a0this\u00a0involves\u00a0a\u00a0large\u00a0number\u00a0of\u00a0genes.\u201d\u00a0\u00a0\n\nAgain\u00a0 the\u00a0 same\u00a0 conclusions\u00a0 are\u00a0 drawn\u00a0 by\u00a0 another\u00a0\n\ncomprehensive\u00a0paper\u00a0of\u00a0a\u00a0large\u00a0international\u00a0collective\u00a0\n\nof\u00a0authors\u00a0(Barros\u00a0et\u00a0al.,\u00a02010):\u00a0\u00a0\n\n\u201cThe\u00a0aim\u00a0of\u00a0this\u00a0study\u00a0was\u00a0to\u00a0evaluate\u00a0the\u00a0use\u00a0of\u00a0four\u00a0non\u2010\n\ntargeted\u00a0 analytical\u00a0 methodologies\u00a0 in\u00a0 the\u00a0 detection\u00a0 of\u00a0\n\nunintended\u00a0 effects\u00a0 that\u00a0 could\u00a0 be\u00a0 derived\u00a0 during\u00a0 genetic\u00a0\n\nmanipulation\u00a0of\u00a0crops.\u00a0Three\u00a0profiling\u00a0technologies\u00a0were\u00a0used\u00a0\n\nto\u00a0compare\u00a0the\u00a0transcriptome,\u00a0proteome\u00a0and\u00a0metabolome\u00a0of\u00a0\n\ntwo\u00a0transgenic\u00a0maize\u00a0lines\u00a0with\u00a0the\u00a0respective\u00a0control\u00a0line.\u00a0By\u00a0\n\ncomparing\u00a0the\u00a0profiles\u00a0of\u00a0the\u00a0two\u00a0transgenic\u00a0 lines\u00a0grown\u00a0 in\u00a0\n\nthe\u00a0 same\u00a0 location\u00a0 over\u00a0 three\u00a0 growing\u00a0 seasons,\u00a0 we\u00a0 could\u00a0\n\ndetermine\u00a0 the\u00a0 extent\u00a0 of\u00a0 environmental\u00a0 variation,\u00a0 while\u00a0 the\u00a0\n\ncomparison\u00a0 with\u00a0 the\u00a0 control\u00a0 maize\u00a0 line\u00a0 allowed\u00a0 the\u00a0\n\ninvestigation\u00a0 of\u00a0 effects\u00a0 caused\u00a0 by\u00a0 a\u00a0 difference\u00a0 in\u00a0 genotype.\u00a0\n\nThe\u00a0 effect\u00a0 of\u00a0 growing\u00a0 conditions\u00a0 as\u00a0 an\u00a0 additional\u00a0\n\nenvironmental\u00a0effect\u00a0was\u00a0also\u00a0evaluated\u00a0by\u00a0comparing\u00a0the\u00a0Bt\n\n\u2010maize\u00a0 line\u00a0with\u00a0the\u00a0control\u00a0 line\u00a0from\u00a0plants\u00a0grown\u00a0 in\u00a0three\u00a0\n\ndifferent\u00a0 locations\u00a0 in\u00a0one\u00a0growing\u00a0season.\u00a0 The\u00a0 environment\u00a0\n\nwas\u00a0shown\u00a0to\u00a0play\u00a0an\u00a0 important\u00a0effect\u00a0 in\u00a0the\u00a0protein,\u00a0gene\u00a0\n\nexpression\u00a0and\u00a0metabolite\u00a0levels\u00a0of\u00a0the\u00a0maize\u00a0samples\u00a0tested\u00a0\n\nwhere\u00a05\u00a0proteins,\u00a065\u00a0genes\u00a0and\u00a015\u00a0metabolites\u00a0were\u00a0found\u00a0to\u00a0\n\nbe\u00a0differentially\u00a0expressed.\u00a0A\u00a0distinct\u00a0separation\u00a0between\u00a0the\u00a0\n\nthree\u00a0 growing\u00a0 seasons\u00a0 was\u00a0 also\u00a0 found\u00a0 for\u00a0 all\u00a0 the\u00a0 samples\u00a0\n\ngrown\u00a0in\u00a0one\u00a0location.\u00a0Together,\u00a0these\u00a0environmental\u00a0factors\u00a0\n\ncaused\u00a0 more\u00a0 variation\u00a0 in\u00a0 the\u00a0 different\u00a0 transcript\u00a0 \u2044 protein\u00a0 \u2044 \nmetabolite\u00a0profiles\u00a0than\u00a0the\u00a0different\u00a0genotypes.\u201d\u00a0(Barros\u00a0et\u00a0\n\nal.,\u00a02010).", "start_char_idx": 18074, "end_char_idx": 20524, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "74d93e8c-2511-410b-b188-93582d067d8e": {"__data__": {"id_": "74d93e8c-2511-410b-b188-93582d067d8e", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cb191bd5-69c2-49a4-b447-5f7f3e59201a", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "ec513a47ec838323a0dbda018c9ab61553b8c53f9566e8df5775e337db6b96f4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f51be9f9-7033-4e41-aa5e-988603f31e94", "node_type": "1", "metadata": {}, "hash": "116b5c126542d0e35c80c0ca68a79d92766f61ecb52d51d87dd31f374d2a603d", "class_name": "RelatedNodeInfo"}}, "text": "Figure\u00a0 2b\u00a0 demonstrates\u00a0 no\u00a0 evident\u00a0 differences\u00a0\n\nbetween\u00a0GM\u00a0\u2013\u00a0and\u00a0non\u2010GM\u00a0maize:\u00a0\n\nInterestingly\u00a0 enough,\u00a0the\u00a0 parallel\u00a0 short\u00a0 report\u00a0 on\u00a0 the\u00a0\n\nwebsite\u00a0of\u00a0USDA\u00a0(www.isb.vt.edu)\u00a0was\u00a0first\u00a0published\u00a0\n\nFig.\u00a02\u00a0Scatter\u00a0plot\u00a0representation\u00a0of\u00a0transcriptome\u00a0comparisons\u00a0of:\u00a0(a)\u00a0transgenic\u00a0B102\u20101\u20101\u00a0line\u00a0vs.\u00a0control\u00a0L88\u201031\u00a0line\u00a0in\u00a0endosperm\u00a0at\u00a014\u00a0dpa\u00a0\n(left),\u00a028\u00a0dpa\u00a0(middle)\u00a0or\u00a0leaf\u00a0at\u00a08\u00a0dpg\u00a0(right);\u00a0(b)\u00a0conventionally\u00a0bred\u00a0L88\u201018\u00a0vs.\u00a0L88\u201031\u00a0line\u00a0in\u00a0endosperm\u00a0at\u00a014\u00a0dpa\u00a0(left),\u00a028\u00a0dpa\u00a0(middle),\u00a0or\u00a0leaf\u00a0\nat\u00a08\u00a0dpg\u00a0(right);\u00a0(c)\u00a0transgenic\u00a0B102\u20101\u20101\u00a0line\u00a0vs.\u00a0conventionally\u00a0bred\u00a0L88\u201018\u00a0line\u00a0in\u00a0endosperm\u00a0at\u00a014\u00a0dpa\u00a0(left),\u00a028\u00a0dpa\u00a0(middle),\u00a0or\u00a0leaf\u00a0at\u00a08\u00a0dpg\u00a0\n(right).\u00a0Dots\u00a0represent\u00a0the\u00a0normalized\u00a0relative\u00a0expression\u00a0level\u00a0of\u00a0each\u00a0arrayed\u00a0gene\u00a0for\u00a0the\u00a0transcriptome\u00a0comparisons\u00a0described.\u00a0Dots\u00a0in\u00a0black\u00a0\nrepresent\u00a0statistically\u00a0significant,\u00a0differentially\u00a0expressed\u00a0genes\u00a0(DEG)\u00a0at\u00a0an\u00a0arbitrary\u00a0cut\u00a0off\u00a0>\u00a01.5.\u00a0The\u00a0inner\u00a0line\u00a0on\u00a0each\u00a0graph\u00a0represents\u00a0no\u00a0\nchange\u00a0in\u00a0expression.\u00a0The\u00a0offset\u00a0dashed\u00a0lines\u00a0are\u00a0set\u00a0at\u00a0a\u00a0relative\u00a0expression\u00a0cut\u2010off\u00a0of\u00a0twofold.\u00a0In\u00a0the\u00a0adjacent\u00a0colored\u00a0bar\u00a0(rectangle\u00a0on\u00a0the\u00a0far\u00a0\nright\u00a0of\u00a0the\u00a0figure),\u00a0the\u00a0vertical\u00a0axis\u00a0represents\u00a0relative\u00a0gene\u00a0expression\u00a0 levels:\u00a0reds\u00a0 indicate\u00a0overexpression,\u00a0yellows\u00a0average\u00a0expression,\u00a0and\u00a0\ngreens\u00a0under\u2010expression.\u00a0Values\u00a0are\u00a0expressed\u00a0as\u00a0n\u2010fold\u00a0changes.\u00a0The\u00a0horizontal\u00a0axis\u00a0of\u00a0this\u00a0bar\u00a0represents\u00a0the\u00a0degree\u00a0to\u00a0which\u00a0data\u00a0can\u00a0be\u00a0\ntrusted:\u00a0dark\u00a0or\u00a0unsaturated\u00a0color\u00a0represents\u00a0low\u00a0trust\u00a0and\u00a0bright\u00a0or\u00a0saturated\u00a0color\u00a0represents\u00a0high\u00a0trust.\u00a0From\u00a0(Baudo\u00a0et\u00a0al.,\u00a02006).\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n36\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n(without\u00a0 notifying\u00a0 the\u00a0 authors)\u00a0 under\u00a0 a\u00a0 clearly\u00a0\n\nmisleading\u00a0 headline\u00a0 \u00a0 \u201cMolecular\u00a0 Profiling\u00a0 Techniques\u00a0\n\nDetect\u00a0 Unintended\u00a0 Effects\u00a0 in\u00a0 Genetically\u00a0 Engineered\u00a0\n\nMaize\u201d,\u00a0it\u00a0was\u00a0subsequently\u00a0corrected\u00a0on\u00a0intervention\u00a0\n\nby\u00a0 the\u00a0 authors\u00a0 to\u00a0 the\u00a0 original\u00a0 headline\u00a0 given\u00a0 in\u00a0 the\u00a0\n\nmanuscript:\u00a0 \u201cMolecular\u00a0 Profiling\u00a0 Techniques\u00a0 as\u00a0 Tools\u00a0\n\nto\u00a0 Detect\u00a0 Potential\u00a0 Unintended\u00a0 Effects\u00a0 in\u00a0 Genetically\u00a0\n\nEngineered\u00a0Maize\u201d\u00a0(Barros,\u00a02010).\u00a0\u00a0\n\nBased\u00a0on\u00a0the\u00a0extensive\u00a0review\u00a0of\u00a0(Wilson\u00a0et\u00a0al.,\u00a02006),\u00a0\n\ntransgenesis\u00a0results\u00a0into\u00a0deletions\u00a0and\u00a0insertions\u00a0in\u00a0the\u00a0\n\ngenome\u00a0of\u00a0considerable\u00a0size,\u00a0just\u00a0as\u00a0radiation\u00a0mutation\u00a0\n\nbreeding\u00a0 can\u00a0 cause:\u00a0 (Meza\u00a0 et\u00a0 al.,\u00a0 2002)\u00a0 show\u00a0 in\u00a0\n\ngenetically\u00a0transformed\u00a0plants:\u00a0\u00a0\n\n\u201cTransgene\u00a0 silencing\u00a0 has\u00a0 been\u00a0 correlated\u00a0 with\u00a0 multiple\u00a0 and\u00a0\n\ncomplex\u00a0 insertions\u00a0 of\u00a0 foreign\u00a0 DNA,\u00a0 e.g.\u00a0 T\u2010DNA\u00a0 and\u00a0 vector\u00a0\n\nbackbone\u00a0 sequences.", "start_char_idx": 20527, "end_char_idx": 23064, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f51be9f9-7033-4e41-aa5e-988603f31e94": {"__data__": {"id_": "f51be9f9-7033-4e41-aa5e-988603f31e94", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "74d93e8c-2511-410b-b188-93582d067d8e", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "61f3632fa3ba1722d81de9586f161f05cdf724a6d8194f3ad5a15a83930379fb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9ec5a26c-0d28-430b-b02e-d2f818a864c8", "node_type": "1", "metadata": {}, "hash": "290b4ddec3ff750e0ad4e885fac11e5c27894cbdfec31cc6594acdf26bc9cbbd", "class_name": "RelatedNodeInfo"}}, "text": "T\u2010DNA\u00a0 and\u00a0 vector\u00a0\n\nbackbone\u00a0 sequences.\u00a0 No\u00a0 striking\u00a0 differences\u00a0 were\u00a0 seen\u00a0\n\nbetween\u00a0the\u00a0TS\u00a0and\u00a0C\u00a0lines.\u00a0The\u00a0majority\u00a0of\u00a0the\u00a0deletions\u00a0are\u00a0\n\n<75\u00a0bp,\u00a0with\u00a0an\u00a0average\u00a0of\u00a036\u00a0bp.\u00a0The\u00a0smallest\u00a0deletion\u00a0was\u00a01\u00a0\n\nbp.\u00a0In\u00a0four\u00a0cases,\u00a0deletions\u00a0of\u00a0>100\u00a0bp\u00a0were\u00a0found,\u00a0the\u00a0largest\u00a0\n\nof\u00a01537\u00a0bp.\u00a0\u00a0Normally,\u00a0the\u00a0deletion\u00a0represented\u00a0a\u00a0continuous\u00a0\n\nstretch\u00a0of\u00a0genomic\u00a0DNA\u00a0(Fig.\u00a02A\u00a0and\u00a0Table\u00a02).\u00a0A\u00a0somewhat\u00a0\n\nmore\u00a0complex\u00a0pattern\u00a0was\u00a0observed\u00a0 in\u00a0only\u00a0one\u00a0 line\u00a0(ex2\u00b14\u00a0\n\nline\u00a08),\u00a0where\u00a0a\u00a0deletion\u00a0of\u00a035\u00a0bp\u00a0at\u00a0the\u00a0integration\u00a0site\u00a0was\u00a0\n\nfollowed\u00a0 by\u00a0 60\u00a0 bp\u00a0 of\u00a0 genomic\u00a0 DNA\u00a0 preceding\u00a0 a\u00a0 second\u00a0\n\ndeletion\u00a0of\u00a0825\u00a0bp.\u201d\u00a0(Meza\u00a0et\u00a0al.,\u00a02002).\u00a0\n\nIt\u00a0is\u00a0one\u00a0of\u00a0the\u00a0most\u00a0frequent\u00a0misunderstandings,\u00a0that\u00a0\n\ntransgenesis\u00a0 causes\u00a0 more\u00a0 genomic\u00a0 disturbance\u00a0 than\u00a0\n\nconventional\u00a0 breeding.\u00a0 It\u00a0 is\u00a0 a\u00a0 very\u00a0 frequently\u00a0\n\nencountered\u00a0 fundamental\u00a0 mistakes\u00a0 of\u00a0 many\u00a0 risk\u00a0\n\nassessment\u00a0 papers\u00a0 related\u00a0 to\u00a0 GMOs:\u00a0 they\u00a0 lack\u00a0 the\u00a0\n\nbaseline\u00a0 comparison\u00a0 \u2013\u00a0 which\u00a0 in\u00a0 the\u00a0 case\u00a0 of\u00a0\n\nenvironmental\u00a0 risk\u00a0 assessment\u00a0 should\u00a0 also\u00a0 comprise\u00a0\n\nthe\u00a0 important\u00a0elements\u00a0of\u00a0agricultural\u00a0practice.\u00a0Here,\u00a0\n\nin\u00a0 chapter\u00a0 3.2.\u00a0 and\u00a0 3.3.\u00a0 we\u00a0 demand\u00a0 a\u00a0 scientifically\u00a0\n\nfounded\u00a0 baseline\u00a0 comparison\u00a0 between\u00a0 the\u00a0 various\u00a0\n\nbreeding\u00a0methods.\u00a0\n\n4.0.\u00a0 Natural\u00a0 Genetically\u00a0 Modified\u00a0\nPlants,\u00a0 DNA\u00a0 as\u00a0 a\u00a0 highly\u00a0 dynamic\u00a0\nsystem\u00a0\nAs\u00a0 a\u00a0 preface\u00a0 to\u00a0 this\u00a0 chapter,\u00a0 one\u00a0 should\u00a0 realize\u00a0 the\u00a0\nfantastic\u00a0 variability\u00a0 of\u00a0 cultivars,\u00a0 here\u00a0 demonstrated\u00a0\nwith\u00a0 an\u00a0 illustration\u00a0 from\u00a0 (Parrott,\u00a0 2010)\u00a0 about\u00a0 the\u00a0\nalready\u00a0ancient\u00a0colorful\u00a0maize\u00a0landraces\u00a0(Fig.\u00a04).\u00a0\n\nIt\u00a0is\u00a0also\u00a0ironic\u00a0and\u00a0a\u00a0clear\u00a0confirmation\u00a0of\u00a0green\u00a0myths,\u00a0\nthat\u00a0 one\u00a0 of\u00a0 the\u00a0 genetically\u00a0 most\u00a0 altered\u00a0 plants,\u00a0 the\u00a0\nsunflower,\u00a0 to\u00a0find\u00a0 it\u00a0 as\u00a0 a\u00a0 symbol\u00a0 of\u00a0 naturalness\u00a0 for\u00a0 a\u00a0\nmajor\u00a0political\u00a0party\u00a0in\u00a0Germany\u00a0(Fig.\u00a05).\u00a0\n\nIt\u00a0is\u00a0also\u00a0worthwhile\u00a0to\u00a0visit\u00a0the\u00a0site\u00a0of\u00a0David\u00a0Tribe\u00a0with\u00a0\nGMO\u00a0 pundit,\u00a0 he\u00a0 offers\u00a0 an\u00a0 extensive\u00a0 site\u00a0 on\u00a0 genomic\u00a0\ncomparison\u00a0 between\u00a0 GMOs\u00a0 and\u00a0 non\u2010GMOs,\u00a0 with\u00a0 an\u00a0\nimpressive\u00a0 collection\u00a0 of\u00a0 \u201cnatural\u00a0 transgenic\u00a0 plants\u201d4.\u00a0\nSee\u00a0 in\u00a0 particular\u00a0 the\u00a0 series\u00a0 of\u00a0 links\u00a0 under\u00a0 Natural\u00a0\nGMOs,\u00a0parts\u00a01\u00a0to\u00a012\u00a0and\u00a013\u00a0to\u00a026.\u00a0\n\nSome\u00a0of\u00a0the\u00a0arguments\u00a0used\u00a0by\u00a0David\u00a0Tribe\u00a0are\u00a0taken\u00a0\nup\u00a0 here\u00a0 and\u00a0 enriched\u00a0 with\u00a0 more\u00a0 arguments\u00a0 and\u00a0\nreferences:\u00a0\n\n4.1.\u00a0Dramatic\u00a0rearrangement\u00a0of\u00a0R\u00a0gene\u00a0loci:\u00a0\nThis\u00a0class\u00a0of\u00a0genes\u00a0diversifies\u00a0more\u00a0rapidly\u00a0\nthan\u00a0other\u00a0genes\u00a0in\u00a0the\u00a0crops\u00a0studies\u00a0\n\nOne\u00a0of\u00a0the\u00a0major\u00a0sources\u00a0of\u00a0genetic\u00a0variability\u00a0(clearly\u00a0\nan\u00a0 evolutionary\u00a0 necessity)\u00a0 is\u00a0 described\u00a0 by\u00a0 (Leister,\u00a0\n2005)\u00a0 on\u00a0 the\u00a0 origin,\u00a0 evolution\u00a0 and\u00a0 genetic\u00a0 effects\u00a0 of\u00a0\nnuclear\u00a0 insertions\u00a0of\u00a0organelle\u00a0DNA,\u00a0 illustrated\u00a0 in\u00a0the\u00a0\nFig.\u00a05.", "start_char_idx": 23023, "end_char_idx": 25624, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9ec5a26c-0d28-430b-b02e-d2f818a864c8": {"__data__": {"id_": "9ec5a26c-0d28-430b-b02e-d2f818a864c8", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f51be9f9-7033-4e41-aa5e-988603f31e94", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "407475f33a4c84db010aed0cf03958010461e25da4c8caaeceb88735095363d8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f5e5279c-4e26-4f10-93d8-fc4c7cdb26fd", "node_type": "1", "metadata": {}, "hash": "526c760d6a7cfab2fcd2d96dedbb97f836b1e55d8f4589030af9a7ab332bb155", "class_name": "RelatedNodeInfo"}}, "text": "In\u00a0Box\u00a01,\u00a0(Leister,\u00a02005)\u00a0describes\u00a0in\u00a0detail\u00a0the\u00a0various\u00a0\npossibilities\u00a0 of\u00a0 gene\u00a0 flow\u00a0 and\u00a0 reasons\u00a0 for\u00a0 genomic\u00a0\nchange:\u00a0\n\n\u201cBox\u00a0 1.\u00a0 DNA\u00a0 flow\u00a0 between\u00a0 different\u00a0 genetic\u00a0 compartments\u00a0\n\nSix\u00a0types\u00a0of\u00a0DNA\u00a0transfer\u00a0are\u00a0conceivable\u00a0between\u00a0the\u00a0three\u00a0\nFig.\u00a03\u00a0\u00a0PCA\u00a0score\u00a0plots\u00a0of\u00a0maize\u00a0grown\u00a0at\u00a0Petit\u00a0over\u00a0three\u00a0consecutive\u00a0\nyears.\u00a0 Separation\u00a0 between\u00a0 the\u00a0 non\u2010GM\u00a0 and\u00a0 GM\u00a0 varieties\u00a0 for\u00a0 (a)\u00a0\nmicroarray\u00a0 data,\u00a0 (b)\u00a0 proteomics\u00a0 data,\u00a0 (c)\u00a0 1H\u2010NMR\u00a0 spectra,\u00a0 (d)\u00a0 gas\u00a0\nchromatographic\u00a0\u2044\u00a0mass\u00a0spectrometric\u00a0(GC\u00a0\u2044\u00a0MS)\u00a0metabolite\u00a0profiles.\u00a0\nFrom\u00a0(Barros\u00a0et\u00a0al.,\u00a02010).\u00a0\n\n4David\u00a0Tribe\u2019s\u00a0blogspot\u00a0on\u00a0Natural\u00a0GMOs:\u00a0http://\n\ngmopundit2.blogspot.com/2005/12/collected\u2010links\u2010to\u2010scientific.html\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n37\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nDNA\u2010containing\u00a0 organelles:\u00a0 nucleus,\u00a0 plastid\u00a0 and\u00a0\n\nmitochondrion.\u00a0 \u00a0 In\u00a0 ptDNAs,\u00a0 no\u00a0 sequence\u00a0 of\u00a0 nuclear\u00a0 or\u00a0\n\nmitochondrial\u00a0 origin\u00a0 has\u00a0 yet\u00a0 been\u00a0 detected,\u00a0 indicating\u00a0 that\u00a0\n\nnucleus\u2010to\u2010plastid\u00a0or\u00a0mitochondrion\u2010to\u2010plastid\u00a0transfer\u00a0occurs\u00a0\n\nextremely\u00a0 rarely\u00a0 or\u00a0 not\u00a0 at\u00a0 all.\u00a0 During\u00a0 the\u00a0 early\u00a0 phase\u00a0 of\u00a0\n\norganelle\u00a0 evolution,\u00a0 organelle\u2010to\u2010nucleus\u00a0 DNA\u00a0 transfer\u00a0\n\n(designated\u00a0in\u00a0Figure\u00a0I\u00a0as\u00a0\u2018a\u2019)\u00a0resulted\u00a0in\u00a0a\u00a0massive\u00a0relocation\u00a0\n\nof\u00a0functional\u00a0genes\u00a0to\u00a0the\u00a0nucleus:\u00a0in\u00a0yeast,\u00a0as\u00a0many\u00a0as\u00a075%\u00a0\n\nof\u00a0 all\u00a0 nuclear\u00a0 genes\u00a0 could\u00a0 derive\u00a0 from\u00a0 proto\u2010mitochondria\u00a0\n\n[62],\u00a0whereas\u00a0w4500\u00a0genes\u00a0in\u00a0the\u00a0nucleus\u00a0of\u00a0Arabidopsis are\u00a0\nof\u00a0 plastid\u00a0 descent\u00a0 [63].\u00a0 Cases\u00a0 of\u00a0 present\u2010day\u00a0 organelle\u2010to\u2010\n\nnucleus\u00a0 DNA\u00a0 transfer,\u00a0 revealed\u00a0 by\u00a0 the\u00a0 presence\u00a0 of\u00a0 NUMTs\u00a0\n\nand\u00a0NUPTs,\u00a0are\u00a0known\u00a0in\u00a0most\u00a0species\u00a0studied\u00a0so\u00a0far.\u00a0\u00a0Among\u00a0\n\nthe\u00a0few\u00a0eukaryotic\u00a0organisms\u00a0in\u00a0which\u00a0norgDNA\u00a0has\u00a0not\u00a0been\u00a0\n\ndetected\u00a0are\u00a0the\u00a0malaria\u00a0mosquito\u00a0(Anopheles\u00a0gambiae)\u00a0and\u00a0\n\nthe\u00a0 honeybee\u00a0 (Apis\u00a0 mellifera).\u00a0 Mitochondrial\u00a0 chromosomes\u00a0\n\ncontain\u00a0 segments\u00a0 homologous\u00a0 to\u00a0 chloroplast\u00a0 sequences,\u00a0 as\u00a0\n\nwell\u00a0 as\u00a0 sequences\u00a0 of\u00a0 nuclear\u00a0 origin,\u00a0 providing\u00a0 indirect\u00a0\n\nevidence\u00a0 for\u00a0 plastid\u2010to\u2010mitochondrion\u00a0 and\u00a0 nucleus\u2010to\u2010\n\nmitochondrion\u00a0transfer\u00a0of\u00a0DNA\u00a0(Figure\u00a0I:\u00a0\u2018b\u2019\u00a0and\u00a0\u2018c\u2019).\u00a0Thus,\u00a0a\u00a0\n\nfew\u00a0 percent\u00a0 of\u00a0the\u00a0 mtDNA\u00a0 of\u00a0 flowering\u00a0 plants\u00a0 derives\u00a0 from\u00a0\n\nptDNA,\u00a0 whereas\u00a0 retrotransposons\u00a0 seem\u00a0 to\u00a0 be\u00a0 the\u00a0 major\u00a0\n\nsource\u00a0 of\u00a0 nucleus\u2010derived\u00a0 mtDNA.", "start_char_idx": 25628, "end_char_idx": 27895, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f5e5279c-4e26-4f10-93d8-fc4c7cdb26fd": {"__data__": {"id_": "f5e5279c-4e26-4f10-93d8-fc4c7cdb26fd", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9ec5a26c-0d28-430b-b02e-d2f818a864c8", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "597e24c5d5dafc435a6668fa28fdc29391ba0a15fdb20a58d0c6d9537112241c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3bac025d-7ae3-4755-bb30-f55bbf3080db", "node_type": "1", "metadata": {}, "hash": "3257a8ee621f541103e3b787363fa8ce649e4e06c35801928e0b9d86406b58d0", "class_name": "RelatedNodeInfo"}}, "text": "Interestingly,\u00a0 although\u00a0\n\nplastid\u2010to\u2010mitochondrion\u00a0 and\u00a0 nucleus\u2010tomitochondrion\u00a0 DNA\u00a0\n\ntransfer\u00a0have\u00a0been\u00a0detected\u00a0in\u00a0almost\u00a0all\u00a0plant\u00a0mitochondrial\u00a0\n\nchromosomes\u00a0sequenced\u00a0so\u00a0far\u00a0[64,65],\u00a0there\u00a0is\u00a0no\u00a0evidence\u00a0\n\nfor\u00a0the\u00a0incorporation\u00a0of\u00a0nDNA\u00a0into\u00a0the\u00a0mitochondrial\u00a0genome\u00a0\n\nof\u00a0maize\u00a0[66].\u201d\u00a0\n\nConclusions\u00a0of\u00a0an\u00a0earlier\u00a0paper\u00a0of\u00a0(Leister\u00a0et\u00a0al.,\u00a01998):\u00a0\n\n\u201cOur\u00a0data\u00a0suggest\u00a0a\u00a0dramatic\u00a0rearrangement\u00a0of\u00a0R\u00a0gene\u00a0loci\u00a0\n\nbetween\u00a0 related\u00a0 species\u00a0 and\u00a0 implies\u00a0 a\u00a0 different\u00a0 mechanism\u00a0\n\nfor\u00a0 nucleotide\u00a0 binding\u00a0 site\u00a0 plus\u00a0 leucine\u2010rich\u00a0 repeat\u00a0 gene\u00a0\n\nevolution\u00a0compared\u00a0with\u00a0the\u00a0rest\u00a0of\u00a0the\u00a0monocot\u00a0genome\u201d\u00a0\n\nAnd\u00a0further\u00a0on\u00a0in\u00a0the\u00a0same\u00a0paper:\u00a0\n\n\u201cHere\u00a0we\u00a0describe\u00a0the\u00a0isolation\u00a0and\u00a0characterization\u00a0of\u00a0NBS\u2010\n\nFig.\u00a04\u00a0 \u00a0Maize\u00a0from\u00a0the\u00a0Guatemalan\u00a0highlands,\u00a0showing\u00a0that\u00a0cross\u00a0pollination\u00a0takes\u00a0place\u00a0naturally\u00a0between\u00a0the\u00a0 landraces.\u00a0Photos\u00a0courtesy\u00a0of\u00a0\nEduardo\u00a0Roesch,\u00a0from\u00a0(Parrott,\u00a02010).\u00a0\n\nFig.\u00a0 5\u00a0 Sunflowers,\u00a0 Helianthus\u00a0 annuus\u00a0 cultivar,\u00a0 one\u00a0 of\u00a0 the\u00a0 most\u00a0\nartificial\u00a0horticultural\u00a0plants\u00a0as\u00a0a\u00a0symbol\u00a0for\u00a0the\u00a0political\u00a0party\u00a0of\u00a0the\u00a0\ngreens\u00a0 from\u00a0 Germany:\u00a0 B\u00fcndnis\u00a0 90,\u00a0 DIE\u00a0 GR\u00dcNEN.\u00a0 http://gruene\u2010\nsenden.de/schlagzeilen/archiv.html\u00a0\n\nFig.\u00a06\u00a0\u00a0Schematic\u00a0overview\u00a0of\u00a0known\u00a0types\u00a0of\u00a0intercompartment\u00a0DNA\u00a0\ntransfer.\u00a0(a)\u00a0Organelle\u2010to\u2010nucleus;\u00a0(b)\u00a0chloroplast\u2010to\u2010mitochondrion;\u00a0\n(c)\u00a0nucleus\u2010to\u2010mitochondrion.\u00a0From\u00a0(Leister,\u00a02005)\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n38\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nWhen\u00a0one\u00a0such\u00a0unit\u00a0is\u00a0incorporated\u00a0at\u00a0the\u00a0locus\u00a0of\u00a0a\u00a0gene,\u00a0it\u00a0\n\nmay\u00a0affect\u00a0genic\u00a0action.\u00a0The\u00a0altered\u00a0action\u00a0 is\u00a0detected\u00a0as\u00a0a\u00a0\n\nmutation.\u00a0Subsequent\u00a0changes\u00a0at\u00a0the\u00a0 locus,\u00a0 initiated\u00a0by\u00a0the\u00a0\n\nextragenic\u00a0unit,\u00a0again\u00a0can\u00a0result\u00a0 in\u00a0change\u00a0 in\u00a0genic\u00a0action\u00a0 ; \nconsequently,\u00a0 a\u00a0 new\u00a0 mutation\u00a0 may\u00a0 be\u00a0 recognized.\u00a0 The\u00a0\n\nextragenic\u00a0units\u00a0undergo\u00a0transposition\u00a0from\u00a0one\u00a0 location\u00a0to\u00a0\n\nanother\u00a0in\u00a0the\u00a0chromosome\u00a0complement.\u00a0It\u00a0is\u00a0this\u00a0mechanism\u00a0\n\nthat\u00a0is\u00a0responsible\u00a0for\u00a0the\u00a0origin\u00a0of\u00a0instability\u00a0at\u00a0the\u00a0locus\u00a0of\u00a0a\u00a0\n\nknown\u00a0 gene;\u00a0 insertion\u00a0 of\u00a0 an\u00a0 extragenic\u00a0 unit\u00a0 adjacent\u00a0 to\u00a0 it\u00a0\n\ninitiates\u00a0 the\u00a0 instability.\u00a0 The\u00a0 extragenic\u00a0 units\u00a0 represent\u00a0\n\nsystems\u00a0in\u00a0the\u00a0nucleus\u00a0that\u00a0are\u00a0responsible\u00a0for\u00a0controlling\u00a0the\u00a0\n\naction\u00a0 of\u00a0 genes.\u00a0 They\u00a0 have\u00a0 specificity\u00a0 in\u00a0 that\u00a0 the\u00a0 mode\u00a0 of\u00a0\n\ncontrol\u00a0of\u00a0genic\u00a0action\u00a0in\u00a0any\u00a0one\u00a0case\u00a0is\u00a0a\u00a0reflection\u00a0of\u00a0the\u00a0\n\nparticular\u00a0system\u00a0in\u00a0operation\u00a0at\u00a0the\u00a0locus\u00a0of\u00a0the\u00a0gene.\u00a0\u00a0One\u00a0\n\nextragenic\u00a0system\u00a0controlling\u00a0genic\u00a0expression\u00a0is\u00a0composed\u00a0of\u00a0\n\ntwo\u00a0interacting\u00a0units.", "start_char_idx": 27899, "end_char_idx": 30366, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3bac025d-7ae3-4755-bb30-f55bbf3080db": {"__data__": {"id_": "3bac025d-7ae3-4755-bb30-f55bbf3080db", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f5e5279c-4e26-4f10-93d8-fc4c7cdb26fd", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "47e0bb0d37208fb744b44a56fc9413e667c0dfe3517b81150ff9e1e4e3f1b759", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f570aacf-9e51-45ef-8b37-673162a9d281", "node_type": "1", "metadata": {}, "hash": "44726724e183423ac729fce0ea8580a575e6a2d0ba2218501488cf973a6f01e4", "class_name": "RelatedNodeInfo"}}, "text": "It\u00a0is\u00a0the\u00a0so\u2010called\u00a0Dissociation\u2010Activator\u00a0\n\n(Ds\u2010Ac)\u00a0system.\u00a0 \u00a0Both\u00a0Ds\u00a0and\u00a0Ac\u00a0undergo\u00a0transposition.\u00a0The\u00a0\n\nDs\u00a0 component,\u00a0 when\u00a0 inserted\u00a0 at\u00a0 the\u00a0 locus\u00a0 of\u00a0 a\u00a0 gene,\u00a0 is\u00a0\n\nresponsible\u00a0for\u00a0modification\u00a0of\u00a0genic\u00a0expression.\u00a0Subsequent\u00a0\n\nchanges\u00a0 at\u00a0 the\u00a0 locus,\u00a0 initiated\u00a0 by\u00a0 Ds,\u00a0 result\u00a0 in\u00a0 further\u00a0\n\nmodification\u00a0 of\u00a0 genic\u00a0 expression.\u00a0 The\u00a0 Ac\u00a0 component\u00a0 in\u00a0 this\u00a0\n\ntwo\u2010unit\u00a0system\u00a0controls\u00a0when\u00a0the\u00a0changes\u00a0at\u00a0Ds\u00a0will\u00a0occur.\u00a0\n\nFrom\u00a0 the\u00a0 conclusions\u00a0 stated\u00a0 above,\u00a0 it\u00a0 was\u00a0 anticipated\u00a0 that\u00a0\n\nthe\u00a0Ds\u2010Ac\u00a0system\u00a0could\u00a0operate\u00a0at\u00a0any\u00a0locus\u00a0of\u00a0known\u00a0genic\u00a0\n\naction.\u00a0 This\u00a0 is\u00a0 because\u00a0 the\u00a0 Ds\u00a0 unit\u00a0 may\u00a0 be\u00a0 transposed\u00a0 to\u00a0\n\nvarious\u00a0locations\u00a0in\u00a0the\u00a0chromosome\u00a0complement.\u00a0To\u00a0obtain\u00a0\n\nthis\u00a0 type\u00a0 of\u00a0 instability\u00a0 at\u00a0 any\u00a0 one\u00a0 locus\u00a0 of\u00a0 kn,own\u00a0 genic\u00a0\n\naction,\u00a0it\u00a0is\u00a0only\u00a0necessary\u00a0to\u00a0provide\u00a0adrquate\u00a0means\u00a0for\u00a0its\u00a0\n\ndetection.\u00a0The\u00a0methods\u00a0used\u00a0to\u00a0obtain\u00a0and\u00a0detect\u00a0this\u00a0type\u00a0of\u00a0\n\ninstability\u00a0 at\u00a0 the\u00a0 AI\u00a0 locus\u00a0 in\u00a0 chromosome\u00a0 3\u00a0 and\u00a0 at\u00a0 the\u00a0 A2 \n\nlocus\u00a0in\u00a0chromosome\u00a05\u00a0are\u00a0described.\u00a0\u00a0A\u00a0detailed\u00a0analysis\u00a0of\u00a0\n\none\u00a0such\u00a0case\u00a0is\u00a0presented\u00a0in\u00a0this\u00a0report.\u201d\u00a0\n\nLater\u00a0commentaries\u00a0of\u00a0Fedoroff\u00a0were\u00a0summarizing\u00a0the\u00a0\nscientific\u00a0 achievements\u00a0 of\u00a0 McClintock,\u00a0 acknowledging\u00a0\nher\u00a0scientific\u00a0merits:\u00a0(Fedoroff,\u00a01992,\u00a01994;\u00a0Fedoroff\u00a0et\u00a0\nal.,\u00a01995;\u00a0Fedoroff,\u00a01984;\u00a0Fedoroff,\u00a01991).\u00a0Especially\u00a0in\u00a0\nthe\u00a0 review\u00a0 published\u00a0 in\u00a0 the\u00a0 Scientific\u00a0 American,\u00a0\ntransposons\u00a0 are\u00a0 well\u00a0 summarized\u00a0 as\u00a0 a\u00a0 generally\u00a0\noccurring\u00a0 phenomenon,\u00a0 having\u00a0 changed\u00a0 considerably\u00a0\nthe\u00a0concept\u00a0of\u00a0genomics,\u00a0this\u00a0is\u00a0well\u00a0illustrated\u00a0in\u00a0the\u00a0\nfact\u00a0of\u00a0the\u00a0multicolored\u00a0maize\u00a0kernels\u00a0(Fig.\u00a07).\u00a0\n\nSee\u00a0 a\u00a0 photo\u00a0 from\u00a0 a\u00a0 landrace\u00a0 preserved\u00a0 as\u00a0 a\u00a0 cultivar\u00a0\nfrom\u00a0 Thusis,\u00a0 Switzerland,\u00a0 visualizing\u00a0 the\u00a0 dynamics\u00a0 of\u00a0\ntransposition\u00a0(Fig.\u00a08).\u00a0\n\nMore\u00a0 comments\u00a0 on\u00a0 McClintocks\u00a0 scientific\u00a0\n\nbreakthrough\u00a0 in\u00a0 (Lewin,\u00a0 1983;\u00a0 Shapiro,\u00a0 1997),\u00a0 the\u00a0\n\nlatter\u00a0 probably\u00a0 the\u00a0 first\u00a0 to\u00a0 coin\u00a0 the\u00a0 term\u00a0 \u2018natural\u00a0\n\ngenetic\u00a0 engineering\u2019.\u00a0 Unfortunately,\u00a0 the\u00a0 dynamics\u00a0 of\u00a0\n\nlife\u00a0DNA\u00a0processes\u00a0was\u00a0not\u00a0taken\u00a0properly\u00a0into\u00a0account\u00a0\n\nwhen\u00a0 the\u00a0 Cartagena\u00a0 protocol\u00a0 on\u00a0 biosafety\u00a0 was\u00a0\n\nconceived.\u00a0\n\nLRR\u00a0homologues\u00a0via\u00a0PCR\u00a0from\u00a0two\u00a0monocot\u00a0species,\u00a0rice\u00a0and\u00a0\n\nbarley,\u00a0based\u00a0on\u00a0structurally\u00a0conserved\u00a0motifs\u00a0 in\u00a0dicot\u00a0NBS\u2010\n\nLRR\u00a0R\u00a0genes.\u00a0We\u00a0have\u00a0analyzed\u00a0their\u00a0sequence\u00a0diversity\u00a0and\u00a0\n\ntheir\u00a0linkage\u00a0to\u00a0genetically\u00a0characterized\u00a0R\u00a0genes.", "start_char_idx": 30367, "end_char_idx": 32677, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f570aacf-9e51-45ef-8b37-673162a9d281": {"__data__": {"id_": "f570aacf-9e51-45ef-8b37-673162a9d281", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3bac025d-7ae3-4755-bb30-f55bbf3080db", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "0151e6519ee51a3ac85fd51d7d3e8657522ffb0de8fde3f2ba3b22832d184f8a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9e3c2165-0f56-4ac7-9c78-6ae037790d74", "node_type": "1", "metadata": {}, "hash": "cf0d306b8cf916c7fa316c975156c918a1107a38669c85f57025814a2c52d5b5", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0results\u00a0\n\nfrom\u00a0a\u00a0comparative\u00a0mapping\u00a0in\u00a0rice,\u00a0barley,\u00a0and\u00a0foxtail\u00a0millet\u00a0\n\nindicates\u00a0 a\u00a0 rapid\u00a0 evolution\u00a0 of\u00a0 R\u00a0 genes\u00a0 in\u00a0 each\u00a0 species\u00a0 and\u00a0\n\nsuggests\u00a0 possible\u00a0 mechanisms\u00a0 to\u00a0 generate\u00a0 diversity\u00a0 in\u00a0\n\nresistance\u00a0loci.\u201d\u00a0And:\u00a0\n\n\u201cAt\u00a0 present,\u00a0 rapid\u00a0 sequence\u00a0 divergence\u00a0 and\u00a0 ectopic\u00a0\n\nrecombination\u00a0 are\u00a0 equally\u00a0 possible\u00a0 mechanisms\u00a0 to\u00a0 explain\u00a0\n\nthe\u00a0 lack\u00a0of\u00a0 intraspecific\u00a0syntenic\u00a0relationships\u00a0detected\u00a0with\u00a0\n\nour\u00a0 set\u00a0 of\u00a0 R\u2010like\u00a0 gene\u00a0 probes.\u00a0 Regardless\u00a0 of\u00a0 whether\u00a0 the\u00a0\n\nformer\u00a0or\u00a0latter\u00a0(or\u00a0both)\u00a0mechanism\u00a0drives\u00a0the\u00a0evolution\u00a0of\u00a0\n\nmonocot\u00a0NBSLRR\u00a0genes,\u00a0the\u00a0data\u00a0shown\u00a0here\u00a0provides\u00a0strong\u00a0\n\nevidence\u00a0that\u00a0this\u00a0class\u00a0of\u00a0genes\u00a0diversifies\u00a0more\u00a0rapidly\u00a0than\u00a0\n\nthe\u00a0 rest\u00a0 of\u00a0 the\u00a0 tested\u00a0 monocot\u00a0 genomes.\u201d\u00a0 \u00a0 (Leister\u00a0 et\u00a0 al.,\u00a0\n\n1998)\u00a0\n\n4.2.\u00a0 Jumping\u00a0 Genes:\u00a0 Their\u00a0 dynamics\u00a0 falsify\u00a0\nthe\u00a0erroneous\u00a0picture\u00a0of\u00a0regulators\u00a0that\u00a0DNA\u00a0\nis\u00a0a\u00a0stable\u00a0string\u00a0of\u00a0genes\u00a0\n\nThe\u00a0seemingly\u00a0absolute\u00a0novelty\u00a0of\u00a0genetic\u00a0engineering\u00a0\n\non\u00a0the\u00a0molecular\u00a0 level\u00a0has\u00a0been\u00a0contested\u00a0already\u00a0 in\u00a0\n\nthe\u00a0 early\u00a0 days\u00a0 of\u00a0 molecular\u00a0 biology\u00a0 in\u00a0 the\u00a0 1930s\u00a0 and\u00a0\n\n1950s\u00a0 with\u00a0 the\u00a0 discovery\u00a0 of\u00a0 cellular\u00a0 systems\u00a0 for\u00a0\n\ngenome\u00a0 restructuring\u00a0 discovered\u00a0 with\u00a0 the\u00a0 classic\u00a0\n\npapers\u00a0of\u00a0McClintock\u00a0(McClintock,\u00a01930,\u00a01953).\u00a0\u00a0\n\n1.\u00a0 A\u00a0 case\u00a0 of\u00a0 semi\u2010sterility\u00a0 in\u00a0 Zea\u00a0 mays\u00a0 was\u00a0 found\u00a0 to\u00a0 be\u00a0\n\nassociated\u00a0 with\u00a0 a\u00a0 reciprocal\u00a0 translocation\u00a0 (segmental\u00a0\n\ninterchange)\u00a0 between\u00a0 the\u00a0 second\u00a0 and\u00a0 third\u00a0 smallest\u00a0\n\nchromosomes.\u00a0\n\n2.\u00a0 Through\u00a0 observations\u00a0 of\u00a0 chromosome\u00a0 synapsis\u00a0 in\u00a0 early\u00a0\n\nmeiotic\u00a0 prophases\u00a0 of\u00a0 plants\u00a0 heterozygous\u00a0 for\u00a0 the\u00a0\n\ninterchange\u00a0it\u00a0has\u00a0been\u00a0possible\u00a0to\u00a0locate\u00a0approximately\u00a0\n\nthe\u00a0 point\u00a0 of\u00a0 interchange\u00a0 in\u00a0 both\u00a0 chromosomes.\u00a0 The\u00a0\n\ninterchange\u00a0was\u00a0found\u00a0to\u00a0be\u00a0unequal.\u00a0\n\n3.\u00a0 An\u00a0 analysis\u00a0 of\u00a0 the\u00a0 chromosome\u00a0 complements\u00a0 in\u00a0 the\u00a0\n\nmicrospores\u00a0of\u00a0plants\u00a0heterozygous\u00a0for\u00a0the\u00a0 interchange\u00a0\n\nindicated\u00a0 that\u00a0 of\u00a0 the\u00a0 four\u00a0 chromosomes\u00a0 constituting\u00a0 a\u00a0\n\nring,\u00a0 those\u00a0 with\u00a0 homologous\u00a0 spindle\u00a0 fiber\u00a0 attachment\u00a0\n\nregions\u00a0can\u00a0pass\u00a0to\u00a0the\u00a0same\u00a0pole\u00a0in\u00a0anaphase\u00a0I\u00a0and\u00a0do\u00a0\n\nso\u00a0in\u00a0a\u00a0considerable\u00a0number\u00a0of\u00a0the\u00a0sporocytes.\u00a0\n\nAnd\u00a0 in\u00a0the\u00a0paper\u00a0of\u00a01953,\u00a0usually\u00a0cited\u00a0as\u00a0the\u00a0classic\u00a0\n\npublication,\u00a0 leading\u00a0 decades\u00a0 later,\u00a0 including\u00a0 her\u00a0\n\nrelentless\u00a0fight\u00a0for\u00a0the\u00a0\u201cjumping\u00a0genes\u00a0concept\u201d:\u00a0Here\u00a0\n\nthe\u00a0full\u00a0summary\u00a0of\u00a0her\u00a0paper:\u00a0\n\n\u201cPrevious\u00a0 studies\u00a0 of\u00a0 the\u00a0 origin\u00a0 and\u00a0 mode\u00a0 of\u00a0 expression\u00a0 of\u00a0\n\ngenic\u00a0instability\u00a0at\u00a0a\u00a0number\u00a0of\u00a0known\u00a0loci\u00a0in\u00a0maize\u00a0led\u00a0to\u00a0the\u00a0\n\nfollowing\u00a0 conclusions.\u00a0 Extragenic\u00a0 units,\u00a0 carried\u00a0 in\u00a0 the\u00a0\n\nchromosomes,\u00a0are\u00a0responsible\u00a0for\u00a0altering\u00a0genic\u00a0expression.\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 32678, "end_char_idx": 35263, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9e3c2165-0f56-4ac7-9c78-6ae037790d74": {"__data__": {"id_": "9e3c2165-0f56-4ac7-9c78-6ae037790d74", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f570aacf-9e51-45ef-8b37-673162a9d281", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "e22592291d2336be660b551e6f0dd737e46c069a4dd10812d3e7d3e328d3201a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b7aa729b-074a-46a9-8b2a-9303f98fbeaf", "node_type": "1", "metadata": {}, "hash": "55f8f5e2883e00e87c8d72bd0ca2dca07f814a428acb5781dda01accef11689d", "class_name": "RelatedNodeInfo"}}, "text": "1:\u00a031\u201048\u00a0\n\n39\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nrepeats\u00a0 and\u00a0 cause\u00a0 no\u00a0 duplication\u00a0 of\u00a0 host\u00a0 genome\u00a0\nsequence\u00a0upon\u00a0insertion.\u00a0\n\n4.4.\u00a0 Polyploids,\u00a0 Alloploids\u00a0 in\u00a0 Flowering\u00a0\nPlants\u00a0\n\nIn\u00a0 his\u00a0 blog\u00a0 series\u00a0 part\u00a0 9,\u00a0 David\u00a0 Tribe\u00a0 sums\u00a0 up\u00a0\npolyploidisation\u00a0dynamics\u00a0of\u00a0higher\u00a0plants6\u00a0:\u00a0\n\u00a0\n\u201cDuring\u00a0 only\u00a0 the\u00a0 past\u00a0 decade\u00a0 [i.e\u00a0 post\u00a0 1985]\u00a0 molecular\u00a0\napproaches\u00a0 have\u00a0 provided\u00a0 a\u00a0 wealth\u00a0 of\u00a0 data\u00a0 that\u00a0 have\u00a0\ndramatically\u00a0reshaped\u00a0views\u00a0of\u00a0polyploid\u00a0evolution,\u00a0providing\u00a0\na\u00a0much\u00a0more\u00a0dynamic\u00a0picture\u00a0than\u00a0traditionally\u00a0espoused.\u00a0In\u00a0\nparticular,\u00a0molecular\u00a0data:\u00a0\n(i)\u00a0 demonstrate\u00a0 that\u00a0 both\u00a0 autopolyploids\u00a0 and\u00a0\n\nallopolyploids\u00a0 exhibit\u00a0 a\u00a0 high\u00a0 frequency\u00a0 of\u00a0 recurrent\u00a0\nformation\u00a0(multiple\u00a0origin),\u00a0\n\n(ii)\u00a0 reveal\u00a0 that\u00a0 multiple\u00a0 polyploidization\u00a0 events\u00a0 within\u00a0\nspecies\u00a0 have\u00a0 significant\u00a0 genetic\u00a0 and\u00a0 evolutionary\u00a0\nimplications,\u00a0and\u00a0\n\n(iii)\u00a0 contradict\u00a0 the\u00a0 traditional\u00a0 view\u00a0 of\u00a0 autoploidy\u00a0 as\u00a0 being\u00a0\nrare\u00a0and\u00a0maladaptive\u00a0(Soltis\u00a0&\u00a0Soltis,\u00a01993).\u00a0\n\n\u00a0\nPerhaps\u00a0one\u00a0of\u00a0the\u00a0most\u00a0important\u00a0contributions\u00a0of\u00a0molecular\u00a0\ndata\u00a0to\u00a0the\u00a0study\u00a0of\u00a0polyploid\u00a0evolution\u00a0is\u00a0the\u00a0documentation\u00a0\nthat\u00a0 a\u00a0 single\u00a0 polyploid\u00a0 species\u00a0 may\u00a0 have\u00a0 separate,\u00a0\nindependent\u00a0 origins\u00a0 from\u00a0 the\u00a0 same\u00a0 diploid\u00a0 progenitor\u00a0\nspecies.\u00a0\n\u00a0\nMultiple\u00a0origins\u00a0of\u00a0polyploids\u00a0have\u00a0now\u00a0been\u00a0documented\u00a0in\u00a0\nbryophytes\u00a0 (Wyatt\u00a0 et\u00a0 al.,\u00a0 1988)\u00a0 and\u00a0 in\u00a0 >40\u00a0 species\u00a0 of\u00a0 ferns\u00a0\n(e.g.,\u00a0 (Werth\u00a0 et\u00a0 al.,\u00a0 1985)\u00a0 and\u00a0 (Ranker\u00a0 et\u00a0 al.,\u00a0 1989)\u00a0 and\u00a0\nangiosperms\u00a0(e.g.,\u00a0refs.\u00a0(Brochmann\u00a0et\u00a0al.,\u00a01992;\u00a0Doyle\u00a0et\u00a0al.,\u00a0\n1990;\u00a0 Soltis\u00a0 et\u00a0 al.,\u00a0 1995;\u00a0 Song\u00a0 &\u00a0 Osborn,\u00a0 1992).\u00a0 In\u00a0 fact,\u00a0\nmolecular\u00a0data\u00a0 indicate\u00a0that\u00a0multiple\u00a0origins\u00a0of\u00a0polyploids\u00a0\nare\u00a0the\u00a0rule\u00a0and\u00a0not\u00a0the\u00a0exception\u00a0(Soltis\u00a0&\u00a0Soltis,\u00a01993).\u00a0In\u00a0\n\n4.3.\u00a0Helitrons\u00a0contribute\u00a0to\u00a0the\u00a0lack\u00a0of\u00a0gene\u00a0\ncolinearity\u00a0 observed\u00a0 in\u00a0 modern\u00a0 maize\u00a0\ninbreds\u00a0\n\nIt\u00a0 was\u00a0 David\u00a0 Tribe\u00a0 in\u00a0 his\u00a0 blogspot\u00a0 on\u00a0 natural\u00a0\n\ntransgenics\u00a0part\u00a07\u00a0linking\u00a0helitrons\u00a0to\u00a0natural\u00a0GMO\u2019s\u00a05\u00a0\u00a0\n\n\u201cUntil\u00a0 recently,\u00a0 it\u00a0 was\u00a0 assumed\u00a0 that\u00a0 the\u00a0 order\u00a0 of\u00a0 gene\u00a0\n\nsequences\u00a0within\u00a0modern\u00a0maize\u00a0would\u00a0be\u00a0virtually\u00a0invariant.\u00a0\n\nRecent\u00a0 discoveries\u00a0 have\u00a0 shown\u00a0 that\u00a0 gene\u00a0 co\u2010linearity\u00a0 is\u00a0 not\u00a0\n\nalways\u00a0the\u00a0case.\u00a0Several\u00a0 laboratories\u00a0(1\u20103)\u00a0have\u00a0found\u00a0DNA\u00a0\n\nregions\u00a0rich\u00a0in\u00a0gene\u00a0sequences\u00a0that\u00a0are\u00a0present\u00a0in\u00a0some\u00a0maize\u00a0\n\ninbred\u00a0 lines\u00a0 but\u00a0 absent\u00a0 at\u00a0 homologous\u00a0 sites\u00a0 in\u00a0 other\u00a0 lines.", "start_char_idx": 35264, "end_char_idx": 37589, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b7aa729b-074a-46a9-8b2a-9303f98fbeaf": {"__data__": {"id_": "b7aa729b-074a-46a9-8b2a-9303f98fbeaf", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9e3c2165-0f56-4ac7-9c78-6ae037790d74", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "7ad57b33482fe6c1d8c1959b0d33aef5a668ae24baafc9d1e588911826833375", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "33361f1e-ee96-411f-8961-214fbfa15225", "node_type": "1", "metadata": {}, "hash": "9330ce19580e470edf491add414e2c9d15c3d9f22b5e4d071554dae67165f354", "class_name": "RelatedNodeInfo"}}, "text": "This\u00a0 variation,\u00a0 termed\u00a0 \"intraspecific\u00a0 violation\u00a0 of\u00a0 genetic\u00a0 co\u2010\n\nlinearity\"\u00a0or\u00a0\"plus/minus\u00a0genetic\u00a0polymorphism,\"\u00a0was\u00a0shown\u00a0\n\nby\u00a0 (Lal\u00a0 &\u00a0 Hannah,\u00a0 2005)\u00a0 in\u00a0 a\u00a0 recent\u00a0 issue\u00a0 of\u00a0 PNAS\u00a0 to\u00a0 be\u00a0\n\ncaused\u00a0 by\u00a0 a\u00a0 newly\u00a0 described\u00a0 transposable\u00a0 element\u00a0 family\u00a0\n\ntermed\u00a0Helitrons.\u201d\u00a0\n\nIn\u00a0 a\u00a0 recent\u00a0 review,\u00a0 (Lal\u00a0 et\u00a0 al.,\u00a0 2009)\u00a0 summarize\u00a0 the\u00a0\n\nimportance\u00a0 of\u00a0 a\u00a0 recently\u00a0 discovered\u00a0 superfamily\u00a0 of\u00a0\n\ntransposable\u00a0 elements.\u00a0 The\u00a0 authors\u00a0 critically\u00a0 analyze\u00a0\n\nthe\u00a0 proposed\u00a0 mechanisms\u00a0 of\u00a0 Helitron\u00a0 transposition,\u00a0\n\ntheir\u00a0impact\u00a0on\u00a0genome\u00a0evolution\u00a0and\u00a0the\u00a0process\u00a0by\u00a0\n\nwhich\u00a0these\u00a0enigmatic\u00a0elements\u00a0capture\u00a0and\u00a0multiply\u00a0\n\nhost\u00a0genes.\u00a0Intriguingly,\u00a0maize\u00a0Helitrons\u00a0share\u00a0striking\u00a0\n\nstructural\u00a0similarity\u00a0\n\nto\u00a0 bacterial\u00a0 integrons.\u00a0 These\u00a0 elements\u00a0 capture\u00a0 gene\u00a0\nsequences\u00a0via\u00a0site\u2010specific\u00a0recombination\u00a0and\u00a0generate\u00a0\ncircular\u00a0 intermediates\u00a0 (Hall\u00a0 &\u00a0 Collis,\u00a0 1995).\u00a0 Both\u00a0\nHelitrons\u00a0 and\u00a0 integrons\u00a0 are\u00a0 mobile,\u00a0 lack\u00a0 terminal\u00a0\n\nFig.\u00a0 7\u00a0 Development\u00a0 time\u00a0 and\u00a0 frequency\u00a0 of\u00a0 transposition\u00a0 differ\u00a0 in\u00a0\nmutations\u00a0 caused\u00a0 by\u00a0 the\u00a0 insertion\u00a0 of\u00a0 different\u00a0 defective\u00a0 Spm\u00a0\nelements.\u00a0 If\u00a0 transposition\u00a0 takes\u00a0 place\u00a0 late\u00a0 in\u00a0 the\u00a0 development,\u00a0 the\u00a0\nclones\u00a0of\u00a0revertent\u00a0cells\u00a0are\u00a0small\u00a0and\u00a0therefore\u00a0so\u00a0are\u00a0the\u00a0pigmented\u00a0\nspots\u00a0(a)\u00a0.\u00a0If\u00a0transposition\u00a0takes\u00a0place\u00a0at\u00a0about\u00a0the\u00a0same\u00a0time\u00a0but\u00a0at\u00a0a\u00a0\nlower\u00a0frequency,\u00a0there\u00a0are\u00a0fewer\u00a0such\u00a0clones\u00a0and\u00a0fewer\u00a0spots\u00a0(b).\u00a0 If\u00a0\nthe\u00a0 transposition\u00a0 that\u00a0 resores\u00a0 gene\u00a0 function\u00a0 takes\u00a0 place\u00a0 earlier,\u00a0 the\u00a0\nrevertant\u00a0clones\u00a0and\u00a0the\u00a0spots\u00a0of\u00a0the\u00a0pigmented\u00a0tissue\u00a0are\u00a0larger\u00a0(c).\u00a0\nFrom\u00a0(Fedoroff,\u00a01984).\u00a0\n\nFig.\u00a0 8\u00a0 \u00a0 Landrace\u00a0 preserved\u00a0 as\u00a0 a\u00a0 cultivar\u00a0 from\u00a0 Thusis,\u00a0 Switzerland,\u00a0\nvisualizing\u00a0 the\u00a0 colorful\u00a0 dynamics\u00a0 of\u00a0 transposition:\u00a0 Photo\u00a0 Klaus\u00a0\nAmmann\u00a0\u00a0\n\n5David\u00a0Tribes\u00a0blogspot\u00a0No.\u00a07:\u00a0http://\n\ngmopundit.blogspot.com/2006/01/natural\u2010gmos\u2010part\u20107\u2010nanobot\u2010\n\ngenetic.html\u00a0\n6David\u00a0Tribes\u00a0blogspot\u00a0No.\u00a09:\u00a0http://\n\ngmopundit.blogspot.com/2006/01/natural\u2010gmos\u2010part\u20109\u2010\n\ndifferent.html\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n40\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nduplicative\u00a0 horizontal\u00a0 gene\u00a0 transfer\u00a0 and\u00a0 differential\u00a0 gene\u00a0\n\nconversion\u00a0is\u00a0proposed\u00a0as\u00a0a\u00a0hitherto\u00a0unrecognized\u00a0source\u00a0of\u00a0\n\ngenetic\u00a0diversity.\u201d\u00a0\n\nThe\u00a0conclusion\u00a0from\u00a0this\u00a0chapter\u00a04.5.\u00a0is\u00a0again\u00a0that\u00a0gene\u00a0\nexchange\u00a0in\u00a0the\u00a0course\u00a0of\u00a0evolution\u00a0has\u00a0been\u00a0proven,\u00a0\nand\u00a0thus\u00a0\u201cevolutionary\u00a0transgenes\u201d\u00a0are\u00a0part\u00a0of\u00a0nature.\u00a0\nThe\u00a0 conclusion\u00a0 again:\u00a0 Transgenesis\u00a0 belongs\u00a0 to\u00a0 nature\u00a0\nand\u00a0 it\u00a0 is\u00a0 scientifically\u00a0 not\u00a0 justified\u00a0 to\u00a0 make\u00a0 a\u00a0\nfundamental\u00a0 distinction\u00a0 between\u00a0 natural\u00a0 organisms\u00a0\n(strictly\u00a0 without\u00a0 transgenes)\u00a0 and\u00a0 artificial\u00a0 organisms\u00a0\ncontaining\u00a0trangenes\u00a0with\u00a0methods\u00a0of\u00a0targeted\u00a0genetic\u00a0\nengineering.\u00a0\u00a0\n\u00a0\n\n5.0.", "start_char_idx": 37592, "end_char_idx": 40309, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "33361f1e-ee96-411f-8961-214fbfa15225": {"__data__": {"id_": "33361f1e-ee96-411f-8961-214fbfa15225", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b7aa729b-074a-46a9-8b2a-9303f98fbeaf", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "724cc12ddfceaadb31f62f44f60b2acd83d1929d069b4f6ee2a7a0120fd9a47b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fc937472-82fa-41b4-87ec-3af548807422", "node_type": "1", "metadata": {}, "hash": "6dd782201a58605858476dfba54e8995e9920f79ac1b7b8e0e3b7453d2d6c871", "class_name": "RelatedNodeInfo"}}, "text": "5.0.\u00a0Some\u00a0conventional\u00a0breeding\u00a0\ncauses\u00a0lots\u00a0of\u00a0genomic\u00a0alteration\u00a0\nOne\u00a0 should\u00a0 also\u00a0 take\u00a0 into\u00a0 account,\u00a0 that\u00a0 many\u00a0 of\u00a0 the\u00a0\nconventional\u00a0breeding\u00a0methods\u00a0such\u00a0as\u00a0colchicination\u00a0\n(Awoleye\u00a0 et\u00a0 al.,\u00a0 1994;\u00a0 Barnab\u00e1s\u00a0 et\u00a0 al.,\u00a0 1999)\u00a0 and\u00a0\nradiation\u00a0 mutation\u00a0 breeding\u00a0 (Reynolds\u00a0 et\u00a0 al.,\u00a0 2000;\u00a0\nShirley\u00a0 et\u00a0 al.,\u00a0 1992)\u00a0 are\u00a0 obviously\u00a0 more\u00a0 damaging\u00a0 to\u00a0\nthe\u00a0genome\u00a0(Schouten\u00a0&\u00a0Jacobsen,\u00a02007),\u00a0and\u00a0 it\u00a0 is\u00a0 in\u00a0\naddition\u00a0not\u00a0possible\u00a0to\u00a0clearly\u00a0define\u00a0what\u00a0impact\u00a0the\u00a0\nun\u2010targeted\u00a0process\u00a0could\u00a0have\u00a0caused.\u00a0(Molnar\u00a0et\u00a0al.,\u00a0\n2009)\u00a0reported\u00a0 in\u00a0detail\u00a0about\u00a0radiation\u00a0treatment\u00a0of\u00a0\nthe\u00a0 chromosome\u00a0 morphology\u00a0 of\u00a0 wheat\u00a0 hybrids:\u00a0\nDicentric\u00a0 chromosomes,\u00a0 fragments,\u00a0 and\u00a0 terminal\u00a0\ntranslocations\u00a0 were\u00a0 most\u00a0 frequently\u00a0 induced\u00a0 by\u00a0\ngamma\u2010radiation,\u00a0 but\u00a0 centric\u00a0 fusions\u00a0 and\u00a0 internal\u00a0\nexchanges\u00a0 were\u00a0 also\u00a0 more\u00a0 abundant\u00a0 in\u00a0 the\u00a0 treated\u00a0\nplants\u00a0than\u00a0 in\u00a0the\u00a0control\u00a0amphiploids.\u00a0The\u00a0 irradiated\u00a0\namphiploids\u00a0 formed\u00a0 fewer\u00a0 seeds\u00a0 than\u00a0 untreated\u00a0\nplants,\u00a0but\u00a0on\u00a0the\u00a0other\u00a0hand\u00a0normal\u00a0levels\u00a0of\u00a0fertility\u00a0\nwere\u00a0recovered\u00a0in\u00a0their\u00a0offspring.\u00a0On\u00a0the\u00a0positive\u00a0side\u00a0\nthe\u00a0 authors\u00a0 are\u00a0 confident\u00a0 that\u00a0 intergenomic\u00a0\ntranslocations\u00a0 will\u00a0 facilitate\u00a0 the\u00a0 successful\u00a0\nintrogression\u00a0 of\u00a0 drought\u00a0 resistance\u00a0 and\u00a0 other\u00a0 alien\u00a0\ntraits\u00a0 in\u00a0 bred\u00a0 wheat.\u00a0 But\u00a0 it\u00a0 has\u00a0 to\u00a0 be\u00a0 admitted\u00a0 that\u00a0\nrepair\u00a0 mechanisms\u00a0 on\u00a0 the\u00a0 DNA\u00a0 level\u00a0 are\u00a0 powerful\u00a0\n(Baarends\u00a0et\u00a0al.,\u00a02001;\u00a0Dong\u00a0et\u00a0al.,\u00a02002;\u00a0Morikawa\u00a0&\u00a0\nShirakawa,\u00a0 2001).\u00a0 It\u00a0 is\u00a0 only\u00a0 logical\u00a0 that\u00a0 opposition\u00a0\nwithin\u00a0organic\u00a0farming\u00a0towards\u00a0genetic\u00a0engineering\u00a0 is\u00a0\nnow\u00a0 expanding\u00a0 also\u00a0 to\u00a0 some\u00a0 of\u00a0 those\u00a0 conventional\u00a0\nbreeding\u00a0 methods,\u00a0 some\u00a0 go\u00a0 even\u00a0 so\u00a0 far\u00a0 as\u00a0 to\u00a0 reject\u00a0\nmarker\u00a0 assisted\u00a0 breeding\u00a0 \u2013\u00a0 typically\u00a0 for\u00a0 the\u00a0 organic\u00a0\nagriculture\u00a0 scene,\u00a0 this\u00a0 trend\u00a0 is\u00a0 based\u00a0 on\u00a0 the\u00a0 myth\u00a0 of\u00a0\n\u201cintrinsic\u00a0integrity\u00a0of\u00a0the\u00a0genome\u201d,\u00a0for\u00a0which\u00a0term\u00a0it\u00a0is\u00a0\nnot\u00a0possible\u00a0in\u00a0the\u00a0literature\u00a0to\u00a0find\u00a0a\u00a0proper\u00a0scientific\u00a0\ndefinition\u00a0based\u00a0on\u00a0comparisons\u00a0(Ammann,\u00a02008).\u00a0The\u00a0\naddition\u00a0 of\u00a0 rejected\u00a0 breeding\u00a0 methods\u00a0 would\u00a0\nultimately\u00a0 lead\u00a0to\u00a0an\u00a0absurd\u00a0situation,\u00a0where\u00a0most\u00a0of\u00a0\n\nseveral\u00a0 species\u00a0 studied\u00a0 in\u00a0 detail\u00a0 with\u00a0 molecular\u00a0 markers,\u00a0\nrecurrent\u00a0 polyploidization\u00a0 was\u00a0 shown\u00a0 to\u00a0 occur\u00a0 with\u00a0 great\u00a0\nfrequency\u00a0 during\u00a0 short\u00a0 time\u00a0 spans\u00a0 and\u00a0 in\u00a0 small\u00a0 geographic\u00a0\nareas\u00a0 ((Brochmann\u00a0 et\u00a0 al.,\u00a0 1992;\u00a0 Soltis\u00a0 et\u00a0 al.,\u00a0 1995).\u00a0 For\u00a0\nexample,\u00a0 Tragopogon\u00a0 mirus\u00a0 and\u00a0 Tragopogon\u00a0 miscellus\u00a0 may\u00a0\nhave\u00a0 formed\u00a0 as\u00a0 many\u00a0 as\u00a0 9\u00a0 and\u00a0 21\u00a0 times,\u00a0 respectively,\u00a0 in\u00a0 a\u00a0\nsmall\u00a0 region\u00a0 of\u00a0 eastern\u00a0 Washington\u00a0 and\u00a0 adjacent\u00a0 Idaho\u00a0\nduring\u00a0 just\u00a0 the\u00a0 past\u00a0 50\u00a0 years\u00a0 (Soltis\u00a0 et\u00a0 al.,\u00a0 1995).", "start_char_idx": 40305, "end_char_idx": 42883, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fc937472-82fa-41b4-87ec-3af548807422": {"__data__": {"id_": "fc937472-82fa-41b4-87ec-3af548807422", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "33361f1e-ee96-411f-8961-214fbfa15225", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "38d0dea53cf6b4b67ae2ebfc0c93b4f2ed06818a61222617e2ee9f10871180b2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bc098ba5-30bc-4870-8591-05777440b758", "node_type": "1", "metadata": {}, "hash": "607ce29819a475d72e2164ac1502f1feb7f2819f7ad02beee1d7436961d8e587", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 frequent\u00a0 recurrence\u00a0 of\u00a0 polyploidization\u00a0 also\u00a0 has\u00a0 major\u00a0\nevolutionary\u00a0 implications,\u00a0 suggesting\u00a0 that\u00a0 polyploids\u00a0 are\u00a0\nmuch\u00a0more\u00a0genetically\u00a0dynamic\u00a0than\u00a0formerly\u00a0envisioned.\u201d\u00a0\n\u00a0\nPolyploidy\u00a0 is\u00a0 one\u00a0 of\u00a0 the\u00a0 most\u00a0 distinctive\u00a0 and\u00a0\nwidespread\u00a0modes\u00a0of\u00a0speciation\u00a0in\u00a0higher\u00a0plants.\u00a0Thirty\u00a0\nto\u00a070%\u00a0\u00a0of\u00a0angiosperms,\u00a0including\u00a0many\u00a0important\u00a0crop\u00a0\nplants,\u00a0 are\u00a0 estimated\u00a0 to\u00a0 have\u00a0 polyploidy\u00a0 in\u00a0 their\u00a0\nlineages\u00a0 (Song\u00a0 et\u00a0 al.,\u00a0 1995),\u00a0 again\u00a0 a\u00a0 strong\u00a0 argument\u00a0\nfor\u00a0the\u00a0high\u00a0dynamics\u00a0of\u00a0the\u00a0genome\u00a0of\u00a0higher\u00a0plants.\u00a0\n\u00a0\n4.5.\u00a0 Horizontal\u00a0 Geneflow\u00a0 between\u00a0 Pro\u00ad\nCaryotes\u00a0and\u00a0Eu\u00adCaryotes\u00a0\n\nThere\u00a0 is\u00a0 a\u00a0 rich\u00a0 literature\u00a0 documenting\u00a0 \u2013\u00a0 on\u00a0 an\u00a0\n\nevolutionary\u00a0 scale\u00a0 \u2013\u00a0 that\u00a0 horizontal\u00a0 transfer\u00a0 of\u00a0 genes\u00a0\n\n(HGT)\u00a0 between\u00a0 pro\u2010caryotes\u00a0 and\u00a0 eu\u2010caryotes\u00a0 are\u00a0 not\u00a0\n\nuncommon:\u00a0However,\u00a0according\u00a0to\u00a0(Keeling\u00a0&\u00a0Palmer,\u00a0\n\n2008)\u00a0many\u00a0records\u00a0of\u00a0HGT\u00a0(Consortium,\u00a02001)\u00a0are\u00a0not\u00a0\n\nconfirmed\u00a0 by\u00a0 phylogenetic\u00a0 analysis\u00a0 proving\u00a0\n\nincongruent\u00a0 sequences\u00a0 (Stanhope\u00a0 et\u00a0 al.,\u00a0 2001).\u00a0 This\u00a0\n\nmeans\u00a0 that\u00a0 potentially,\u00a0 molecular\u00a0 processes\u00a0 can\u00a0\n\ntransfer\u00a0 foreign\u00a0 genes,\u00a0 so\u00a0 \u2013\u00a0 actually,\u00a0 all\u00a0 living\u00a0\n\norganisms\u00a0 are\u00a0 in\u00a0 that\u00a0 sense\u00a0 \u201ctransgenic\u00a0 organisms\u201d,\u00a0\n\nbut\u00a0 only\u00a0 considering\u00a0 evolutionary\u00a0 time\u00a0 scales\u00a0 of\u00a0\n\nmillions\u00a0of\u00a0years\u00a0time\u00a0span\u00a0for\u00a0the\u00a0transfer\u00a0event.\u00a0To\u00a0\n\nbe\u00a0 clear,\u00a0 there\u00a0 is\u00a0 no\u00a0 evidence\u00a0 of\u00a0 horizontal\u00a0 gene\u00a0\n\ntransfer\u00a0 coming\u00a0 from\u00a0 the\u00a0 relatively\u00a0 new\u00a0 practice\u00a0 in\u00a0\n\nmodern\u00a0 breeding\u00a0 methods\u00a0 of\u00a0 genetic\u00a0 engineering\u00a0\n\n(Smalla\u00a0&\u00a0Sobecky,\u00a02002;\u00a0Smalla\u00a0&\u00a0Vogel,\u00a02007).\u00a0Even\u00a0\n\nthe\u00a0much\u00a0publicized\u00a0case\u00a0of\u00a0HGT\u00a0with\u00a0a\u00a0transgene\u00a0 in\u00a0\n\nthe\u00a0 human\u00a0 guts\u00a0 is\u00a0 based\u00a0 on\u00a0 clearly\u00a0 wrong\u00a0\n\ninterpretation\u00a0 and\u00a0 false\u00a0 claims\u00a0 (Ammann,\u00a0 2002).\u00a0\n\nHowever,\u00a0for\u00a0mitochondrial\u00a0DNA\u00a0things\u00a0are\u00a0different:\u00a0\n\nAccording\u00a0 to\u00a0 (Archibald\u00a0 &\u00a0 Richards,\u00a0 2010),\u00a0\n\nmitrochondrial\u00a0 DNA\u00a0 can\u00a0 be\u00a0 exchanges\u00a0 rather\u00a0\n\nfrequently:\u00a0\u00a0\n\n\u201cParasitic\u00a0 plants\u00a0 and\u00a0 their\u00a0 hosts\u00a0 have\u00a0 proven\u00a0 remarkably\u00a0\n\nadept\u00a0 at\u00a0 exchanging\u00a0 fragments\u00a0 of\u00a0 mitochondrial\u00a0 DNA.\u00a0 Two\u00a0\n\nrecent\u00a0 studies\u00a0 (Mower\u00a0 et\u00a0 al.,\u00a0 2010;\u00a0 Richardson\u00a0 &\u00a0 Palmer,\u00a0\n\n2007)\u00a0 \u00a0 provide\u00a0 important\u00a0 mechanistic\u00a0 insights\u00a0 into\u00a0 the\u00a0\n\npattern,\u00a0 process\u00a0 and\u00a0 consequences\u00a0 of\u00a0 horizontal\u00a0 gene\u00a0\n\ntransfer,\u00a0 demonstrating\u00a0 that\u00a0 genes\u00a0 can\u00a0 be\u00a0 transferred\u00a0 in\u00a0\n\nlarge\u00a0chunks\u00a0and\u00a0that\u00a0gene\u00a0conversion\u00a0between\u00a0foreign\u00a0and\u00a0\n\nnative\u00a0genes\u00a0leads\u00a0to\u00a0intragenic\u00a0mosaicism.\u00a0A\u00a0model\u00a0involving\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 42885, "end_char_idx": 45303, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bc098ba5-30bc-4870-8591-05777440b758": {"__data__": {"id_": "bc098ba5-30bc-4870-8591-05777440b758", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fc937472-82fa-41b4-87ec-3af548807422", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "a2bd4ccb854fff1d82b5a12ad4d958473c3872016c168e62626bdc683ffb2b09", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "635c1c46-71b9-4225-90b3-55303029ff24", "node_type": "1", "metadata": {}, "hash": "a618687c9f9aaea53035422a4b867cb60a1453579dd04ec2338c6ab5c4d0c914", "class_name": "RelatedNodeInfo"}}, "text": "1:\u00a031\u201048\u00a0\n\n41\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nIn\u00a0 a\u00a0 letter7\u00a0 to\u00a0 the\u00a0 executives\u00a0 of\u00a0 the\u00a0 Convention\u00a0 on\u00a0\nBiological\u00a0 Diversity\u00a0 (CBD),\u00a0 the\u00a0 Public\u00a0 Research\u00a0 and\u00a0\nRegulation\u00a0 Initiative\u00a0 (PRRI)\u00a0 is\u00a0 asking\u00a0 for\u00a0 a\u00a0 scientific\u00a0\ndiscussion\u00a0in\u00a0order\u00a0to\u00a0exempt\u00a0a\u00a0list\u00a0of\u00a0GM\u00a0crops\u00a0from\u00a0\nthe\u00a0 expensive\u00a0 regulatory\u00a0 process\u00a0 for\u00a0 approval,\u00a0 here\u00a0\nonly\u00a0the\u00a0final\u00a0statement:\u00a0\n\n\u201cBearing\u00a0in\u00a0mind\u00a0that\u00a0the\u00a0method\u00a0of\u00a0transformation\u00a0itself\u00a0is\u00a0\nneutral,\u00a0 i.e.\u00a0 that\u00a0 there\u00a0 are\u00a0 no\u00a0 risks\u00a0 related\u00a0 to\u00a0 process\u00a0 of\u00a0\ntransformation,\u00a0PRRI\u00a0believes\u00a0that\u00a0there\u00a0are\u00a0several\u00a0types\u00a0of\u00a0\nLMOs\u00a0and\u00a0traits\u00a0for\u00a0which\u00a0\u2010\u00a0on\u00a0the\u00a0basis\u00a0of\u00a0the\u00a0characteristics\u00a0\nof\u00a0the\u00a0host\u00a0plant,\u00a0the\u00a0functioning\u00a0of\u00a0the\u00a0 inserted\u00a0genes\u00a0and\u00a0\nexperience\u00a0with\u00a0the\u00a0resulting\u00a0GMO\u00a0\u2010\u00a0it\u00a0can\u00a0be\u00a0concluded\u00a0that\u00a0\nthey\u00a0are\u00a0as\u00a0safe\u00a0as\u00a0its\u00a0conventional\u00a0counterpart\u00a0with\u00a0respect\u00a0\nto\u00a0 potential\u00a0 effects\u00a0 on\u00a0 the\u00a0 environment,\u00a0 taking\u00a0 also\u00a0 into\u00a0\naccount\u00a0human\u00a0health.\u00a0\u201c\u00a0\n\nIn\u00a0 a\u00a0 recent\u00a0 paper,\u00a0 an\u00a0 indiscriminate\u00a0 continuation\u00a0 of\u00a0\n\nfood\u00a0biosafety\u00a0research\u00a0 is\u00a0questioned\u00a0on\u00a0the\u00a0basis\u00a0of\u00a0\n\nall\u00a0the\u00a0above\u00a0arguments\u00a0by\u00a0Herman\u00a0et\u00a0al.\u00a0(Herman\u00a0et\u00a0\n\nal.,\u00a02009)\u00a0with\u00a0good\u00a0reason:\u00a0\n\n\u201cCompositional\u00a0studies\u00a0comparing\u00a0transgenic\u00a0crops\u00a0with\u00a0non\u2010\ntransgenic\u00a0 crops\u00a0 are\u00a0 almost\u00a0 universally\u00a0 required\u00a0 by\u00a0\ngovernmental\u00a0 regulatory\u00a0 bodies\u00a0 to\u00a0 support\u00a0 the\u00a0 safety\u00a0\nassessment\u00a0 of\u00a0 new\u00a0 transgenic\u00a0 crops.\u00a0 Here\u00a0 we\u00a0 discuss\u00a0 the\u00a0\nassumptions\u00a0 that\u00a0 led\u00a0 to\u00a0 this\u00a0 requirement\u00a0 and\u00a0 lay\u00a0 out\u00a0 the\u00a0\ntheoretical\u00a0 and\u00a0 empirical\u00a0 evidence\u00a0 suggesting\u00a0 that\u00a0 such\u00a0\nstudies\u00a0are\u00a0no\u00a0more\u00a0necessary\u00a0for\u00a0evaluating\u00a0the\u00a0safety\u00a0of\u00a0\ntransgenic\u00a0crops\u00a0than\u00a0they\u00a0are\u00a0for\u00a0traditionally\u00a0bred\u00a0crops.\u201d\u00a0\n\n6.1.\u00a0 Perspectives\u00a0 for\u00a0 a\u00a0 dissolution\u00a0 of\u00a0 this\u00a0\ndivide\u00a0\n\nThese\u00a0new\u00a0perspectives\u00a0create\u00a0hope,\u00a0that\u00a0solutions\u00a0can\u00a0\n\nbe\u00a0found:\u00a0\n\nIn\u00a0 a\u00a0 first\u00a0 phase\u00a0 some\u00a0 of\u00a0 the\u00a0 widespread\u00a0 transgenic\u00a0\ncrops\u00a0like\u00a0transgenic\u00a0maize\u00a0with\u00a0the\u00a0Cry1Ab\u00a0endotoxin\u00a0\nshould\u00a0 be\u00a0 exempt\u00a0 from\u00a0 regulation,\u00a0 which\u00a0 is\u00a0 indeed\u00a0\npossible\u00a0 according\u00a0 to\u00a0 art.\u00a0 7.4\u00a0 in\u00a0 the\u00a0 Cartagena\u00a0\nProtocol.\u00a0 In\u00a0 COP\u2010MOP58\u00a0 2010\u00a0 in\u00a0 Japan\u00a0 it\u00a0 should\u00a0 be\u00a0\npossible,\u00a0to\u00a0amend\u00a0the\u00a0protocol\u00a0with\u00a0the\u00a0introduction\u00a0\nof\u00a0 a\u00a0 dynamics\u00a0 which\u00a0 allows\u00a0 to\u00a0 start\u00a0 the\u00a0 regulatory\u00a0\nprocess\u00a0with\u00a0an\u00a0initial\u00a0phase\u00a0focusing\u00a0on\u00a0the\u00a0process\u00a0of\u00a0\ntransgenesis,\u00a0 first\u00a0 following\u00a0 procedures\u00a0 proposed\u00a0 for\u00a0\nnon\u2010target\u00a0 insects\u00a0 by\u00a0 (Raybould,\u00a0 2010;\u00a0 Romeis\u00a0 et\u00a0 al.,\u00a0\n2008),\u00a0but\u00a0 in\u00a0due\u00a0time\u00a0shifting\u00a0 later\u00a0the\u00a0focus\u00a0on\u00a0the\u00a0\nproduct,\u00a0 making\u00a0 it\u00a0 possible\u00a0 to\u00a0 abbreviate\u00a0 the\u00a0\nregulatory\u00a0process\u00a0wherever\u00a0possible\u00a0and\u00a0feasible.\u00a0\u00a0\n\nthe\u00a0modern\u00a0time\u00a0traits\u00a0would\u00a0have\u00a0to\u00a0be\u00a0rejected\u00a0and\u00a0\nbreeding\u00a0would\u00a0be\u00a0forced\u00a0to\u00a0start\u00a0from\u00a0scratch.", "start_char_idx": 45304, "end_char_idx": 47930, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "635c1c46-71b9-4225-90b3-55303029ff24": {"__data__": {"id_": "635c1c46-71b9-4225-90b3-55303029ff24", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bc098ba5-30bc-4870-8591-05777440b758", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "74dc93af2dc6ddebc0a1750762680f7ae884c1eaf158719840b6ba8dfa04b7b1", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1d150305-bf4e-4bfb-8cd2-aa2c5a88c3a8", "node_type": "1", "metadata": {}, "hash": "5815783cc605061916e6e903863f061d5696cac9481572cd31a09e70ee1c3553", "class_name": "RelatedNodeInfo"}}, "text": "Basically,\u00a0many\u00a0of\u00a0the\u00a0first\u00a0generation\u00a0GM\u00a0crops\u00a0should\u00a0\nbe\u00a0 today\u00a0 subject\u00a0 to\u00a0 a\u00a0 professional\u00a0 debate\u00a0 on\u00a0\nderegulation,\u00a0and\u00a0there\u00a0 is\u00a0good\u00a0and\u00a0sturdy\u00a0reason\u00a0to\u00a0\nstate\u00a0 that\u00a0 many\u00a0 of\u00a0 these\u00a0 GM\u00a0 crops\u00a0 should\u00a0 not\u00a0 have\u00a0\nbeen\u00a0 treated\u00a0 in\u00a0 such\u00a0 a\u00a0 special\u00a0 way\u00a0 in\u00a0 the\u00a0 first\u00a0 place,\u00a0\nthey\u00a0can\u00a0be\u00a0compared\u00a0 in\u00a0their\u00a0risk\u00a0potential\u00a0to\u00a0many\u00a0\ncrops\u00a0created\u00a0with\u00a0traditional\u00a0methods.\u00a0\n\nThis\u00a0should\u00a0not\u00a0be\u00a0misunderstood\u00a0as\u00a0a\u00a0plea\u00a0for\u00a0general\u00a0\nderegulation\u00a0 of\u00a0 GM\u00a0 crops,\u00a0 rather\u00a0 for\u00a0 a\u00a0 strict\u00a0 and\u00a0\nscience\u00a0 based\u00a0 risk\u00a0 based\u00a0 regulation\u00a0 focusing\u00a0 also\u00a0 on\u00a0\nproducts,\u00a0not\u00a0on\u00a0processes\u00a0alone.\u00a0\n\nSomatic\u00a0 hybridization\u00a0 also\u00a0 deserves\u00a0 a\u00a0 short\u00a0 mention\u00a0\nhere,\u00a0 the\u00a0 method\u00a0 enabled\u00a0 the\u00a0 artificial\u00a0\nhybridization\u00a0of\u00a0crops\u00a0which\u00a0have\u00a0no\u00a0genomic\u00a0natural\u00a0\ncompatibility,\u00a0 see\u00a0 the\u00a0 review\u00a0 of\u00a0 (Waara\u00a0 &\u00a0 Glimelius,\u00a0\n1995),\u00a0 Progeny\u00a0 analysis\u00a0 of\u00a0 some\u00a0 hybrid\u00a0 combinations\u00a0\nalso\u00a0 reveals\u00a0 inter\u2010genomic\u00a0 translocations\u00a0 which\u00a0 may\u00a0\nlead\u00a0 to\u00a0 the\u00a0 introgression\u00a0 of\u00a0 the\u00a0 alien\u00a0 genes\u00a0 .\u00a0\nFurthermore,\u00a0fusion\u00a0techniques\u00a0enable\u00a0the\u00a0resynthesis\u00a0\nof\u00a0 allopolyploid\u00a0 crops\u00a0 to\u00a0 increase\u00a0 their\u00a0 genetic\u00a0\nvariability\u00a0 and\u00a0 to\u00a0 restore\u00a0 ploidy\u00a0 level\u00a0 and\u00a0\nheterozygosity\u00a0after\u00a0breeding\u00a0at\u00a0reduced\u00a0ploidy\u00a0level\u00a0in\u00a0\npolyploid\u00a0crops.\u00a0\n\n6.0.\u00a0 Regulatory\u00a0 dissent\u00a0 over\u00a0\nmolecular\u00a0 differences\u00a0 causes\u00a0\ntransatlantic\u00a0divide\u00a0\nThis\u00a0actually\u00a0includes\u00a0a\u00a0critical\u00a0questioning\u00a0about\u00a0some\u00a0\n\nbasic\u00a0 rules\u00a0 of\u00a0 the\u00a0 United\u00a0 Nations\u00a0 Convention\u00a0 on\u00a0\n\nBiological\u00a0Diversity\u00a0(CBD):\u00a0transgenic\u00a0crops\u00a0of\u00a0the\u00a0first\u00a0\n\ngeneration\u00a0 should\u00a0 not\u00a0 have\u00a0 been\u00a0 generally\u00a0 subjected\u00a0\n\nto\u00a0 regulation\u00a0 purely\u00a0 based\u00a0 on\u00a0 the\u00a0 process\u00a0 of\u00a0\n\ntransgenesis\u00a0alone;\u00a0rather\u00a0it\u00a0would\u00a0have\u00a0been\u00a0wiser\u00a0to\u00a0\n\nhave\u00a0a\u00a0close\u00a0look\u00a0at\u00a0the\u00a0products\u00a0in\u00a0each\u00a0case,\u00a0as\u00a0John\u00a0\n\nMaddox\u00a0 already\u00a0 proposed\u00a0 in\u00a0 1992\u00a0 in\u00a0 an\u00a0 editorial\u00a0 in\u00a0\n\nNature\u00a0 (Anonymous,\u00a0 1992).\u00a0 This\u00a0 is\u00a0 also\u00a0 the\u00a0 view\u00a0 of\u00a0\n\nCanadian\u00a0 regulators\u00a0 (Andree,\u00a0 2002;\u00a0 Berwald\u00a0 et\u00a0 al.,\u00a0\n\n2006;\u00a0Macdonald\u00a0&\u00a0Yarrow,\u00a02002),\u00a0where\u00a0the\u00a0novelty\u00a0\n\nof\u00a0 the\u00a0 crop\u00a0 is\u00a0 the\u00a0 primary\u00a0 trigger\u00a0 for\u00a0 regulation.\u00a0 This\u00a0\n\ntransatlantic\u00a0 (and\u00a0 transoceanic)\u00a0 contrast\u00a0 has\u00a0 been\u00a0\n\ncommented\u00a0by\u00a0many\u00a0(Aerni\u00a0et\u00a0al.,\u00a02009;\u00a0Bennett\u00a0et\u00a0al.,\u00a0\n\n1986;\u00a0Kalaitzandonakes\u00a0et\u00a0al.,\u00a02005;\u00a0Ramjoue,\u00a02007a,\u00a0\n\nb;\u00a0 Snyder\u00a0 et\u00a0 al.,\u00a0 2008;\u00a0 Thro,\u00a0 2004),\u00a0 and\u00a0 although\u00a0 for\u00a0\n\nmany\u00a0years\u00a0a\u00a0solution\u00a0and\u00a0mediation\u00a0seemed\u00a0to\u00a0be\u00a0too\u00a0\n\ndifficult,\u00a0contrasts\u00a0can\u00a0be\u00a0overcome:\u00a0\n\n7PRRI\u00a0 letter\u00a0 :\u00a0 http://www.pubresreg.org/index.php?\n\noption=com_docman&task=doc_download&gid=490\u00a0\n8Fifth\u00a0meeting\u00a0of\u00a0the\u00a0Conference\u00a0of\u00a0the\u00a0Parties\u00a0serving\u00a0as\u00a0the\u00a0Meet\u2010\n\ning\u00a0of\u00a0the\u00a0Parties\u00a0to\u00a0the\u00a0Cartagena\u00a0Protocol\u00a0on\u00a0Biosafety\u00a0(COP\u2010MOP\u00a0\n\n5),\u00a0 \u00a0 11\u00a0 \u2013\u00a0 15.\u00a0 10.", "start_char_idx": 47933, "end_char_idx": 50549, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1d150305-bf4e-4bfb-8cd2-aa2c5a88c3a8": {"__data__": {"id_": "1d150305-bf4e-4bfb-8cd2-aa2c5a88c3a8", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "635c1c46-71b9-4225-90b3-55303029ff24", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "083e79f3d94da94ee97815f888cbbaa9dca864b9073e9cfbfbea814072a2ae9a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "177f4ec6-383e-4c35-850b-c85ad74ff263", "node_type": "1", "metadata": {}, "hash": "1f5967ddf86e0aa6a2724d84e2754216bd0eb3ec89f11d37aa09475781bc454e", "class_name": "RelatedNodeInfo"}}, "text": "10.\u00a0 2010\u00a0 Nagoya,\u00a0 Japan\u00a0 http://bch.cbd.int/protocol/\n\nmeetings/\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n42\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nThe\u00a0difficulties\u00a0start\u00a0there,\u00a0where\u00a0a\u00a0clear\u00a0definition\u00a0of\u00a0\n\nPNTs\u00a0 is\u00a0 needed\u00a0 to\u00a0 come\u00a0 to\u00a0 a\u00a0 decision:\u00a0 It\u00a0 means\u00a0 that\u00a0\n\nplants\u00a0 produced\u00a0 using\u00a0 recombinant\u00a0 DNA\u00a0 techniques,\u00a0\n\nchemical\u00a0 mutagenesis,\u00a0 cell\u00a0 fusion,\u00a0 cis\u2010genics\u00a0 or\u00a0 any\u00a0\n\nother\u00a0 in\u2010vitro\u00a0technique\u00a0 leading\u00a0to\u00a0a\u00a0novel\u00a0trait,\u00a0need\u00a0\n\nto\u00a0undergo\u00a0risk\u00a0assessment\u00a0in\u00a0the\u00a0Canadian\u00a0system.\u00a0No\u00a0\n\nwonder\u00a0the\u00a0Canadian\u00a0definition\u00a0of\u00a0novel\u00a0traits\u00a0is\u00a0rather\u00a0\n\nwordy,\u00a0but\u00a0remains\u00a0broad\u00a0minded:\u00a0\n\n\u201cA\u00a0 plant\u00a0 variety/genotype\u00a0 possessing\u00a0 characteristics\u00a0 that\u00a0\n\ndemonstrate\u00a0 neither\u00a0 familiarity\u00a0 nor\u00a0 substantial\u00a0 equivalence\u00a0\n\nto\u00a0those\u00a0present\u00a0in\u00a0a\u00a0distinct,\u00a0stable\u00a0population\u00a0of\u00a0a\u00a0cultivated\u00a0\n\nseed\u00a0 in\u00a0 Canada\u00a0 and\u00a0 that\u00a0 have\u00a0 been\u00a0 intentionally\u00a0 selected,\u00a0\n\ncreated\u00a0 or\u00a0 introduced\u00a0 into\u00a0 a\u00a0 population\u00a0 of\u00a0 that\u00a0 species\u00a0\n\nthrough\u00a0a\u00a0specific\u00a0genetic\u00a0change.\u201d\u00a0\n\n7.0.\u00a0Conclusions\u00a0\nThere\u00a0can\u00a0be\u00a0no\u00a0doubt\u00a0that\u00a0product\u2010based\u00a0regulatory\u00a0\n\napproaches\u00a0 are\u00a0 truest\u00a0 to\u00a0 the\u00a0 scientific\u00a0 principle\u00a0 that\u00a0\n\nbiotechnology\u00a0 is\u00a0not\u00a0 inherently\u00a0more\u00a0risky\u00a0than\u00a0other\u00a0\n\ntechnologies\u00a0that\u00a0have\u00a0a\u00a0long\u00a0and\u00a0accepted\u00a0history\u00a0of\u00a0\n\napplication\u00a0in\u00a0agriculture\u00a0and\u00a0food\u00a0production,\u00a0it\u00a0is\u00a0also\u00a0\n\nless\u00a0 prescriptive\u00a0 than\u00a0 process\u2010based\u00a0 systems,\u00a0 see\u00a0\n\nMcLean\u00a0et\u00a0al.\u00a0(McLean\u00a0et\u00a0al.,\u00a02002).\u00a0\n\nA\u00a0conceptual\u00a0framework\u00a0is\u00a0proposed\u00a0by\u00a0IFPRI/ISNAR\u00a0\n\nin\u00a0 2002,\u00a0 the\u00a0 International\u00a0 Service\u00a0 for\u00a0 National\u00a0\n\nAgricultural\u00a0 Research\u00a0 (McLean\u00a0 et\u00a0 al.,\u00a0 2002),\u00a0 a\u00a0 careful\u00a0\n\nevaluation\u00a0 of\u00a0 process\u2010based\u00a0 versus\u00a0 product\u2010based\u00a0\n\ntriggers\u00a0in\u00a0regulatory\u00a0action\u00a0can\u00a0also\u00a0lead\u00a0to\u00a0a\u00a0merger\u00a0\n\nof\u00a0 both\u00a0 seemingly\u00a0 so\u00a0 contrasting\u00a0 concepts\u00a0 into\u00a0 a\u00a0\n\nlegalized\u00a0 decision\u00a0 making\u00a0 process\u00a0 on\u00a0 which\u00a0 trigger\u00a0\n\nshould\u00a0be\u00a0chosen\u00a0in\u00a0a\u00a0case\u00a0by\u00a0case\u00a0strategy:\u00a0\n\n\u201cProcess\u2010based\u00a0 triggers\u00a0 are\u00a0 the\u00a0 rule\u00a0 in\u00a0 almost\u00a0 all\u00a0 countries\u00a0\n\nthat\u00a0 have\u00a0 developed\u00a0 national\u00a0 biosafety\u00a0 regulatory\u00a0 systems;\u00a0\n\nthere\u00a0are\u00a0exceptions,\u00a0however,\u00a0where\u00a0the\u00a0novelty\u00a0of\u00a0the\u00a0trait\u00a0\n\ndetermines\u00a0 the\u00a0 extent\u00a0 of\u00a0 regulatory\u00a0 oversight\u00a0 and\u00a0 not\u00a0 the\u00a0\n\nprocess\u00a0 by\u00a0 which\u00a0 the\u00a0 trait\u00a0 was\u00a0 introduced.", "start_char_idx": 50546, "end_char_idx": 52748, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "177f4ec6-383e-4c35-850b-c85ad74ff263": {"__data__": {"id_": "177f4ec6-383e-4c35-850b-c85ad74ff263", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1d150305-bf4e-4bfb-8cd2-aa2c5a88c3a8", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "1dfa9dfcd7652ee67cc45fc21d64ccac8c49a232cd0ef371d1cdb681702867fc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5c11db01-cdb7-4541-9417-8b173c3e5269", "node_type": "1", "metadata": {}, "hash": "8d332b5f7cceafb788575c71d6e703f10b48a926b9fdf096415fb1541bdbca9f", "class_name": "RelatedNodeInfo"}}, "text": "While\u00a0 such\u00a0 a\u00a0\n\nproduct\u2010based\u00a0approach\u00a0to\u00a0defining\u00a0the\u00a0object\u00a0of\u00a0regulation\u00a0\n\nis\u00a0 truest\u00a0 to\u00a0 the\u00a0 scientific\u00a0 principle\u00a0 that\u00a0 biotechnology\u00a0 is\u00a0 not\u00a0\n\ninherently\u00a0 more\u00a0 risky\u00a0 than\u00a0 other\u00a0 technologies\u00a0 that\u00a0 have\u00a0 a\u00a0\n\nlong\u00a0 and\u00a0 accepted\u00a0 history\u00a0 of\u00a0 application\u00a0 in\u00a0 agriculture\u00a0 and\u00a0\n\nfood\u00a0 production,\u00a0 it\u00a0 is\u00a0 less\u00a0 prescriptive\u00a0 than\u00a0 process\u2010based\u00a0\n\nregulatory\u00a0systems.\u201d\u00a0\n\nMany\u00a0 of\u00a0 the\u00a0 debates\u00a0 on\u00a0 those\u00a0 two\u00a0 concepts\u00a0 suffer\u00a0\n\nfrom\u00a0a\u00a0lack\u00a0of\u00a0clear\u2010cut\u00a0definitions,\u00a0it\u00a0will\u00a0be\u00a0important\u00a0\n\nto\u00a0have\u00a0a\u00a0close\u00a0look\u00a0at\u00a0the\u00a0Canadian\u00a0regulatory\u00a0system\u00a0\n\nand\u00a0the\u00a0definition\u00a0of\u00a0PNTs\u00a0(Plants\u00a0with\u00a0Novel\u00a0Traits).\u00a0In\u00a0\n\nCanada,\u00a0the\u00a0trigger\u00a0for\u00a0risk\u2010assessment\u00a0is\u00a0the\u00a0novelty\u00a0of\u00a0\n\nthe\u00a0plant\u00a0rather\u00a0than\u00a0the\u00a0methods\u00a0used\u00a0to\u00a0produce\u00a0it.\u00a0\n\n8.0.\u00a0Cited\u00a0literature\u00a0\nAerni,\u00a0P.,\u00a0Rae,\u00a0A.,\u00a0&\u00a0Lehmann,\u00a0B.\u00a0(2009)\u00a0Nostalgia\u00a0versus\u00a0\n\nPragmatism?\u00a0How\u00a0attitudes\u00a0and\u00a0interests\u00a0shape\u00a0the\u00a0\nterm\u00a0sustainable\u00a0agriculture\u00a0in\u00a0Switzerland\u00a0and\u00a0New\u00a0\nZealand.\u00a0Food\u00a0Policy,\u00a034,\u00a02,\u00a0pp\u00a0\u00a0227\u2010235\u00a0http://\nwww.sciencedirect.com/science/article/B6VCB\u2010\n4V1MFKR\u20101/2/b72610f6397bc5572a076cbe0ae3e599\u00a0\nAND\u00a0http://www.botanischergarten.ch/Sustainability/\nAerni\u2010Nostalgia\u2010versus\u2010Pragmatism\u20102009.pdf\u00a0\n\n\u00a0\nAmmann,\u00a0K.\u00a0(2002)\u00a0University\u00a0of\u00a0Newcastel\u00a0report\u00a0\n\nsummaries:\u00a0no\u00a0significant\u00a0horizontal\u00a0transgene\u00a0transfer\u00a0\ndetected\u00a0in\u00a0human\u00a0guts.\u00a0In\u00a0Berne\u00a0Debates\u00a0blog,\u00a0\nUniversity\u00a0of\u00a0Newcastle\u00a0report\u00a0summaries.\u00a0Klaus\u00a0\nAmmann,\u00a0Bern\u00a0\n\n\u00a0 http://www.ask\u2010force.org/web/HorizontalGT/Ammann\u2010\nNewcastle\u2010Human\u2010Guts\u20102002.PDF\u00a0\n\n\u00a0\nAmmann,\u00a0K.\u00a0(2008)\u00a0Feature:\u00a0Integrated\u00a0farming:\u00a0Why\u00a0\n\norganic\u00a0farmers\u00a0should\u00a0use\u00a0transgenic\u00a0crops.\u00a0New\u00a0\nBiotechnology,\u00a025,\u00a02,\u00a0pp\u00a0\u00a0101\u00a0\u2010\u00a0107\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/NewBiotech/\nAmmann\u2010Integrated\u2010Farming\u2010Organic\u20102008.publ.pdf\u00a0\u00a0\nAND\u00a0DOI:\u00a0http://dx.doi.org/10.1016/j.nbt.2008.08.012\u00a0\n\n\u00a0\nAmmann,\u00a0K.\u00a0(2009)\u00a0Feature:\u00a0Why\u00a0farming\u00a0with\u00a0high\u00a0tech\u00a0\n\nmethods\u00a0should\u00a0integrate\u00a0elements\u00a0of\u00a0organic\u00a0\nagriculture.\u00a0accepted,\u00a0corrected\u00a0proof.\u00a0New\u00a0\n\nBiotechnology,\u00a04,\u00a0\u00a0pp\u00a0\u00a0\u00a0\n\u00a0 http://dx.doi.org/10.1016/j.nbt.2009.06.933\u00a0AND\u00a0\n\nhttp://www.botanischergarten.ch/NewBiotech/\nIntegrated\u2010Farming\u2010Biotech\u2010Org\u201020090724\u2010publ.pdf\u00a0\n\nAndree,\u00a0P.\u00a0(2002)\u00a0The\u00a0biopolitics\u00a0of\u00a0genetically\u00a0modified\u00a0\norganisms\u00a0in\u00a0Canada.\u00a0Journal\u00a0of\u00a0Canadian\u00a0Studies\u2010Revue\u00a0\nD\u00a0Etudes\u00a0Canadiennes,\u00a037,\u00a03,\u00a0pp\u00a0\u00a0162\u2010191\u00a0\u00a0<Go\u00a0to\u00a0ISI>://\nWOS:000236751100009\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Andree\u2010\nBiopolitics\u2010\u2010GMO\u2010Canada.pdf\u00a0\n\n\u00a0\nAnonymous\u00a0(1992)\u00a0Products\u00a0pose\u00a0no\u00a0special\u00a0risks\u00a0just\u00a0\n\nbecause\u00a0of\u00a0the\u00a0processes\u00a0used\u00a0to\u00a0make\u00a0them.", "start_char_idx": 52750, "end_char_idx": 55212, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5c11db01-cdb7-4541-9417-8b173c3e5269": {"__data__": {"id_": "5c11db01-cdb7-4541-9417-8b173c3e5269", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "177f4ec6-383e-4c35-850b-c85ad74ff263", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "77f86a028cee4dc8f2e77966a4b0d2a1aabe820145b8510b0a9ac4c6b8a2f568", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0e8af144-aea5-492d-800e-fac809241807", "node_type": "1", "metadata": {}, "hash": "ea1bb29ae46bf0b730bd1d9ee86a32906a66fd6292df8888f09d75f88ccab292", "class_name": "RelatedNodeInfo"}}, "text": "Nature,\u00a0\n356,\u00a06364,\u00a0pp\u00a0\u00a01\u20102\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/356001b0\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Anonymous\u2010US\u2010\nRegulation\u2010Nature\u20101992.pdf\u00a0\n\nArber,\u00a0W.\u00a0(1994)\u00a0\n\u00a0 MOLECULAR\u00a0EVOLUTION:\u00a0COMPARISON\u00a0OF\u00a0NATURAL\u00a0\n\nAND\u00a0ENGINEERED\u00a0GENETIC\u00a0VARIATIONS.\u00a0Pontifical\u00a0\nAcademy\u00a0of\u00a0Sciences\u00a0Scripta\u00a0Varia,\u00a0103,\u00a0\u00a0pp\u00a0\u00a090\u2010101\u00a0\u00a0\n\n\u00a0 http://as8978.http.sasm3.net/roman_curia/\npontifical_academies/acdscien/archivio/s.v.103_chagas/\npart3.pdf\u00a0AND\u00a0http://www.botanischergarten.ch/\nGenomics/Arber\u2010Molecular\u2010Evolution\u2010Comparison\u2010PAS\u2010\n1994.pdf\u00a0\n\nArber,\u00a0W.\u00a0(2000)\u00a0\u00a0Genetic\u00a0variation:\u00a0molecular\u00a0mechanisms\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n43\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nmicrospores\u00a0cultured\u00a0in\u00a0anthero.\u00a0Plant\u00a0Cell\u00a0Reports,\u00a018,\u00a0\n10,\u00a0pp\u00a0\u00a0858\u2010862\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1007/s002990050674\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Mutations/Barnabas\u2010\nColchicine\u2010Genome\u2010Doubling\u20101999.pdf\u00a0\n\nBarros,\u00a0E.\u00a0(2010)\u00a0Molecular\u00a0Profiling\u00a0Techniques\u00a0as\u00a0Tools\u00a0to\u00a0\nDetect\u00a0Potential\u00a0Unintended\u00a0Effects\u00a0in\u00a0Genetically\u00a0\nEngineered\u00a0Maize\u00a0(revised\u00a0title),\u00a0Molecular\u00a0Profiling\u00a0\nTechniques\u00a0Detect\u00a0Unintended\u00a0Effects\u00a0in\u00a0Genetically\u00a0\nEngineered\u00a0Maize\u00a0(old\u00a0title).\u00a0ISB\u00a0News\u00a0Report,\u00a0May\u00a0\n2010,\u00a0\u00a0pp\u00a0\u00a04\u20107\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/Genomics/Barros\u2010ISB\n\u2010News\u2010Report\u2010Genomics\u2010Maisze\u2010old\u2010201005.pdf\u00a0AND\u00a0\nhttp://www.botanischergarten.ch/Genomics/Barros\u2010ISB\n\u2010News\u2010Report\u2010Genomics\u2010Maize\u2010new\u20104\u20107.pdf\u00a0\n\nBarros,\u00a0E.,\u00a0Lezar,\u00a0S.,\u00a0Anttonen,\u00a0M.J.,\u00a0Dijk,\u00a0J.P.v.,\u00a0R\u00f6hlig,\u00a0R.M.,\u00a0\nKok,\u00a0E.J.,\u00a0&\u00a0Engel,\u00a0K.\u2010H.\u00a0(2010)\u00a0\n\n\u00a0 Comparison\u00a0of\u00a0two\u00a0GM\u00a0maize\u00a0varieties\u00a0with\u00a0a\u00a0near\u2010\nisogenic\u00a0non\u2010GM\u00a0variety\u00a0using\u00a0transcriptomics,\u00a0\nproteomics\u00a0and\u00a0metabolomics.\u00a0Plant\u00a0Biotechnology\u00a0\nJournal,\u00a08,\u00a04,\u00a0pp\u00a0\u00a0436\u2010451\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1111/j.1467\u20107652.2009.00487.x\u00a0\nAND\u00a0http://www.botanischergarten.ch/Genomics/\nBarros\u2010Comparison\u2010GM\u2010crops\u20102010.pdf\u00a0\n\nBatista,\u00a0R.,\u00a0Saibo,\u00a0N.,\u00a0Lourenco,\u00a0T.,\u00a0&\u00a0Oliveira,\u00a0M.M.\u00a0(2008)\u00a0\nMicroarray\u00a0analyses\u00a0reveal\u00a0that\u00a0plant\u00a0mutagenesis\u00a0may\u00a0\ninduce\u00a0more\u00a0transcriptomic\u00a0changes\u00a0than\u00a0transgene\u00a0\ninsertion.", "start_char_idx": 55213, "end_char_idx": 57244, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0e8af144-aea5-492d-800e-fac809241807": {"__data__": {"id_": "0e8af144-aea5-492d-800e-fac809241807", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5c11db01-cdb7-4541-9417-8b173c3e5269", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "8636bcdacb7158e70a135fbe02b7d8e9925f6e18a551b129325bdb5a17f8190f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b2c8d7e8-2c83-484a-ba0a-04797c1b0ced", "node_type": "1", "metadata": {}, "hash": "ee69b5046b652657a767f7c3327b4e92a80757fd0164aed35c35605edbebb633", "class_name": "RelatedNodeInfo"}}, "text": "Proceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0\nSciences\u00a0of\u00a0the\u00a0United\u00a0States\u00a0of\u00a0America,\u00a0105,\u00a09,\u00a0pp\u00a0\u00a0\n3640\u20103645\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000253846500082\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Batista\u2010\nMicroarray\u2010Analysis\u20102008.pdf\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Transgenesis\u2010\nComparison\u2010Slides.pdf\u00a0AND\u00a0Http://\nwww.botanischergarten.ch/Genomics/Transgenesis\u2010\nComparison\u2010Slides.ppt\u00a0\n\nBaudo,\u00a0M.M.,\u00a0Lyons,\u00a0R.,\u00a0Powers,\u00a0S.,\u00a0Pastori,\u00a0G.M.,\u00a0Edwards,\u00a0\nK.J.,\u00a0Holdsworth,\u00a0M.J.,\u00a0&\u00a0Shewry,\u00a0P.R.\u00a0(2006)\u00a0\nTransgenesis\u00a0has\u00a0less\u00a0impact\u00a0on\u00a0the\u00a0transcriptome\u00a0of\u00a0\nwheat\u00a0grain\u00a0than\u00a0conventional\u00a0breeding.\u00a0Plant\u00a0\nBiotechnology\u00a0Journal,\u00a04,\u00a04,\u00a0pp\u00a0\u00a0369\u2010380\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000238256500001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Organic/Baudo\u2010Impact\u2010\n2006.pdf\u00a0\n\n\u00a0\nBennett,\u00a0D.,\u00a0Glasner,\u00a0P.,\u00a0&\u00a0Travis,\u00a0D.\u00a0(1986)\u00a0\u00a0The\u00a0Politics\u00a0of\u00a0\n\nUncertainty\u00a0Routledge\u00a0&\u00a0Kegan\u00a0Paul\u00a0plc.,\u00a0\u00a0IS:\u00a00\u20107102\u2010\n0503\u20101,\u00a0pp\u00a0218\u00a0\u00a0\n\n\u00a0 \u00a0\nBerg,\u00a0P.,\u00a0Baltimore,\u00a0D.,\u00a0Brenner,\u00a0S.,\u00a0Roblin,\u00a0R.O.,\u00a0&\u00a0Singer,\u00a0\n\nM.F.\u00a0(1975)\u00a0SUMMARY\u00a0STATEMENT\u00a0OF\u00a0ASILOMAR\u00a0\nCONFERENCE\u00a0ON\u00a0RECOMBINANT\u00a0DNA\u2010MOLECULES.\u00a0\nProceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0Sciences\u00a0of\u00a0the\u00a0\nUnited\u00a0States\u00a0of\u00a0America,\u00a072,\u00a06,\u00a0pp\u00a0\u00a01981\u20101984\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1975AG70300001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Berg\u2010Summary\u2010\nStatement\u2010Asilomar\u20101975.pdf\u00a0\n\n\u00a0\nBerg,\u00a0P.\u00a0&\u00a0Singer,\u00a0M.\u00a0(1995)\u00a0THE\u00a0RECOMBINANT\u2010DNA\u00a0\n\nand\u00a0impact\u00a0on\u00a0microbial\u00a0evolution.\u00a0Fems\u00a0Microbiology\u00a0\nReviews,\u00a024,\u00a01,\u00a0pp\u00a0\u00a01\u20107\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000084915900001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Mutations/Arber\u2010Gen\u2010\nVariation\u2010FEMS\u20102000.pdf\u00a0\n\n\u00a0\nArber,\u00a0W.\u00a0(2002)\u00a0Roots,\u00a0strategies\u00a0and\u00a0prospects\u00a0of\u00a0\n\nfunctional\u00a0genomics.\u00a0Current\u00a0Science,\u00a083,\u00a07,\u00a0pp\u00a0\u00a0826\u2010828\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://000178662800019\u00a0and\u00a0http://\n\nwww.botanischergarten.ch/Mutations/Arber\u2010\nComparison\u20102002.pdf\u00a0\n\n\u00a0\nArber,\u00a0W.\u00a0(2003)\u00a0Elements\u00a0for\u00a0a\u00a0theory\u00a0of\u00a0molecular\u00a0\n\nevolution.\u00a0Gene,\u00a0317,\u00a01\u20102,\u00a0pp\u00a0\u00a03\u201011\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://000186667000002\u00a0and\u00a0http://\n\nwww.botanischergarten.ch/Mutations/Arber\u2010Gene\u2010317\u2010\n2003.pdf\u00a0\n\n\u00a0\nArber,\u00a0W.\u00a0(2004)\u00a0Biological\u00a0evolution:\u00a0Lessons\u00a0to\u00a0be\u00a0learned\u00a0\n\nfrom\u00a0microbial\u00a0population\u00a0biology\u00a0and\u00a0genetics.", "start_char_idx": 57245, "end_char_idx": 59304, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b2c8d7e8-2c83-484a-ba0a-04797c1b0ced": {"__data__": {"id_": "b2c8d7e8-2c83-484a-ba0a-04797c1b0ced", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0e8af144-aea5-492d-800e-fac809241807", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "7dd9810b7c3ccde261b6fe29b7d30a49a4de8e109fbd8b0474007fdfa56b8426", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "845da421-bb3a-44be-9937-4e762e84041a", "node_type": "1", "metadata": {}, "hash": "39035e529a59d899d3df72a7082a0815aecab2912f4537c13f3b695bf5b9b578", "class_name": "RelatedNodeInfo"}}, "text": "Research\u00a0in\u00a0Microbiology,\u00a0155,\u00a05,\u00a0pp\u00a0\u00a0297\u2010300\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000222736200001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Mutations/Arber\u2010Evolution\u2010\nLessons\u20102004.pdf\u00a0\n\n\u00a0\nArber,\u00a0W.\u00a0(2010)\u00a0Genetic\u00a0engineering\u00a0compared\u00a0to\u00a0natural\u00a0\n\ngenetic\u00a0variations.\u00a0New\u00a0Biotechnology,\u00a0In\u00a0Press,\u00a0\nCorrected\u00a0Proof,\u00a0\u00a0pp\u00a0\u00a0\u00a0\n\n\u00a0 http://www.sciencedirect.com/science/article/B8JG4\u2010\n504JYNT\u20102/2/a7e6edd02959e1b3167158dd264f24a2\u00a0\nAND\u00a0http://www.ask\u2010force.org/web/Vatican\u2010PAS\u2010NBT\u2010\npubl/Arber\u2010Genetic\u2010Engineering\u2010PAS\u20102010.pdf\u00a0\n\nArchibald,\u00a0J.\u00a0&\u00a0Richards,\u00a0T.\u00a0(2010)\u00a0Gene\u00a0transfer:\u00a0anything\u00a0\ngoes\u00a0in\u00a0plant\u00a0mitochondria.\u00a0Bmc\u00a0Biology,\u00a08,\u00a01,\u00a0pp\u00a0\u00a0147\u00a0\u00a0\n\n\u00a0 http://www.biomedcentral.com/1741\u20107007/8/147\u00a0AND\u00a0\nhttp://www.ask\u2010force.org/web/HorizontalGT/Archibald\u2010\nGene\u2010Transfer\u2010Mitochondria\u20102010.pdf\u00a0\n\nAwoleye,\u00a0F.,\u00a0Vanduren,\u00a0M.,\u00a0Dolezel,\u00a0J.,\u00a0&\u00a0Novak,\u00a0F.J.\u00a0(1994)\u00a0\nNUCLEAR\u2010DNA\u00a0CONTENT\u00a0AND\u00a0IN\u2010VITRO\u00a0INDUCED\u00a0\nSOMATIC\u00a0POLYPLOIDIZATION\u00a0CASSAVA\u00a0(MANIHOT\u2010\nESCULENTA\u00a0CRANTZ)\u00a0BREEDING.\u00a0Euphytica,\u00a076,\u00a03,\u00a0pp\u00a0\u00a0\n195\u2010202\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1994PK33000005\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Radiation\u2010Mutants/Awoleye\n\u2010Nuclear\u2010DNA\u2010content\u20101994.pdf\u00a0\n\n\u00a0\nBaarends,\u00a0W.M.,\u00a0van\u00a0der\u00a0Laan,\u00a0R.,\u00a0&\u00a0Grootegoed,\u00a0J.A.\u00a0(2001)\u00a0\n\nDNA\u00a0repair\u00a0mechanisms\u00a0and\u00a0gametogenesis.\u00a0\nReproduction,\u00a0121,\u00a01,\u00a0pp\u00a0\u00a031\u201039\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000168328900004\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Baarends\u2010DNA\u2010\nRepair\u2010Mechanisms\u20102001.pdf\u00a0\n\n\u00a0\nBanks,\u00a0P.M.,\u00a0Xu,\u00a0S.J.,\u00a0Wang,\u00a0R.R.C.,\u00a0&\u00a0Larkin,\u00a0P.J.\u00a0(1993)\u00a0\n\nVARYING\u00a0CHROMOSOME\u00a0COMPOSITION\u00a0OF\u00a056\u2010\nCHROMOSOME\u00a0WHEAT\u00a0X\u00a0THINOPYRUM\u2010INTERMEDIUM\u00a0\nPARTIAL\u00a0AMPHIPLOIDS.\u00a0Genome,\u00a036,\u00a02,\u00a0pp\u00a0\u00a0207\u2010215\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://A1993LA74500001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Banks\u2010Varying\u2010\nChromosome\u2010Composition\u20101993.pdf\u00a0\n\n\u00a0\nBarnab\u00e1s,\u00a0B.,\u00a0Obert,\u00a0B.,\u00a0&\u00a0Kov\u00e1cs,\u00a0G.\u00a0(1999)\u00a0Colchicine,\u00a0an\u00a0\n\nefficient\u00a0genome\u2010doubling\u00a0agent\u00a0for\u00a0maize\u00a0(Zea\u00a0mays\u00a0L.)\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n44\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n\u00a0\nFedoroff,\u00a0N.\u00a0(1994)\u00a0MCCLINTOCK,BARBARA\u00a0(JUNE\u00a016,\u00a01902\u00a0\n\nSEPTEMBER\u00a02,\u00a01992).", "start_char_idx": 59306, "end_char_idx": 61325, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "845da421-bb3a-44be-9937-4e762e84041a": {"__data__": {"id_": "845da421-bb3a-44be-9937-4e762e84041a", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b2c8d7e8-2c83-484a-ba0a-04797c1b0ced", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "f4a05799e2b1d1c8529938696ea7ca515f0870d70ce848fdaf5e12fbf499cb63", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "274634fa-adc4-4378-a32a-f44a0617fe40", "node_type": "1", "metadata": {}, "hash": "24ac83ea41a96ff8504074ba0fd7ff35c116684800cee22d3b79adbd96209d9f", "class_name": "RelatedNodeInfo"}}, "text": "Proceedings\u00a0of\u00a0the\u00a0American\u00a0\nPhilosophical\u00a0Society,\u00a0138,\u00a03,\u00a0pp\u00a0\u00a0431\u2010445\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1994PH68500011\u00a0AND\u00a0NEBIS\u00a0\n20090920\u00a0\n\n\u00a0\nFedoroff,\u00a0N.,\u00a0Schlappi,\u00a0M.,\u00a0&\u00a0Raina,\u00a0R.\u00a0(1995)\u00a0EPIGENETIC\u00a0\n\nREGULATION\u00a0OF\u00a0THE\u00a0MAIZE\u00a0SPM\u00a0TRANSPOSON.\u00a0\nBioessays,\u00a017,\u00a04,\u00a0pp\u00a0\u00a0291\u2010297\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1995QV74600004\u00a0\n\u00a0\nFedoroff,\u00a0N.V.\u00a0(1984)\u00a0Transposable\u00a0genetic\u00a0elements\u00a0in\u00a0\n\nmaize\u00a0[Corn,\u00a0Zea\u00a0mays].\u00a0Scientific\u00a0American,\u00a0250,\u00a0\u00a0pp\u00a0\u00a064\n\u201074\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/Genomics/Fedoroff\u2010\ntransposable\u2010Elements\u2010Maize\u20101984.pdf\u00a0\n\n\u00a0\nFedoroff,\u00a0N.V.\u00a0(1991)\u00a0The\u00a0Restless\u00a0Gene\u00a0\u2010\u00a0How\u00a0the\u00a0Colors\u00a0of\u00a0\n\nIndian\u00a0Corn\u00a0Have\u00a0Led\u00a0to\u00a0an\u00a0Understanding\u00a0of\u00a0Wandering\u00a0\nDNA.\u00a0Sciences\u2010New\u00a0York,\u00a031,\u00a01,\u00a0pp\u00a0\u00a022\u201028\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1991EM86800018\u00a0\u00a0\n\u00a0\nFerreira,\u00a0S.J.,\u00a0Senning,\u00a0M.,\u00a0Sonnewald,\u00a0S.,\u00a0Kessling,\u00a0P.M.,\u00a0\n\nGoldstein,\u00a0R.,\u00a0&\u00a0Sonnewald,\u00a0U.\u00a0(2010)\u00a0Comparative\u00a0\ntranscriptome\u00a0analysis\u00a0coupled\u00a0to\u00a0X\u2010ray\u00a0CT\u00a0reveals\u00a0\nsucrose\u00a0supply\u00a0and\u00a0growth\u00a0velocity\u00a0as\u00a0major\u00a0\ndeterminants\u00a0of\u00a0potato\u00a0tuber\u00a0starch\u00a0biosynthesis.\u00a0BMC\u00a0\nGenomics,\u00a011,\u00a0\u00a0pp\u00a0\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000275291800004\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Ferreira\u2010\nComparative\u2010Transcriptome\u2010Analysis\u2010Potato\u20102010.pdf\u00a0\n\n\u00a0\nFriedberg,\u00a0E.C.\u00a0(2007)\u00a0The\u00a0writing\u00a0life\u00a0of\u00a0James\u00a0D.\u00a0Watson.\u00a0\n\nAdler\u00a0Museum\u00a0Bulletin,\u00a033,\u00a02,\u00a0pp\u00a0\u00a03\u201016\u00a0\u00a0\n\u00a0 http://www.botanischergarten.ch/History/Friedberg\u2010\n\nWriting\u2010Life\u2010James\u2010Watson\u20102007.pdf\u00a0\n\u00a0\nGhatnekar,\u00a0L.,\u00a0Jaarola,\u00a0M.,\u00a0&\u00a0Bengtsson,\u00a0B.O.\u00a0(2006)\u00a0The\u00a0\n\nintrogression\u00a0of\u00a0a\u00a0functional\u00a0nuclear\u00a0gene\u00a0from\u00a0Poa\u00a0to\u00a0\nFestuca\u00a0ovina.\u00a0Proceedings:\u00a0Biological\u00a0Sciences,\u00a0273,\u00a0\n1585,\u00a0pp\u00a0\u00a0395\u00a0\u2010\u00a0399\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/Genomics/Ghatnekar\n\u2010Transgen\u2010Festuca.pdf\u00a0\n\n\u00a0\nHackett,\u00a0P.\u00a0(2002)\u00a0Genetic\u00a0Engineering:\u00a0What\u00a0are\u00a0We\u00a0\n\nFearing?\u00a0Transgenic\u00a0Research,\u00a011,\u00a02,\u00a0pp\u00a0\u00a097\u201099\u00a0\u00a0\n\u00a0 http://dx.doi.org/10.1023/A:1015292423744\u00a0AND\u00a0\n\nhttp://www.botanischergarten.ch/Genomics/Hackett\u2010\nGE\u2010What\u2010are\u2010we\u2010fearing\u20102002.pdf\u00a0\n\nHall,\u00a0R.M.\u00a0&\u00a0Collis,\u00a0C.M.\u00a0(1995)\u00a0Mobile\u00a0gene\u00a0cassettes\u00a0and\u00a0\nintegrons:\u00a0capture\u00a0and\u00a0spread\u00a0of\u00a0genes\u00a0by\u00a0site\u2010specific\u00a0\nrecombination.", "start_char_idx": 61326, "end_char_idx": 63320, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "274634fa-adc4-4378-a32a-f44a0617fe40": {"__data__": {"id_": "274634fa-adc4-4378-a32a-f44a0617fe40", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "845da421-bb3a-44be-9937-4e762e84041a", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "f40fd8c7660bd2c0d70abfb671a1a15f12fb0c04c33201dcbcc6902c3b43e0ad", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7b438baa-e7db-46f6-8098-869649ce43c1", "node_type": "1", "metadata": {}, "hash": "146a90b36fb4045b5168c318e800f59574ca1cc2c126ce820651e55388f471ad", "class_name": "RelatedNodeInfo"}}, "text": "Molecular\u00a0Microbiology,\u00a015,\u00a04,\u00a0pp\u00a0\u00a0593\u2010\n600\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1111/j.1365\u20102958.1995.tb02368.x\u00a0\nAND\u00a0http://www.ask\u2010force.org/web/Genomics/Hall\u2010\nMobile\u2010Gene\u2010Cassettes\u20101995.pdf\u00a0\n\nHerman,\u00a0R.A.,\u00a0Chassy,\u00a0B.M.,\u00a0&\u00a0Parrott,\u00a0W.\u00a0(2009)\u00a0\nCompositional\u00a0assessment\u00a0of\u00a0transgenic\u00a0crops:\u00a0an\u00a0idea\u00a0\nwhose\u00a0time\u00a0has\u00a0passed.\u00a0Trends\u00a0in\u00a0Biotechnology,\u00a0In\u00a0\nPress,\u00a0Corrected\u00a0Proof,\u00a0\u00a0pp\u00a0\u00a0\u00a0\n\n\u00a0 http://www.sciencedirect.com/science/article/B6TCW\u2010\n4X26XP1\u20101/2/bcfd547d5f12695fc76c4fc5886ba9fc\u00a0AND\u00a0\n\nCONTROVERSY\u00a0\u2010\u00a020\u00a0YEARS\u00a0LATER.\u00a0Bio\u2010Technology,\u00a013,\u00a0\n10,\u00a0pp\u00a0\u00a01132\u20101134\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1995RY31800031\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Berg\u2010Recombinant\u2010\nDNA\u2010Twenty\u2010years\u2010later\u20101995.pdf\u00a0\n\n\u00a0\nBerwald,\u00a0D.,\u00a0Carter,\u00a0C.A.,\u00a0&\u00a0Gruere,\u00a0G.P.\u00a0(2006)\u00a0Rejecting\u00a0\n\nnew\u00a0technology:\u00a0The\u00a0case\u00a0of\u00a0genetically\u00a0modified\u00a0wheat.\u00a0\nAmerican\u00a0Journal\u00a0of\u00a0Agricultural\u00a0Economics,\u00a088,\u00a02,\u00a0pp\u00a0\u00a0\n432\u2010447\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000236716200012\u00a0\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Berwald\u2010\nRejecting\u2010New\u2010Technology\u20102006.pdf\u00a0\n\n\u00a0\nBrochmann,\u00a0C.,\u00a0Soltis,\u00a0P.S.,\u00a0&\u00a0Soltis,\u00a0D.E.\u00a0(1992)\u00a0RECURRENT\u00a0\n\nFORMATION\u00a0AND\u00a0POLYPHYLY\u00a0OF\u00a0NORDIC\u00a0POLYPLOIDS\u00a0\nIN\u00a0DRABA\u00a0(BRASSICACEAE).\u00a0American\u00a0Journal\u00a0of\u00a0Botany,\u00a0\n79,\u00a06,\u00a0pp\u00a0\u00a0673\u2010688\u00a0\u00a0<Go\u00a0to\u00a0ISI>://\nWOS:A1992HZ92600009\u00a0\n\n\u00a0\nChassy,\u00a0B.M.\u00a0(2007)\u00a0The\u00a0history\u00a0and\u00a0future\u00a0of\u00a0GMOs\u00a0in\u00a0food\u00a0\n\nand\u00a0agriculture.\u00a0Cereal\u00a0Foods\u00a0World,\u00a052,\u00a04,\u00a0pp\u00a0\u00a0169\u2010172\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://000248207400002\u00a0AND\u00a0http://\n\nwww.botanischergarten.ch/History/Chassy\u2010History\u2010\nFuture\u20102007.pdf\u00a0\n\n\u00a0\nColl,\u00a0A.,\u00a0Nadal,\u00a0A.,\u00a0Collado,\u00a0R.,\u00a0Capellades,\u00a0G.,\u00a0Messeguer,\u00a0J.,\u00a0\n\nMel\u00e9,\u00a0E.,\u00a0Palaudelm\u00e0s,\u00a0M.,\u00a0&\u00a0Pla,\u00a0M.\u00a0(2009)\u00a0Gene\u00a0\nexpression\u00a0profiles\u00a0of\u00a0MON810\u00a0and\u00a0comparable\u00a0non\u2010GM\u00a0\nmaize\u00a0varieties\u00a0cultured\u00a0in\u00a0the\u00a0field\u00a0are\u00a0more\u00a0similar\u00a0\nthan\u00a0are\u00a0those\u00a0of\u00a0conventional\u00a0lines.\u00a0Transgenic\u00a0\nResearch,\u00a018,\u00a05,\u00a0pp\u00a0\u00a0801\u2010808\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1007/s11248\u2010009\u20109266\u2010z\u00a0AND\u00a0\nhttp://www.botanischergarten.ch/Genomics/Coll\u2010Gene\u2010\nExpressioin\u2010Profiles\u2010Comparable\u20102009.pdf\u00a0\n\nConsortium,\u00a0I.H.G.S.\u00a0(2001)\u00a0Initial\u00a0sequencing\u00a0and\u00a0analysis\u00a0of\u00a0\nthe\u00a0human\u00a0genome.", "start_char_idx": 63321, "end_char_idx": 65313, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7b438baa-e7db-46f6-8098-869649ce43c1": {"__data__": {"id_": "7b438baa-e7db-46f6-8098-869649ce43c1", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "274634fa-adc4-4378-a32a-f44a0617fe40", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "865353a04fda5afbce97606ce75cd92399007d936ccdb715fb00fff7e4ab0311", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "19a79b29-b2d6-4f0a-b624-330c3bd0bb0a", "node_type": "1", "metadata": {}, "hash": "19b3008c0e62832404d3de6c6c5435689ab3899e584a5b47f1dad5f0a3fca202", "class_name": "RelatedNodeInfo"}}, "text": "Nature,\u00a0409,\u00a06822,\u00a0pp\u00a0\u00a0860\u2010921\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/35057062\u00a0AND\u00a0http://\nwww.nature.com/nature/journal/v409/n6822/\nsuppinfo/409860a0_S1.html\u00a0AND\u00a0http://www.ask\u2010\nforce.org/web/HorizontalGT/International\u2010Sequencing\u2010\nHuman\u2010Genome\u2010Nature\u20102001.pdf\u00a0\n\nDong,\u00a0C.M.,\u00a0Whitford,\u00a0R.,\u00a0&\u00a0Langridge,\u00a0P.\u00a0(2002)\u00a0A\u00a0DNA\u00a0\nmismatch\u00a0repair\u00a0gene\u00a0links\u00a0to\u00a0the\u00a0Ph2\u00a0locus\u00a0in\u00a0wheat.\u00a0\nGenome,\u00a045,\u00a01,\u00a0pp\u00a0\u00a0116\u2010124\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000173553000016\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Dong\u2010DNA\u2010\nMismatch\u2010Repair\u2010Gene\u20102002.pdf\u00a0\n\n\u00a0\nDoyle,\u00a0J.J.,\u00a0Doyle,\u00a0J.L.,\u00a0Brown,\u00a0A.H.,\u00a0&\u00a0Grace,\u00a0J.P.\u00a0(1990)\u00a0\n\nMultiple\u00a0origins\u00a0of\u00a0polyploids\u00a0in\u00a0the\u00a0Glycine\u00a0tabacina\u00a0\ncomplex\u00a0inferred\u00a0from\u00a0chloroplast\u00a0DNA\u00a0polymorphism.\u00a0\nProceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0Sciences\u00a0of\u00a0the\u00a0\nUnited\u00a0States\u00a0of\u00a0America,\u00a087,\u00a02,\u00a0pp\u00a0\u00a0714\u2010717\u00a0\u00a0\n\n\u00a0 http://www.pnas.org/content/87/2/714.abstract\u00a0AND\u00a0\nhttp://www.botanischergarten.ch/Genomics/Doyle\u2010\nMultiple\u2010Origins\u20101990.pdf\u00a0\n\nFedoroff,\u00a0N.\u00a0(1992)\u00a0MCCLINTOCK,BARBARA,\u00a0THE\u00a0GENETICIST,\u00a0\nTHE\u00a0GENIUS,\u00a0THE\u00a0WOMAN\u00a0\u2010\u00a0OBITUARY.\u00a0Cell,\u00a071,\u00a02,\u00a0pp\u00a0\u00a0\n181\u2010182\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1992JU39500001\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n45\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n\u00a0\nLeister,\u00a0D.,\u00a0Kurth,\u00a0J.,\u00a0Laurie,\u00a0D.A.,\u00a0Yano,\u00a0M.,\u00a0Sasaki,\u00a0T.,\u00a0\n\nDevos,\u00a0K.,\u00a0Graner,\u00a0A.,\u00a0&\u00a0Schulze\u2010Lefert,\u00a0P.\u00a0(1998)\u00a0Rapid\u00a0\nreorganization\u00a0of\u00a0resistance\u00a0gene\u00a0homologues\u00a0in\u00a0cereal\u00a0\ngenomes.\u00a0Proceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0\nSciences\u00a0of\u00a0the\u00a0United\u00a0States\u00a0of\u00a0America,\u00a095,\u00a01,\u00a0pp\u00a0\u00a0370\u2010\n375\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000071429500070\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Leister\u2010Rapid\u2010\nReorganization\u2010Reisitance.1998.pdf\u00a0\n\n\u00a0\nLewin,\u00a0R.\u00a0(1983)\u00a0A\u00a0Naturalist\u00a0of\u00a0the\u00a0Genome.\u00a0Science,\u00a0222,\u00a0\n\n4622,\u00a0pp\u00a0\u00a0402\u2010405\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1983RM23800018\u00a0\u00a0AND\u00a0http://\n\nwww.botanischergarten.ch/Genomics/Lewin\u2010Naturalist\u2010\nGenome\u2010McClintock\u20101983.pdf\u00a0\n\n\u00a0\nMacdonald,\u00a0P.\u00a0&\u00a0Yarrow,\u00a0S.\u00a0(2002)\u00a0Regulation\u00a0of\u00a0Bt\u00a0crops\u00a0in\u00a0\n\nCanada,\u00a0Iguassu\u00a0Falls,\u00a0Brazil\u00a0Academic\u00a0Press\u00a0Inc\u00a0Elsevier\u00a0\nScience\u00a08th\u00a0International\u00a0Colloquium\u00a0on\u00a0Invertebrate\u00a0\nPathology\u00a0and\u00a0Microbial\u00a0Control/35th\u00a0Annual\u00a0Meeting\u00a0\nof\u00a0the\u00a0SIP/6th\u00a0International\u00a0Conference\u00a0on\u00a0Bacillus\u00a0\nThuringiensis\u00a0Ed.", "start_char_idx": 65314, "end_char_idx": 67472, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "19a79b29-b2d6-4f0a-b624-330c3bd0bb0a": {"__data__": {"id_": "19a79b29-b2d6-4f0a-b624-330c3bd0bb0a", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7b438baa-e7db-46f6-8098-869649ce43c1", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "b802e4cbe186dc181b68d59e89168c847f9a3580491a33e3cb077abf77be0155", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2845f672-9954-4379-80af-bbab928a5c4b", "node_type": "1", "metadata": {}, "hash": "308aaf91f527a82c50c77e62363afa24a0b4d2eaf08bd356b59736d6ea6f3694", "class_name": "RelatedNodeInfo"}}, "text": "pp\u00a093\u201099\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000183491000002\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/MacDonald\u2010\nRegulation\u2010Bt\u2010crops\u2010Canada\u20102003.pdf\u00a0\n\n\u00a0\nMcClintock,\u00a0B.\u00a0(1930)\u00a0A\u00a0cytological\u00a0demonstration\u00a0of\u00a0the\u00a0\n\nlocation\u00a0of\u00a0an\u00a0interchange\u00a0between\u00a0two\u00a0non\u2010\nhomologous\u00a0chromosomes\u00a0of\u00a0Zea\u00a0mays.\u00a0Proceedings\u00a0of\u00a0\nthe\u00a0National\u00a0Academy\u00a0of\u00a0Sciences\u00a0of\u00a0the\u00a0United\u00a0States\u00a0\nof\u00a0America,\u00a016,\u00a0\u00a0pp\u00a0\u00a0791\u2010796\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000201970600143\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/McClintock\u2010\nCytological\u2010Interchange\u2010Location\u20101930.pdf\u00a0\n\n\u00a0\nMcClintock,\u00a0B.\u00a0(1953)\u00a0Induction\u00a0of\u00a0Instability\u00a0at\u00a0Selected\u00a0Loci\u00a0\n\nin\u00a0Maize.\u00a0Genetics,\u00a038,\u00a06,\u00a0pp\u00a0\u00a0579\u2010599\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1953XW86100004\u00a0\u00a0AND\u00a0http://\n\nwww.botanischergarten.ch/Genomics/McClintock\u2010\nInstability\u2010Maize\u20101953.pdf\u00a0\n\n\u00a0\nMcLean,\u00a0M.A.,\u00a0Frederick,\u00a0R.J.,\u00a0Traynor,\u00a0P.L.,\u00a0Cohen,\u00a0J.I.,\u00a0&\u00a0\n\nKomen,\u00a0J.\u00a0(2002)\u00a0A\u00a0Conceptual\u00a0Framework\u00a0for\u00a0\nImplementing\u00a0Biosafety:\u00a0Linking\u00a0Policy,\u00a0Capacity,\u00a0and\u00a0\nRegulation,\u00a0ISNAR,\u00a0International\u00a0Service\u00a0for\u00a0National\u00a0\nAgricultural\u00a0Research\u00a0pp\u00a01\u201012\u00a0ISNAR\u00a0Briefing\u00a0Papers\u00a0\u00a0\nWashington\u00a0DC.\u00a0(Report)\u00a0\n\n\u00a0 ftp://ftp.cgiar.org/isnar/publicat/bp\u201047.pdf\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/McLean\u2010\nConceptual\u2010Framework\u2010ISNAR\u201047\u20102002.pdf\u00a0\n\n\u00a0\nMeza,\u00a0T.J.,\u00a0Stangeland,\u00a0B.,\u00a0Mercy,\u00a0I.S.,\u00a0Skarn,\u00a0M.,\u00a0Nymoen,\u00a0\n\nD.A.,\u00a0Berg,\u00a0A.,\u00a0Butenko,\u00a0M.A.,\u00a0Hakelien,\u00a0A.M.,\u00a0Haslekas,\u00a0\nC.,\u00a0Meza\u2010Zepeda,\u00a0L.A.,\u00a0&\u00a0Aalen,\u00a0R.B.\u00a0(2002)\u00a0Analyses\u00a0of\u00a0\nsingle\u2010copy\u00a0Arabidopsis\u00a0T\u2010DNA\u2010transformed\u00a0lines\u00a0show\u00a0\nthat\u00a0the\u00a0presence\u00a0of\u00a0vector\u00a0backbone\u00a0sequences,\u00a0short\u00a0\ninverted\u00a0repeats\u00a0and\u00a0DNA\u00a0methylation\u00a0is\u00a0not\u00a0sufficient\u00a0\nor\u00a0necessary\u00a0for\u00a0the\u00a0induction\u00a0of\u00a0transgene\u00a0silencing.\u00a0\nNucleic\u00a0Acids\u00a0Research,\u00a030,\u00a020,\u00a0pp\u00a0\u00a04556\u20104566\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000178826700034\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Meza\u2010Analyses\u2010\nArabidopsis\u20102002.pdf\u00a0\n\n\u00a0\n\nhttp://www.botanischergarten.ch/Regulation/Herman\u2010\nCompositional\u2010Analysis\u20102009.pdf\u00a0\n\nKalaitzandonakes,\u00a0N.,\u00a0Marks,\u00a0L.,\u00a0&\u00a0Vickner,\u00a0S.S.\u00a0(2005)\nSentiments\u00a0and\u00a0acts\u00a0towards\u00a0genetically\u00a0modified\u00a0foods.\u00a0\nInternational\u00a0Journal\u00a0of\u00a0Biotechnology,\u00a07,\u00a01\u20103,\u00a0pp\u00a0\u00a0161\u2010\n177\u00a0\u00a0\n\n\u00a0 http://www.inderscience.com/search/index.php?\naction=record&rec_id=6452&prevQuery=&ps=10&m=or\u00a0\nAND\u00a0http://www.botanischergarten.ch/Regulation/\nKalaitzandonakes\u2010Sentiments\u2010Acts\u20102005.pdf\u00a0\n\nKeeling,\u00a0P.J.\u00a0&\u00a0Palmer,\u00a0J.D.\u00a0(2008)\u00a0Horizontal\u00a0gene\u00a0transfer\u00a0in\u00a0\neukaryotic\u00a0evolution.", "start_char_idx": 67474, "end_char_idx": 69780, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2845f672-9954-4379-80af-bbab928a5c4b": {"__data__": {"id_": "2845f672-9954-4379-80af-bbab928a5c4b", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "19a79b29-b2d6-4f0a-b624-330c3bd0bb0a", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "955790b5eceb0f857a27bba8f565bb19830fb966203d116cf8ff056c338a404f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fe08369c-da8a-4ec2-8348-5fcc7fd81cd5", "node_type": "1", "metadata": {}, "hash": "f2a2abfde61e2ef9fe499faa3786836398dbe1548f11e0729e2baff8ff5da46f", "class_name": "RelatedNodeInfo"}}, "text": "Nature\u00a0Reviews\u00a0Genetics,\u00a09,\u00a08,\u00a0pp\u00a0\u00a0\n605\u2010618\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000257758400011\u00a0AND\u00a0http://\nwww.botanischergarten.ch/HorizontalGT/Keeling\u2010HGT\u2010\nEukaryotic\u2010Evolution\u20102008.pdf\u00a0\n\n\u00a0\nKlug,\u00a0A.\u00a0(2004)\u00a0The\u00a0Discovery\u00a0of\u00a0the\u00a0DNA\u00a0Double\u00a0Helix.\u00a0\n\nJournal\u00a0of\u00a0Molecular\u00a0Biology,\u00a0335,\u00a01,\u00a0pp\u00a0\u00a03\u201026\u00a0\u00a0\n\u00a0 http://www.sciencedirect.com/science/article/B6WK7\u2010\n\n4B41W2K\u20104/2/181d9023626e6f1c425f384c9f3d31a4\u00a0\nAND\u00a0http://www.botanischergarten.ch/History/Klug\u2010\nDiscovery\u2010DNA\u2010Double\u2010Helix\u20102004.pdf\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Klug\u2010Discovery\u2010DNA\n\u2010Double\u2010Helix\u2010corrigendum\u20102004.pdf\u00a0\n\n\u00a0\nKogel,\u00a0K.\u2010H.,\u00a0Voll,\u00a0L.M.,\u00a0Schaefer,\u00a0P.,\u00a0Jansen,\u00a0C.,\u00a0Wu,\u00a0Y.,\u00a0\n\nLangen,\u00a0G.,\u00a0Imani,\u00a0J.,\u00a0Hofmann,\u00a0J.r.,\u00a0Schmiedl,\u00a0A.,\u00a0\nSonnewald,\u00a0S.,\u00a0von\u00a0Wettstein,\u00a0D.,\u00a0Cook,\u00a0R.J.,\u00a0&\u00a0\nSonnewald,\u00a0U.\u00a0(2010)\u00a0Transcriptome\u00a0and\u00a0metabolome\u00a0\nprofiling\u00a0of\u00a0field\u2010grown\u00a0transgenic\u00a0barley\u00a0lack\u00a0induced\u00a0\ndifferences\u00a0but\u00a0show\u00a0cultivar\u2010specific\u00a0variances.\u00a0\nProceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0Sciences,\u00a0107,\u00a0\n14,\u00a0pp\u00a0\u00a06198\u20106203\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000276374400016\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Kogel\u2010\nTranscriptome\u2010Metabolome\u20102010.pdf\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Kogel\u2010\nTranscriptome\u2010Metabolome\u2010Supporting\u20102010.pdf\u00a0\n\nLal,\u00a0S.,\u00a0Oetjens,\u00a0M.,\u00a0&\u00a0Hannah,\u00a0L.C.\u00a0(2009)\u00a0Helitrons:\u00a0\nEnigmatic\u00a0abductors\u00a0and\u00a0mobilizers\u00a0of\u00a0host\u00a0genome\u00a0\nsequences.\u00a0Plant\u00a0Science,\u00a0176,\u00a02,\u00a0pp\u00a0\u00a0181\u2010186\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000262566600003\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Lal\u2010Helitrons\u2010\nEnigmatic\u2010Abductors\u20102009.pdf\u00a0\n\n\u00a0\nLal,\u00a0S.K.\u00a0&\u00a0Hannah,\u00a0L.C.\u00a0(2005)\u00a0Helitrons\u00a0contribute\u00a0to\u00a0the\u00a0\n\nlack\u00a0of\u00a0gene\u00a0colinearity\u00a0observed\u00a0in\u00a0modern\u00a0maize\u00a0\ninbreds.\u00a0Proceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0\nSciences\u00a0of\u00a0the\u00a0United\u00a0States\u00a0of\u00a0America,\u00a0102,\u00a029,\u00a0pp\u00a0\u00a0\n9993\u20109994\u00a0\u00a0\n\n\u00a0 http://www.pnas.org/content/102/29/9993.short\u00a0AND\u00a0\nhttp://www.botanischergarten.ch/Genomics/Lal\u2010\nHelitrons\u2010contribute\u20102005.pdf\u00a0\n\nLeister,\u00a0D.\u00a0(2005)\u00a0Origin,\u00a0evolution\u00a0and\u00a0genetic\u00a0effects\u00a0of\u00a0\nnuclear\u00a0insertions\u00a0of\u00a0organelle\u00a0DNA.\u00a0Trends\u00a0in\u00a0Genetics,\u00a0\n21,\u00a012,\u00a0pp\u00a0\u00a0655\u2010663\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000233672700005\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Leister\u2010Origin\u2010\nEvolution\u2010Genetic\u2010Effects\u20102005.pdf\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 69781, "end_char_idx": 71948, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fe08369c-da8a-4ec2-8348-5fcc7fd81cd5": {"__data__": {"id_": "fe08369c-da8a-4ec2-8348-5fcc7fd81cd5", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2845f672-9954-4379-80af-bbab928a5c4b", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "2ccf7611a9f50dfe33e3c5c263e00ca18c8a61a09b7996bedb1ba4ffce8826d0", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "69d01884-f4c5-4896-90f6-397fd5eb2c20", "node_type": "1", "metadata": {}, "hash": "0e8230184c6249b733b14295d72515494200ed48498ea9ea377def22362ad1e7", "class_name": "RelatedNodeInfo"}}, "text": "1:\u00a031\u201048\u00a0\n\n46\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n\u00a0 GENETIC\u2010EVIDENCE\u00a0FOR\u00a0ALLOPOLYPLOIDY\u00a0IN\u00a0THE\u00a0\nNEOTROPICAL\u00a0FERN\u00a0HEMIONITIS\u2010PINNATIFIDA\u00a0\n(ADIANTACEAE)\u00a0AND\u00a0THE\u00a0RECONSTRUCTION\u00a0OF\u00a0AN\u00a0\nANCESTRAL\u00a0GENOME.\u00a0Systematic\u00a0Botany,\u00a014,\u00a04,\u00a0pp\u00a0\u00a0439\u2010\n447\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1989AW04900001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Ranker\u2010Genetic\u2010\nEvidence\u2010Allopolyploidy\u20101989.pdf\u00a0\n\n\u00a0\nRaybould,\u00a0A.F.\u00a0(2010)\u00a0Reducing\u00a0uncertainty\u00a0in\u00a0regulatory\u00a0\n\ndecision\u2010making\u00a0for\u00a0transgenic\u00a0crops:\u00a0More\u00a0ecological\u00a0\nresearch\u00a0or\u00a0shrewder\u00a0environmental\u00a0risk\u00a0assessment?\u00a0\nGM\u00a0crops,\u00a01,\u00a01,\u00a0pp\u00a0\u00a01\u20107\u00a0\u00a0\n\n\u00a0 http://www.landesbioscience.com/journals/gmcrops/\narticle/9776\u00a0AND\u00a0http://www.botanischergarten.ch/\nRegulation/Raybould\u2010Reducing\u2010uncertainty\u20102010.pdf\u00a0\n\nReynolds,\u00a0M.P.,\u00a0van\u00a0Ginkel,\u00a0M.,\u00a0&\u00a0Ribaut,\u00a0J.M.\u00a0(2000)\nAvenues\u00a0for\u00a0genetic\u00a0modification\u00a0of\u00a0radiation\u00a0use\u00a0\nefficiency\u00a0in\u00a0wheat.\u00a0Journal\u00a0of\u00a0Experimental\u00a0Botany,\u00a051,\u00a0\u00a0\npp\u00a0\u00a0459\u2010473\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000085386600017\u00a0AND\u00a0http://\nwww.botanischergarten.ch/DroughtResistance/\nReynolds\u2010Avenues\u2010Genetic\u2010Modification\u20102000.pdf\u00a0\n\n\u00a0\nRichardson,\u00a0A.O.\u00a0&\u00a0Palmer,\u00a0J.D.\u00a0(2007)\u00a0Horizontal\u00a0gene\u00a0\n\ntransfer\u00a0in\u00a0plants.\u00a0Journal\u00a0of\u00a0Experimental\u00a0Botany,\u00a058,\u00a01,\u00a0\npp\u00a0\u00a01\u20109\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000243063800002\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/HorizontalGT/Richardson\u2010\nHorizonta\u2010Gene\u2010Transfer\u20102007.pdf\u00a0\n\n\u00a0\nRomeis,\u00a0J.,\u00a0Bartsch,\u00a0D.,\u00a0Bigler,\u00a0F.,\u00a0Candolfi,\u00a0M.,\u00a0Gielkens,\u00a0\n\nM.C.,\u00a0Hartley,\u00a0S.E.,\u00a0Hellmich,\u00a0R.,\u00a0Huesing,\u00a0J.E.,\u00a0Jepson,\u00a0\nP.C.,\u00a0Layton,\u00a0R.J.,\u00a0Quemada,\u00a0H.,\u00a0Raybould,\u00a0A.,\u00a0Rose,\u00a0R.,\u00a0\nSchiemann,\u00a0J.,\u00a0Sears,\u00a0M.K.,\u00a0Shelton,\u00a0M.,\u00a0Sweet,\u00a0J.,\u00a0\nVaituzis,\u00a0Z.,\u00a0&\u00a0Wolt,\u00a0J.D.\u00a0(2008)\u00a0Assessment\u00a0of\u00a0risk\u00a0of\u00a0\ninsect\u2010resistant\u00a0transgenic\u00a0crops\u00a0to\u00a0nontarget\u00a0\narthropods.\u00a0Nature\u00a0Biotechnology,\u00a026,\u00a02,\u00a0pp\u00a0\u00a0203\u2010208\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/nbt1381\u00a0AND\u00a0http://\nwww.nature.com/nbt/journal/v26/n2/suppinfo/\nnbt1381_S1.html\u00a0\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Bt/Romeis\u2010Nontarget\u2010\n2008.pdf\u00a0\n\nSchouten,\u00a0H.J.\u00a0&\u00a0Jacobsen,\u00a0E.\u00a0(2007)\u00a0Are\u00a0mutations\u00a0in\u00a0\ngenetically\u00a0modified\u00a0plants\u00a0dangerous?\u00a0Journal\u00a0of\u00a0\nBiomedicine\u00a0and\u00a0Biotechnology,\u00a0\u00a0pp\u00a0\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000252054100001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Schouten\u2010\nMutations\u2010Dangerous\u20102007.pdf\u00a0\n\n\u00a0\nShapiro,\u00a0J.A.", "start_char_idx": 71949, "end_char_idx": 74139, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "69d01884-f4c5-4896-90f6-397fd5eb2c20": {"__data__": {"id_": "69d01884-f4c5-4896-90f6-397fd5eb2c20", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fe08369c-da8a-4ec2-8348-5fcc7fd81cd5", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9d479d428a40cf5df282a7f82db8f0fe01ea4185bef87a074f41325a9d94df4d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "40bf7598-1d98-43fa-9daf-abfa086b2cb6", "node_type": "1", "metadata": {}, "hash": "07eb9f41fed49f9d8eb1bd21a3345b63942bfadea45ba9c4e87d8ffa285402a8", "class_name": "RelatedNodeInfo"}}, "text": "(1997)\u00a0Genome\u00a0organization,\u00a0natural\u00a0genetic\u00a0\n\nengineering\u00a0and\u00a0adaptive\u00a0mutation.\u00a0Trends\u00a0in\u00a0Genetics,\u00a0\n13,\u00a03,\u00a0pp\u00a0\u00a098\u2010104\u00a0\u00a0\n\n\u00a0 http://www.sciencedirect.com/science/article/B6TCY\u2010\n3RH119N\u201013/2/8e4f005e42c9889d6cc09ab2f3f2fd25\u00a0\nAND\u00a0http://www.botanischergarten.ch/Genomics/\nShapiro\u2010Natural\u2010Genetic\u2010Engineering\u20101997.pdf\u00a0\n\nShewry,\u00a0P.R.,\u00a0Baudo,\u00a0M.,\u00a0Lovegrove,\u00a0A.,\u00a0&\u00a0Powers,\u00a0S.\u00a0(2007)\u00a0\n\u00a0 Are\u00a0GM\u00a0and\u00a0conventionally\u00a0bred\u00a0cereals\u00a0really\u00a0different?\u00a0\n\nTrends\u00a0in\u00a0Food\u00a0Science\u00a0&\u00a0Technology,\u00a018,\u00a04,\u00a0pp\u00a0\u00a0201\u2010209\u00a0\u00a0\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000245784600003\u00a0AND\u00a0http://\n\nMiller,\u00a0H.I.\u00a0&\u00a0Conko,\u00a0G.\u00a0(2004)\u00a0Chasing\u00a0'transgenic'\u00a0shadows.\u00a0\nNat\u00a0Biotech,\u00a022,\u00a06,\u00a0pp\u00a0\u00a0654\u2010655\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/nbt0604\u2010654\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Bt/Miller\u2010Chasing\u2010\ntransgenic\u2010shadows\u20102004.pdf\u00a0\n\nMolnar,\u00a0I.,\u00a0Benavente,\u00a0E.,\u00a0&\u00a0Molnar\u2010Lang,\u00a0M.\u00a0(2009)\nDetection\u00a0of\u00a0intergenomic\u00a0chromosome\u00a0rearrangements\u00a0\nin\u00a0irradiated\u00a0Triticum\u00a0aestivum\u00a0\u2010\u00a0Aegilops\u00a0biuncialis\u00a0\namphiploids\u00a0by\u00a0multicolour\u00a0genomic\u00a0in\u00a0situ\u00a0\nhybridization.\u00a0Genome,\u00a052,\u00a02,\u00a0pp\u00a0\u00a0156\u2010165\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000265606300006\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Mutations/Molnar\u2010\nDetection\u2010Interngenomic\u2010rearrangements\u20102009.pdf\u00a0\n\n\u00a0\nMorikawa,\u00a0K.\u00a0&\u00a0Shirakawa,\u00a0M.\u00a0(2001)\u00a0Three\u2010dimensional\u00a0\n\nstructural\u00a0views\u00a0of\u00a0damaged\u2010DNA\u00a0recognition:\u00a0T4\u00a0\nendonuclease\u00a0V,\u00a0E.\u00a0coli\u00a0Vsr\u00a0protein,\u00a0and\u00a0human\u00a0\nnucleotide\u00a0excision\u00a0repair\u00a0factor\u00a0XPA\u00a0(vol\u00a0460,\u00a0pg\u00a0257,\u00a0\n2000).\u00a0Mutation\u00a0Research\u2010DNA\u00a0Repair,\u00a0485,\u00a03,\u00a0pp\u00a0\u00a0267\u2010\n268\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000167838200009\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Morikawa\u2010\nStructural\u2010Views\u2010Damaged\u2010DNA\u20102000.pdf\u00a0\n\n\u00a0\nMower,\u00a0J.,\u00a0Stefanovic,\u00a0S.,\u00a0Hao,\u00a0W.,\u00a0Gummow,\u00a0J.,\u00a0Jain,\u00a0K.,\u00a0\n\nAhmed,\u00a0D.,\u00a0&\u00a0Palmer,\u00a0J.\u00a0(2010)\u00a0Horizontal\u00a0acquisition\u00a0of\u00a0\nmultiple\u00a0mitochondrial\u00a0genes\u00a0from\u00a0a\u00a0parasitic\u00a0plant\u00a0\nfollowed\u00a0by\u00a0gene\u00a0conversion\u00a0with\u00a0host\u00a0mitochondrial\u00a0\ngenes.\u00a0Bmc\u00a0Biology,\u00a08,\u00a01,\u00a0pp\u00a0\u00a0150\u00a0\u00a0\n\n\u00a0 http://www.biomedcentral.com/1741\u20107007/8/150\u00a0AND\u00a0\nhttp://www.ask\u2010force.org/web/HorizontalGT/Mower\u2010\nHorizontal\u2010acquisition\u2010Mitochondrial\u20102010.pdf\u00a0\n\nNational\u2010Research\u2010Council\u00a0(1989)\u00a0Field\u00a0Testing\u00a0Genetically\u00a0\nModified\u00a0Organism.\u00a0Framework\u00a0for\u00a0Decisions,\u00a0\nCommittee\u00a0on\u00a0Scientific\u00a0Evaluation\u00a0of\u00a0the\u00a0Introduction\u00a0\nof\u00a0Genetically\u00a0Modified\u00a0Microorganisms\u00a0and\u00a0Plants\u00a0into\u00a0\nthe\u00a0Environment,\u00a0National\u00a0Research\u00a0Council\u00a0edn.", "start_char_idx": 74140, "end_char_idx": 76381, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "40bf7598-1d98-43fa-9daf-abfa086b2cb6": {"__data__": {"id_": "40bf7598-1d98-43fa-9daf-abfa086b2cb6", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "69d01884-f4c5-4896-90f6-397fd5eb2c20", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "643aa1a854c38f8f07eee43a0c3b03b700bbbf199b75592d4adc456042891071", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "524f2222-bbc3-447c-9f7c-2b0b9fdf3831", "node_type": "1", "metadata": {}, "hash": "9fc4b07fb4cb4b3d55f670d6894b4665da93f2f82e74ad615d308b46aa6dcbd5", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0\nNational\u00a0Academy\u00a0Press,\u00a0\u00a0\u00a0pp\u00a0184\u00a0\u00a0\n\n\u00a0 free\u00a0online\u00a0reading\u00a0http://www.nap.edu/\ncatalog/1431.html\u00a0\n\n\u00a0\nParrott,\u00a0W.\u00a0(2010)\u00a0Genetically\u00a0modified\u00a0myths\u00a0and\u00a0realities.\u00a0\n\nNew\u00a0Biotechnology,\u00a027,\u00a05,\u00a0pp\u00a0\u00a0545\u2010551\u00a0\u00a0\n\u00a0 http://www.sciencedirect.com/science/article/B8JG4\u2010\n\n506RN94\u20102/2/41a40cb121ad20dd44db6f76d34f1bd5\u00a0\nAND\u00a0\u00a0http://www.ask\u2010force.org/web/Vatican\u2010PAS\u2010\nStudyweek\u2010Elsevier\u2010publ\u201020101130/Parrott\u2010Wayne\u2010PAS\u2010\nGenetically\u2010Modified\u2010Myths\u201020101130\u2010publ.pdf\u00a0\n\n\u00a0\nRamjoue,\u00a0C.\u00a0(2007a)\u00a0The\u00a0transatlantic\u00a0rift\u00a0in\u00a0genetically\u00a0\n\nmodified\u00a0food\u00a0policy.\u00a0Journal\u00a0of\u00a0Agricultural\u00a0&\u00a0\nEnvironmental\u00a0Ethics,\u00a020,\u00a05,\u00a0pp\u00a0\u00a0419\u2010436\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:000248855000003\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Ramjoue\u2010\nTransatlantic\u2010Rift\u20102007.pdf\u00a0\n\n\u00a0\nRamjoue,\u00a0C.\u00a0(2007b)\u00a0The\u00a0transatlantic\u00a0rift\u00a0in\u00a0genetically\u00a0\n\nmodified\u00a0food\u00a0policy.\u00a0Doctoral\u00a0Thesis,\u00a0University\u00a0of\u00a0\nZurich,\u00a0Zurich\u00a0Thesis,\u00a0pp\u00a0\u00a0263\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/Regulation/Ramjoue\u2010\nThesis\u2010Transatlantic\u2010Rift\u20102007.pdf\u00a0\n\n\u00a0\nRanker,\u00a0T.A.,\u00a0Haufler,\u00a0C.H.,\u00a0Soltis,\u00a0P.S.,\u00a0&\u00a0Soltis,\u00a0D.E.\u00a0(1989)\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n47\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nRapid\u2010Genome\u2010Change\u2010Brassica\u20101995.pdf\u00a0\n\n\u00a0\nSong,\u00a0K.\u00a0&\u00a0Osborn,\u00a0T.C.\u00a0(1992)\u00a0POLYPHYLETIC\u00a0ORIGINS\u00a0OF\u00a0\n\nBRASSICA\u2010NAPUS\u00a0\u2010\u00a0NEW\u00a0EVIDENCE\u00a0BASED\u00a0ON\u00a0\nORGANELLE\u00a0AND\u00a0NUCLEAR\u00a0RFLP\u00a0ANALYSES.\u00a0Genome,\u00a0\n35,\u00a06,\u00a0pp\u00a0\u00a0992\u20101001\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1992KD50000014\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Song\u2010\nPolyphlyletic\u2010Origins\u2010Brassica\u20101962.pdf\u00a0\n\n\u00a0\nStanhope,\u00a0M.J.,\u00a0Lupas,\u00a0A.,\u00a0Italia,\u00a0M.J.,\u00a0Koretke,\u00a0K.K.,\u00a0Volker,\u00a0\n\nC.,\u00a0&\u00a0Brown,\u00a0J.R.\u00a0(2001)\u00a0Phylogenetic\u00a0analyses\u00a0do\u00a0not\u00a0\nsupport\u00a0horizontal\u00a0gene\u00a0transfers\u00a0from\u00a0bacteria\u00a0to\u00a0\nvertebrates.\u00a0Nature,\u00a0411,\u00a06840,\u00a0pp\u00a0\u00a0940\u2010944\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/35082058\u00a0AND\u00a0http://\nwww.nature.com/nature/journal/v411/n6840/\nsuppinfo/411940a0_S1.html\u00a0AND\u00a0http://www.ask\u2010\nforce.org/web/HorizontalGT/Stanhope\u2010Phylogenetic\u2010\nAnalysis\u20102001.pdf\u00a0\n\nThro,\u00a0A.M.\u00a0(2004)\u00a0Europe\u00a0on\u00a0transgenic\u00a0crops:\u00a0How\u00a0public\u00a0\nplant\u00a0breeding\u00a0and\u00a0eco\u2010transgenics\u00a0can\u00a0help\u00a0in\u00a0the\u00a0\ntransatlantic\u00a0debate.\u00a0Commentary.\u00a0In\u00a0AgBioForum,\u00a0Vol.\u00a0\n7,\u00a0pp.", "start_char_idx": 76382, "end_char_idx": 78497, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "524f2222-bbc3-447c-9f7c-2b0b9fdf3831": {"__data__": {"id_": "524f2222-bbc3-447c-9f7c-2b0b9fdf3831", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "40bf7598-1d98-43fa-9daf-abfa086b2cb6", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "a3bfbe5c6f30efa161edd5d4298bb69d83bb8a6b8d429ae5f240297b9332f7c4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ef10d940-7bda-4b05-aca3-1f3a1d466636", "node_type": "1", "metadata": {}, "hash": "0c92093b81b9b2daa3fa6f843c3743a434ac883218d099b31a04d91828e65dcb", "class_name": "RelatedNodeInfo"}}, "text": "7,\u00a0pp.\u00a0142\u2010148\u00a0\n\n\u00a0 http://www.agbioforum.org/v7n3/v7n3a06\u2010thro.htm\u00a0\nAND\u00a0http://www.botanischergarten.ch/Thro\u2010Europe\u2010\nTransgenic\u2010Crops\u20102004.pdf\u00a0\n\n\u00a0\nvan\u00a0Bueren,\u00a0E.T.L.,\u00a0Ostergard,\u00a0H.,\u00a0Goldringer,\u00a0I.,\u00a0&\u00a0Scholten,\u00a0\n\nO.\u00a0(2008)\u00a0Plant\u00a0breeding\u00a0for\u00a0organic\u00a0and\u00a0sustainable,\u00a0\nlow\u2010input\u00a0agriculture:\u00a0dealing\u00a0with\u00a0genotype\u2010\nenvironment\u00a0interactions.\u00a0Euphytica,\u00a0163,\u00a03,\u00a0pp\u00a0\u00a0321\u2010\n322\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000258654800001\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Organic/Lammerts\u2010Plant\u2010\nBreeding\u2010Interactions\u20102008.pdf\u00a0\n\n\u00a0\nVan\u00a0Bueren,\u00a0E.T.L.\u00a0&\u00a0Struik,\u00a0P.C.\u00a0(2004)\u00a0The\u00a0consequences\u00a0of\u00a0\n\nthe\u00a0concept\u00a0of\u00a0naturalness\u00a0for\u00a0organic\u00a0plant\u00a0breeding\u00a0\nand\u00a0propagation.\u00a0Njas\u2010Wageningen\u00a0Journal\u00a0of\u00a0Life\u00a0\nSciences,\u00a052,\u00a01,\u00a0pp\u00a0\u00a085\u201095\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000226051800007\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Organic/Van\u2010Bueren\u2010\nConsequences\u20102004.pdf\u00a0\n\n\u00a0\nVan\u00a0Bueren,\u00a0E.T.L.\u00a0&\u00a0Struik,\u00a0P.C.\u00a0(2005)\u00a0Integrity\u00a0and\u00a0rights\u00a0\n\nof\u00a0plants:\u00a0Ethical\u00a0notions\u00a0in\u00a0organic\u00a0plant\u00a0breeding\u00a0and\u00a0\npropagation.\u00a0Journal\u00a0of\u00a0Agricultural\u00a0&\u00a0Environmental\u00a0\nEthics,\u00a018,\u00a05,\u00a0pp\u00a0\u00a0479\u2010493\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000231949300003\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Organic/Van\u2010Bueren\u2010Ethical\n\u20102005.pdf\u00a0\n\n\u00a0\nVan\u00a0Bueren,\u00a0E.T.L.,\u00a0Struik,\u00a0P.C.,\u00a0Tiemens\u2010Hulscher,\u00a0M.,\u00a0&\u00a0\n\nJacobsen,\u00a0E.\u00a0(2003)\u00a0Concepts\u00a0of\u00a0intrinsic\u00a0value\u00a0and\u00a0\nintegrity\u00a0of\u00a0plants\u00a0in\u00a0organic\u00a0plant\u00a0breeding\u00a0and\u00a0\npropagation.\u00a0Crop\u00a0Science,\u00a043,\u00a06,\u00a0pp\u00a0\u00a01922\u20101929\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000186477700003\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Organic/Van\u2010Bueren\u2010\nIntrinsic\u20102003.pdf\u00a0\n\n\u00a0\nWaara,\u00a0S.\u00a0&\u00a0Glimelius,\u00a0K.\u00a0(1995)\u00a0THE\u00a0POTENTIAL\u00a0OF\u00a0\n\nSOMATIC\u00a0HYBRIDIZATION\u00a0IN\u00a0CROP\u00a0BREEDING.\u00a0\nEuphytica,\u00a085,\u00a01\u20103,\u00a0pp\u00a0\u00a0217\u2010233\u00a0\u00a0\n\nwww.botanischergarten.ch/Wheat/Shewry\u2010Are\u2010GM\u2010\nConvent\u2010Cereals\u2010different\u20102007.pdf\u00a0\n\n\u00a0\nShirley,\u00a0B.W.,\u00a0Hanley,\u00a0S.,\u00a0&\u00a0Goodman,\u00a0H.M.\u00a0(1992)\u00a0EFFECTS\u00a0\n\nOF\u00a0IONIZING\u2010RADIATION\u00a0ON\u00a0A\u00a0PLANT\u00a0GENOME\u00a0\u2010\u00a0\nANALYSIS\u00a0OF\u00a02\u00a0ARABIDOPSIS\u00a0TRANSPARENT\u2010TESTA\u00a0\nMUTATIONS.\u00a0Plant\u00a0Cell,\u00a04,\u00a03,\u00a0pp\u00a0\u00a0333\u2010347\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1992HL04000011\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Shirley\u2010Inonizing\u2010\nRadiation\u2010Arabidopsis\u20101992.pdf\u00a0\n\n\u00a0\nSinigovets,\u00a0M.E.\u00a0(1987)\u00a0THE\u00a0CYTOGENETIC\u00a0STRUCTURE\u00a0OF\u00a0A\u00a0\n\n56\u2010CHROMOSOME\u00a0TRITICUM\u2010AESTIVUM\u00a0\u2010\u00a0ELYTRIGIA\u00a0\nINTERMEDIA\u00a0HYBRIDS.", "start_char_idx": 78491, "end_char_idx": 80608, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ef10d940-7bda-4b05-aca3-1f3a1d466636": {"__data__": {"id_": "ef10d940-7bda-4b05-aca3-1f3a1d466636", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "524f2222-bbc3-447c-9f7c-2b0b9fdf3831", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "680c75d4375ca5a1ee85bf96d7817eaf99b1da01c1216935d7982623eb26abfe", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f6831f8c-af35-436f-b6a4-58daf162302b", "node_type": "1", "metadata": {}, "hash": "63b8aee5bfde1ea3de06335eda7764ca1800afb6d5c4d14198707cbfc42a6d7a", "class_name": "RelatedNodeInfo"}}, "text": "Genetika,\u00a023,\u00a05,\u00a0pp\u00a0\u00a0854\u2010862\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1987H772100012\u00a0\n\u00a0\nSmalla,\u00a0K.\u00a0&\u00a0Sobecky,\u00a0P.A.\u00a0(2002)\u00a0The\u00a0prevalence\u00a0and\u00a0\n\ndiversity\u00a0of\u00a0mobile\u00a0genetic\u00a0elements\u00a0in\u00a0bacterial\u00a0\ncommunities\u00a0of\u00a0different\u00a0environmental\u00a0habitats:\u00a0\ninsights\u00a0gained\u00a0from\u00a0different\u00a0methodological\u00a0\napproaches.\u00a0Fems\u00a0Microbiology\u00a0Ecology,\u00a042,\u00a02,\u00a0pp\u00a0\u00a0165\u2010\n175\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://000179450100002\u00a0AND\u00a0http://\nwww.botanischergarten.ch/HorizontalGT/Smalla\u2010\nReview\u2010HGT\u20102002.pdf\u00a0\n\n\u00a0\nSmalla,\u00a0K.\u00a0&\u00a0Vogel,\u00a0T.M.\u00a0(2007)\u00a0Presentation\u00a0of\u00a0the\u00a0thematic\u00a0\n\nissue\u00a0on\u00a0horizontal\u00a0gene\u00a0transfer.\u00a0Environmental\u00a0\nBiosafety\u00a0Research,\u00a06,\u00a01\u20102,\u00a0pp\u00a0\u00a01\u20102\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://BIOSIS:PREV200700602019\u00a0AND\u00a0http://\nwww.botanischergarten.ch/HorizontalGT/Smalla\u2010\nHorizontal\u2010Geneflow\u20102007.pdf\u00a0\n\n\u00a0\nSnyder,\u00a0L.U.,\u00a0Gallo,\u00a0M.,\u00a0Fulford,\u00a0S.G.,\u00a0Irani,\u00a0T.,\u00a0Rudd,\u00a0R.,\u00a0\n\nDiFino,\u00a0S.M.,\u00a0&\u00a0Durham,\u00a0T.C.\u00a0(2008)\u00a0European\u00a0Union's\u00a0\nMoratorium\u00a0Impact\u00a0on\u00a0Food\u00a0Biotechnology:\u00a0A\u00a0\nDiscussion\u2010Based\u00a0Scenario.\u00a0Journal\u00a0of\u00a0Natural\u00a0Resources\u00a0\nand\u00a0Life\u00a0Sciences\u00a0Education,\u00a037,\u00a0\u00a0pp\u00a0\u00a027\u201031\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://BIOSIS:PREV200900043152\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Regulation/Snyder\u2010Unruh\u2010\nEuropean\u2010Moratorium\u20102008.pdf\u00a0\n\n\u00a0\nSoltis,\u00a0D.E.\u00a0&\u00a0Soltis,\u00a0P.S.\u00a0(1993)\u00a0MOLECULAR\u2010DATA\u00a0AND\u00a0THE\u00a0\n\nDYNAMIC\u00a0NATURE\u00a0OF\u00a0POLYPLOIDY.\u00a0Critical\u00a0Reviews\u00a0in\u00a0\nPlant\u00a0Sciences,\u00a012,\u00a03,\u00a0pp\u00a0\u00a0243\u2010273\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1993LJ92200004\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Soltis\u2010Molecular\u2010\nData\u2010Dynamic\u20101993.pdf\u00a0\n\n\u00a0\nSoltis,\u00a0P.S.,\u00a0Plunkett,\u00a0G.M.,\u00a0Novak,\u00a0S.J.,\u00a0&\u00a0Soltis,\u00a0D.E.\u00a0(1995)\u00a0\n\u00a0 GENETIC\u2010VARIATION\u00a0IN\u00a0TRAGOPOGON\u00a0SPECIES\u00a0\u2010\u00a0\n\nADDITIONAL\u00a0ORIGINS\u00a0OF\u00a0THE\u00a0ALLOTETRAPLOIDS\u00a0T\u2010\nMIRUS\u00a0AND\u00a0T\u2010MISCELLUS\u00a0(COMPOSITAE).\u00a0American\u00a0\nJournal\u00a0of\u00a0Botany,\u00a082,\u00a010,\u00a0pp\u00a0\u00a01329\u20101341\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1995TA45600015\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Soltis\u2010Genetic\u2010\nVariation\u2010Tragopogon\u20101995.pdf\u00a0\n\n\u00a0\nSong,\u00a0K.,\u00a0Lu,\u00a0P.,\u00a0Tang,\u00a0K.,\u00a0&\u00a0Osborn,\u00a0T.C.\u00a0(1995)\u00a0Rapid\u00a0\n\ngenome\u00a0change\u00a0in\u00a0synthetic\u00a0polyploids\u00a0of\u00a0Brassica\u00a0and\u00a0\nits\u00a0implications\u00a0for\u00a0polyploid\u00a0evolution.\u00a0Proceedings\u00a0of\u00a0\nthe\u00a0National\u00a0Academy\u00a0of\u00a0Sciences\u00a0of\u00a0the\u00a0United\u00a0States\u00a0\nof\u00a0America,\u00a092,\u00a017,\u00a0pp\u00a0\u00a07719\u20107723\u00a0\u00a0\n\n\u00a0 http://www.pnas.org/content/92/17/7719.abstract\u00a0\nAND\u00a0http://www.ask\u2010force.org/web/Genomics/Song\u2010", "start_char_idx": 80609, "end_char_idx": 82757, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f6831f8c-af35-436f-b6a4-58daf162302b": {"__data__": {"id_": "f6831f8c-af35-436f-b6a4-58daf162302b", "embedding": null, "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-54", "node_type": "4", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "9f82881298c31a001ac3affd74fa042ec8b8bd426576a262b8b6af0bda337ae8", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ef10d940-7bda-4b05-aca3-1f3a1d466636", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "d5c7855edb81e157f463416880f5a7cc48c3ac2371470c6ec88751832ebc0404", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9dda38a0-c9e9-45d2-b054-0928c7c96ab5", "node_type": "1", "metadata": {}, "hash": "81b56e65d7357cd9d85e0406a2eb0746b5404bf8d354f79deda961d2cef15c42", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a031\u201048\u00a0\n\n48\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1995TF37600028\u00a0AND\u00a0http://\nwww.ask\u2010force.org/web/Genomics/Waara\u2010Potential\u2010\nSomatic\u2010Hybridization\u20101995.pdf\u00a0\n\n\u00a0\nWatson,\u00a0J.D.\u00a0&\u00a0Crick,\u00a0F.H.C.\u00a0(1953a)\u00a0GENETICAL\u00a0\n\nIMPLICATIONS\u00a0OF\u00a0THE\u00a0STRUCTURE\u00a0OF\u00a0\nDEOXYRIBONUCLEIC\u00a0ACID.\u00a0Nature,\u00a0171,\u00a04361,\u00a0pp\u00a0\u00a0964\u2010\n967\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1953UA43900005\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Watson\u2010Crick\u2010\nGenetical\u2010Implications\u2010DNA\u20101953.pdf\u00a0\n\n\u00a0\nWatson,\u00a0J.D.\u00a0&\u00a0Crick,\u00a0F.H.C.\u00a0(1953b)\u00a0MOLECULAR\u00a0STRUCTURE\u00a0\n\nOF\u00a0NUCLEIC\u00a0ACIDS\u00a0\u2010\u00a0A\u00a0STRUCTURE\u00a0FOR\u00a0DEOXYRIBOSE\u00a0\nNUCLEIC\u00a0ACID.\u00a0Nature,\u00a0171,\u00a04356,\u00a0pp\u00a0\u00a0737\u2010738\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1953UA43400007\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Watson\u2010Crick\u2010\nMolecular\u2010Structure\u2010Nucleic\u2010Acids\u20101953.pdf\u00a0\n\n\u00a0\nWerth,\u00a0C.R.,\u00a0Guttman,\u00a0S.I.,\u00a0&\u00a0Eshbaugh,\u00a0W.H.\u00a0(1985)\u00a0\n\nRECURRING\u00a0ORIGINS\u00a0OF\u00a0ALLOPOLYPLOID\u00a0SPECIES\u00a0IN\u00a0\nASPLENIUM.\u00a0Science,\u00a0228,\u00a04700,\u00a0pp\u00a0\u00a0731\u2010733\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1985AGH4200034\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Werth\u2010Recurring\u2010\n\nOrigins\u2010Asplenium\u20101965.pdf\u00a0\n\u00a0\nWilkins,\u00a0M.H.F.,\u00a0Seeds,\u00a0W.E.,\u00a0Stokes,\u00a0A.R.,\u00a0&\u00a0Wilson,\u00a0H.R.\u00a0\n\n(1953)\u00a0Helical\u00a0Structure\u00a0of\u00a0Crystalline\u00a0Deoxypentose\u00a0\nNucleic\u00a0Acid.\u00a0Nature,\u00a0172,\u00a04382,\u00a0pp\u00a0\u00a0759\u2010762\u00a0\u00a0\n\n\u00a0 http://dx.doi.org/10.1038/172759b0\u00a0AND\u00a0http://\nwww.botanischergarten.ch/History/Wilkins\u2010Helical\u2010\nStructure\u2010Desoxypentose\u20101953.pdf\u00a0\n\n\u00a0\nWilson,\u00a0A.,\u00a0Latham,\u00a0J.,\u00a0&\u00a0Steinbrecher,\u00a0R.\u00a0(2006)\u00a0\n\nTransformation\u2010induced\u00a0mutations\u00a0in\u00a0transgenic\u00a0plants:\u00a0\nAnalysis\u00a0and\u00a0biosafety\u00a0implications.\u00a0.\u00a0Biotechnology\u00a0and\u00a0\nGenetic\u00a0Engineering\u00a0Reviews\u00a023,\u00a011,\u00a0pp\u00a0\u00a01\u201026\u00a0\u00a0\n\n\u00a0 http://www.botanischergarten.ch/Radiation\u2010Mutants/\nWilson\u2010Transformation\u2010Induced\u2010Mutations\u20102006.pdf\u00a0\n\n\u00a0\nWyatt,\u00a0R.,\u00a0Odrzykoski,\u00a0I.J.,\u00a0Stoneburner,\u00a0A.,\u00a0Bass,\u00a0H.W.,\u00a0&\u00a0\n\nGalau,\u00a0G.A.\u00a0(1988)\u00a0ALLOPOLYPLOIDY\u00a0IN\u00a0BRYOPHYTES\u00a0\u2010\u00a0\nMULTIPLE\u00a0ORIGINS\u00a0OF\u00a0PLAGIOMNIUM\u2010MEDIUM.\u00a0\nProceedings\u00a0of\u00a0the\u00a0National\u00a0Academy\u00a0of\u00a0Sciences\u00a0of\u00a0the\u00a0\nUnited\u00a0States\u00a0of\u00a0America,\u00a085,\u00a015,\u00a0pp\u00a0\u00a05601\u20105604\u00a0\u00a0\n\n\u00a0 <Go\u00a0to\u00a0ISI>://WOS:A1988P574600056\u00a0AND\u00a0http://\nwww.botanischergarten.ch/Genomics/Wyatt\u2010\nAllopolyploidy\u2010Bryophytes\u20101988.pdf", "start_char_idx": 82761, "end_char_idx": 84877, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9dda38a0-c9e9-45d2-b054-0928c7c96ab5": {"__data__": {"id_": "9dda38a0-c9e9-45d2-b054-0928c7c96ab5", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f6831f8c-af35-436f-b6a4-58daf162302b", "node_type": "1", "metadata": {"identifier": "njb-54", "author": "Ammann, Klaus", "title": "Molecular differences between GM- and non-GM crops over-estimated?", "date": "2022-07-11", "file": "njb-54.pdf"}, "hash": "0887ab1fe1549ffbb44cc78e4c3909b2aabfb57e68ee3d6ac33b4d306bf30f11", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "75b6f36b-1567-442b-a617-9268f486aeac", "node_type": "1", "metadata": {}, "hash": "1383f88ba532438938a59767df2bd3147b1557e46391087eb48da67f012d53d8", "class_name": "RelatedNodeInfo"}}, "text": "4\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n22\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nORIGINAL\u00a0RESEARCH\u00a0ARTICLE\u00a0\n\nDevelopment\u00a0of\u00a0PCR\u00a0assay\u00a0for\u00a0targeting\u00a0partial\u00a0lipL21\u00a0\nand\u00a0lipL41\u00a0gene\u00a0of\u00a0leptospira\u00a0\n\nS.\u00a0Chandan1,\u00a0S.\u00a0Umesha1,\u00a0S.K.\u00a0Bhure2,\u00a0N.\u00a0Haraprasad3,\u00a0S.\u00a0Chandrashekar1\u00a0\n1\u00a0Department\u00a0of\u00a0Studies\u00a0in\u00a0Biotechnology,\u00a0University\u00a0of\u00a0Mysore,\u00a0Manasagangotri,\u00a0Mysore\u2010570006,\u00a0Karnataka,\u00a0India.\u00a02\u00a0IVRI,\u00a0Izatnagar,\u00a0Bareilly\u2010243\u00a0\n\n122,\u00a0Uttar\u00a0Pradesh,\u00a0India.\u00a03\u00a0Department\u00a0of\u00a0Biotechnology,\u00a0SJ\u00a0College\u00a0of\u00a0Engineering,\u00a0Manasagangotri,\u00a0Mysore\u2010570006,\u00a0Karnataka,\u00a0India.\u00a0\n\nAbstract\u00a0\n\nLeptospirosis\u00a0is\u00a0a\u00a0bacterial\u00a0zoonotic\u00a0disease\u00a0caused\u00a0by\u00a0spirochetes\u00a0of\u00a0the\u00a0genus\u00a0Leptospira\u00a0that\u00a0affects\u00a0human\u00a0\n\nand\u00a0a\u00a0wide\u00a0range\u00a0of\u00a0animals.\u00a0The\u00a0direct\u00a0method\u00a0of\u00a0diagnosis\u00a0of\u00a0leptospirosis,\u00a0has\u00a0been\u00a0so\u00a0far\u00a0by\u00a0culture\u00a0isolation\u00a0\n\nbut\u00a0it\u00a0is\u00a0time\u00a0consuming\u00a0and\u00a0potentially\u00a0biohazardous.\u00a0Another\u00a0traditional\u00a0method\u00a0is\u00a0the\u00a0detection\u00a0of\u00a0antibodies\u00a0\n\n(Serological\u00a0 tests)\u00a0 which\u00a0 is\u00a0 also\u00a0 a\u00a0 time\u00a0 consuming\u00a0 method\u00a0 and\u00a0 fails\u00a0 to\u00a0 identify\u00a0 the\u00a0 infecting\u00a0 serovar.\u00a0 To\u00a0\n\novercome\u00a0 these\u00a0 limitations\u00a0 associated\u00a0 with\u00a0 the\u00a0 cultivation\u00a0 and\u00a0 serology,\u00a0 we\u00a0 developed\u00a0 PCR\u00a0 assay\u00a0 targeting\u00a0\n\npartial\u00a0lipL21\u00a0gene\u00a0and\u00a0lipL41\u00a0gene\u00a0of\u00a0Leptospires\u00a0using\u00a0in\u2010house\u00a0designed\u00a0P28/29\u00a0and\u00a0P30/31\u00a0primers,\u00a0with\u00a0a\u00a0\n\nproduct\u00a0size\u00a0of\u00a0385bp\u00a0and\u00a0427bp.\u00a0The\u00a0amplicons\u00a0were\u00a0subjected\u00a0to\u00a0restriction\u00a0enzyme\u00a0digestion\u00a0using\u00a0RsaI,\u00a0Pvu\u00a0\n\nII\u00a0and\u00a0HindIII\u00a0for\u00a0product\u00a0of\u00a0P28/29\u00a0and\u00a0ClaI,\u00a0TaqI\u00a0and\u00a0RsaI\u00a0were\u00a0used\u00a0for\u00a0product\u00a0of\u00a0P30/31.\u00a0The\u00a0protocols\u00a0were\u00a0\n\nstandardized\u00a0 and\u00a0 the\u00a0 assay\u00a0 targeting\u00a0 the\u00a0 partial\u00a0 lipL21\u00a0 and\u00a0 lipL41\u00a0 gene\u00a0 was\u00a0 found\u00a0 to\u00a0 be\u00a0 specific\u00a0 for\u00a0 eight\u00a0\n\npathogenic\u00a0Leptospires\u00a0out\u00a0of\u00a0nine\u00a0leptospires\u00a0tested.\u00a0The\u00a0products\u00a0were\u00a0then\u00a0cloned\u00a0in\u00a0pGEMT\u00a0Easy\u00a0vector\u00a0and\u00a0\n\nsequenced\u00a0to\u00a0facilitate\u00a0further\u00a0studies.\u00a0\u00a0PCR\u00a0could\u00a0detect\u00a0the\u00a0target\u00a0bacterial\u00a0gene\u00a0without\u00a0any\u00a0ambiguity\u00a0and\u00a0\n\nshowed\u00a0 good\u00a0 efficiency\u00a0 in\u00a0 detection\u00a0 of\u00a0 targeted\u00a0 species\u00a0 in\u00a0 the\u00a0 sample.\u00a0 This\u00a0 simple,\u00a0 rapid\u00a0 and\u00a0 cost\u2010effective\u00a0\n\nmethod\u00a0can\u00a0be\u00a0applicable\u00a0in\u00a0a\u00a0prediction\u00a0system\u00a0to\u00a0prevent\u00a0disease\u00a0outbreak\u00a0by\u00a0these\u00a0Leptospira\u00a0species\u00a0and\u00a0\n\ncan\u00a0be\u00a0considered\u00a0as\u00a0an\u00a0effective\u00a0tool\u00a0for\u00a0disease\u00a0diagnosis\u00a0of\u00a0Leptospira\u00a0species.", "start_char_idx": 45, "end_char_idx": 2261, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "75b6f36b-1567-442b-a617-9268f486aeac": {"__data__": {"id_": "75b6f36b-1567-442b-a617-9268f486aeac", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9dda38a0-c9e9-45d2-b054-0928c7c96ab5", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "7dd54f48fe415959da3cebf1abdfa490d20bc0ddeb4279d7e667245423e83d6c", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5a45cd5f-56ce-454f-855e-6de1332651b0", "node_type": "1", "metadata": {}, "hash": "5a59a7bb2042e16e62d6ed31d946d783de14a942a74864f9134be295ec834856", "class_name": "RelatedNodeInfo"}}, "text": "Key\u00a0words:\u00a0PCR,\u00a0lipL21,\u00a0lipL41,\u00a0Molecular\u00a0Diagnostics,\u00a0Transformation.\u00a0\u00a0\n\n\u00a0\nCorrespondence\u00a0Author:\u00a0\u00a0\n\nSharanaiah\u00a0Umesha,\u00a0DOS\u00a0in\u00a0Biotechnology,\u00a0University\u00a0of\u00a0Mysore,\u00a0Karnataka,\u00a0India.\u00a0\nE\u2010mail:\u00a0umeshgroup@yahoo.co.in\u00a0\n\nIntroduction\u00a0\nThe\u00a0 economic\u00a0 importance\u00a0 of\u00a0 bovine\u00a0 leptospirosis\u00a0\n\nincludes\u00a0 loss\u00a0due\u00a0to\u00a0abortion,\u00a0 loss\u00a0of\u00a0milk\u00a0production\u00a0\n\nand\u00a0 elevated\u00a0 veterinary\u00a0 costs\u00a0 and\u00a0 predominantly\u00a0\n\nhuman\u00a0 infection\u00a0 (Levett\u00a0 et\u00a0 al.,\u00a0 2005).\u00a0 \u00a0 Leptospirosis\u00a0\n\nalso\u00a0known\u00a0as\u00a0Weil's\u00a0disease,\u00a0canicola\u00a0fever,\u00a0canefield\u00a0\n\nfever,\u00a0nanukayami\u00a0fever,\u00a07\u2010day\u00a0fever\u00a0and\u00a0many\u00a0more.\u00a0\u00a0\n\nLeptospirosis\u00a0recognized\u00a0as\u00a0one\u00a0of\u00a0the\u00a0most\u00a0common\u00a0\nzoonoses.\u00a0 Leptospirosis\u00a0 is\u00a0 commonly\u00a0 transmitted\u00a0 to\u00a0\nhumans\u00a0through\u00a0contact\u00a0of\u00a0animal\u00a0urine\u00a0with\u00a0unhealed\u00a0\nbreaks\u00a0 in\u00a0 the\u00a0 skin,\u00a0 eyes\u00a0 or\u00a0 with\u00a0 the\u00a0 mucous\u00a0\nmembranes.\u00a0Outside\u00a0tropical\u00a0areas,\u00a0leptospirosis\u00a0cases\u00a0\nhave\u00a0a\u00a0relatively\u00a0distinct\u00a0seasonality\u00a0with\u00a0most\u00a0of\u00a0them\u00a0\noccurring\u00a0during\u00a0August\u2010September/February\u2010March\u00a0in\u00a0\u00a0\nyear\u00a0(Zhang\u00a0et\u00a0al.,\u00a01992).\u00a0\u00a0\n\nThe\u00a0spectrum\u00a0of\u00a0human\u00a0disease\u00a0caused\u00a0by\u00a0leptospires\u00a0\n\nis\u00a0extremely\u00a0wide,\u00a0ranging\u00a0from\u00a0subclinical\u00a0infection\u00a0to\u00a0\n\na\u00a0 severe\u00a0 syndrome\u00a0 of\u00a0 multi\u00a0 organ\u00a0 infection\u00a0 resulting\u00a0\n\nhigh\u00a0mortality.\u00a0This\u00a0syndrome,\u00a0icteric\u00a0leptospirosis\u00a0with\u00a0\n\nrenal\u00a0 failure,\u00a0 was\u00a0 first\u00a0 reported\u00a0 more\u00a0 than\u00a0 100\u00a0 years\u00a0\n\nago\u00a0 by\u00a0 Adolf\u00a0 Weil\u00a0 in\u00a0 Heidelberg.\u00a0 However,\u00a0 an\u00a0\n\napparently\u00a0 identical\u00a0 syndrome\u00a0 occurring\u00a0 in\u00a0 sewer\u00a0\n\nworkers\u00a0 was\u00a0 described\u00a0 several\u00a0 years\u00a0 earlier.\u00a0 Earlier\u00a0\n\ndescriptions\u00a0 of\u00a0 diseases\u00a0 that\u00a0 were\u00a0 probably\u00a0\n\nleptospirosis\u00a0 were\u00a0 reviewed\u00a0 recently\u00a0 (Serres\u00a0 et\u00a0 al.,\u00a0\n\n1995).\u00a0\n\nLeptospira\u00a0(from\u00a0the\u00a0Greek\u00a0word\u00a0leptos\u00a0means\u00a0fine\u00a0or\u00a0\n\nthin\u00a0and\u00a0Latin\u00a0word\u00a0spira\u00a0means\u00a0saprophytic\u00a0species).\u00a0\n\nLeptospira\u00a0was\u00a0first\u00a0observed\u00a0 in\u00a01907\u00a0 in\u00a0kidney\u00a0tissue\u00a0\n\nslices\u00a0of\u00a0a\u00a0leptospirosis\u00a0victim\u00a0who\u00a0was\u00a0reported\u00a0to\u00a0die\u00a0\n\nof\u00a0\u201cyellow\u00a0fever\u201d\u00a0(Abdollahpour,\u00a01990).\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The\u00a0direct\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n23\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nin\u00a0 artificial\u00a0 medium,\u00a0 with\u00a0 Leptospira\u00a0 present\u00a0 in\u00a0 an\u00a0\n\ninfected\u00a0 animal.\u00a0 Several\u00a0 leptospiral\u00a0 outer\u00a0 membrane\u00a0\n\nproteins\u00a0 have\u00a0 been\u00a0 shown\u00a0 to\u00a0 attach\u00a0 to\u00a0 the\u00a0 host\u00a0\n\nextracellular\u00a0matrix\u00a0 and\u00a0 to\u00a0 factor\u00a0H,\u00a0 suggesting\u00a0 these\u00a0\n\nproteins\u00a0may\u00a0be\u00a0important\u00a0for\u00a0adhesion\u00a0of\u00a0Leptospira\u00a0\n\nto\u00a0 host\u00a0 tissues\u00a0 and\u00a0 in\u00a0 resisting\u00a0 complement,\u00a0\n\nrespectively.\u00a0 The\u00a0 outer\u00a0 membrane\u00a0 of\u00a0 Leptospira,\u00a0 like\u00a0\n\nthose\u00a0of\u00a0most\u00a0other\u00a0Gram\u2010negative\u00a0bacteria,\u00a0contains\u00a0\n\nlipopolysaccharide\u00a0 (LPS).", "start_char_idx": 2266, "end_char_idx": 4757, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5a45cd5f-56ce-454f-855e-6de1332651b0": {"__data__": {"id_": "5a45cd5f-56ce-454f-855e-6de1332651b0", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "75b6f36b-1567-442b-a617-9268f486aeac", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "06d9660f3662fcfa256dc58a043b9bd245b077669f2d175d840ddac0e8695669", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c9c3f336-86a8-4cb9-8430-516d6ea109c0", "node_type": "1", "metadata": {}, "hash": "42d646b8a54739dae7b2b396b0e63f895a833a441ea065704e8efd2a6393703b", "class_name": "RelatedNodeInfo"}}, "text": "Differences\u00a0 in\u00a0 the\u00a0 highly\u00a0\n\nimmunogenic\u00a0LPS\u00a0structure\u00a0account\u00a0for\u00a0the\u00a0numerous\u00a0\n\nserovars\u00a0 of\u00a0 Leptospira.\u00a0 Consequently,\u00a0 immunity\u00a0 is\u00a0\n\nserovar\u00a0 specific;\u00a0 current\u00a0 leptospiral\u00a0 vaccines,\u00a0 which\u00a0\n\nconsist\u00a0 of\u00a0 one\u00a0 or\u00a0 several\u00a0 serovars\u00a0 of\u00a0 Leptospira\u00a0\n\nendemic\u00a0 in\u00a0 the\u00a0 population\u00a0 to\u00a0 be\u00a0 immunized,\u00a0 protect\u00a0\n\nonly\u00a0 against\u00a0 the\u00a0 serovars\u00a0 contained\u00a0 in\u00a0 the\u00a0 vaccine\u00a0\n\npreparation.\u00a0Leptospiral\u00a0LPS\u00a0has\u00a0low\u00a0endotoxin\u00a0activity.\u00a0\u00a0\n\nAn\u00a0unusual\u00a0feature\u00a0of\u00a0leptospiral\u00a0LPS\u00a0is\u00a0that\u00a0it\u00a0activates\u00a0\n\nhost\u00a0 cells\u00a0 via\u00a0 TLR2\u00a0 rather\u00a0 than\u00a0 TLR4.\u00a0 \u00a0 The\u00a0 unique\u00a0\n\nstructure\u00a0 of\u00a0 the\u00a0 lipid\u00a0 A\u00a0 portion\u00a0 of\u00a0 the\u00a0 LPS\u00a0 molecule\u00a0\n\nmay\u00a0 account\u00a0 for\u00a0 this\u00a0 observation.\u00a0 Finally,\u00a0 the\u00a0 LPS\u00a0 O\u00a0\n\nantigen\u00a0content\u00a0of\u00a0L.\u00a0 interrogans\u00a0differs\u00a0 in\u00a0an\u00a0acutely\u00a0\n\ninfected\u00a0versus\u00a0a\u00a0chronically\u00a0infected\u00a0animal.\u00a0\u00a0The\u00a0role\u00a0\n\nof\u00a0 O\u00a0 antigen\u00a0 changes\u00a0 in\u00a0 the\u00a0 establishment\u00a0 or\u00a0\n\nmaintenance\u00a0 of\u00a0 acute\u00a0 or\u00a0 chronic\u00a0 infection,\u00a0 if\u00a0 any,\u00a0 is\u00a0\n\nunknown\u00a0 (Zhang\u00a0 et\u00a0 al,.\u00a0 1992;\u00a0 Bharti\u00a0 et\u00a0 al.,\u00a0 2003).\u00a0\n\nLeptospires\u00a0 enter\u00a0 into\u00a0 the\u00a0 body\u00a0 of\u00a0 a\u00a0 susceptible\u00a0 host\u00a0\n\nthrough\u00a0mucous\u00a0membrane\u00a0or\u00a0abraded\u00a0skin.\u00a0\u00a0\u00a0After\u00a04\u00a0\n\nto\u00a010\u00a0days,\u00a0the\u00a0host\u00a0becomes\u00a0bacteraemic,\u00a0this\u00a0period\u00a0\n\nlasting\u00a0from\u00a0hours\u00a0to\u00a07\u00a0days,\u00a0and\u00a0may\u00a0be\u00a0characterised\u00a0\n\nby\u00a0pyrexia\u00a0and\u00a0anorexia\u00a0(Abdollahpour,\u00a01990).\u00a0\u00a0\n\nMaterials\u00a0and\u00a0Methods\u00a0\nLeptospira\u00a0 Serovars.\u00a0 A\u00a0 total\u00a0 of\u00a0 9\u00a0 Leptospira\u00a0 serovars\u00a0\n\nnamely,\u00a0 L.\u00a0 icterohaemorrhagiae,\u00a0 L.\u00a0 canicola,\u00a0 L.\u00a0\n\npamona,\u00a0 L.\u00a0 autumnalis,\u00a0 L.\u00a0 javanica,\u00a0 L.\u00a0 pyrogenes,\u00a0 L.\u00a0\n\naustralis,\u00a0 L.\u00a0 hardjo\u00a0 and\u00a0 L.\u00a0 inadai\u00a0 were\u00a0 procured\u00a0 from\u00a0\n\nthe\u00a0 repository\u00a0 of\u00a0 PD_ADMAS(Project\u00a0 Directorate\u00a0 on\u00a0\n\nAnimal\u00a0 Disease\u00a0 Monitoring\u00a0 and\u00a0 surveillance),\u00a0 UAS,\u00a0\n\nBangalore,\u00a0India.\u00a0\u00a0\n\nCultivation\u00a0 of\u00a0 Leptospira.\u00a0 Leptospira\u00a0 serovars\u00a0\n\nmentioned\u00a0above\u00a0were\u00a0grown\u00a0in\u00a0Luria\u2010Bertani\u00a0(LB)\u00a0agar\u00a0\n\n(Difco\u00a0Detroit,\u00a0MI,\u00a0USA)\u00a0supplemented\u00a0with\u00a01\u20103%\u00a0NaCl\u00a0\n\nat\u00a0370C\u00a0overnight.\u00a0\n\nDNA\u00a0 Preparation.\u00a0 Template\u00a0 Genomic\u00a0 DNA\u00a0 was\u00a0\n\nextracted/isolated\u00a0from\u00a0pure\u00a0culture\u00a0of\u00a0corresponding\u00a0\n\nLeptospires,\u00a0 using\u00a0 QIAamp\u00a0 DNA\u00a0 minikit\u00a0 (QIAGEN,\u00a0\n\nGermany)\u00a0and\u00a0other\u00a0alternative\u00a0method\u00a0described\u00a0by\u00a0\u00a0\u00a0\u00a0\n\nHoshino\u00a0 et\u00a0 al.,\u00a0 1998.\u00a0 For\u00a0 it\u00a0 \u00a0 freshly\u00a0 grown\u00a0 bacterial\u00a0\n\nmethod\u00a0for\u00a0diagnosis\u00a0of\u00a0leptospirosis,\u00a0has\u00a0been\u00a0so\u00a0far\u00a0\n\nby\u00a0 culture\u00a0 isolation\u00a0 but\u00a0 it\u00a0 is\u00a0 time\u00a0 consuming\u00a0 and\u00a0\n\npotentially\u00a0 hazardous.", "start_char_idx": 4759, "end_char_idx": 7032, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c9c3f336-86a8-4cb9-8430-516d6ea109c0": {"__data__": {"id_": "c9c3f336-86a8-4cb9-8430-516d6ea109c0", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5a45cd5f-56ce-454f-855e-6de1332651b0", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "8b44be91f9a71cbc8853b8c2b5c394319a1c579ab1da77bfe2351aa882eef8b2", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "cbfdeecb-1e03-4fbd-8a0d-aa1e197186cd", "node_type": "1", "metadata": {}, "hash": "77e95ee1e24ec241299a10a81ee02ea25a0cc2e2d8c9bd72a302ed9c0da05788", "class_name": "RelatedNodeInfo"}}, "text": "Another\u00a0 traditional\u00a0 method\u00a0 is\u00a0\n\nthe\u00a0detection\u00a0of\u00a0antibodies\u00a0(serological\u00a0tests)\u00a0which\u00a0is\u00a0\n\nalso\u00a0a\u00a0time\u00a0consuming\u00a0method\u00a0and\u00a0fails\u00a0to\u00a0identify\u00a0the\u00a0\n\ninfecting\u00a0 serovar.\u00a0 \u00a0 Classification\u00a0 and\u00a0 identification\u00a0 of\u00a0\n\nLeptospires\u00a0 using\u00a0 serology\u00a0 is\u00a0 a\u00a0 difficult\u00a0 and\u00a0 tedious\u00a0\n\nprocedure.\u00a0 \u00a0 Recently\u00a0 molecular\u00a0 biology\u00a0 techniques\u00a0\n\nhave\u00a0been\u00a0introduced\u00a0for\u00a0detection.\u00a0\u00a0These\u00a0techniques\u00a0\n\nare\u00a0predominantly\u00a0used\u00a0for\u00a0facilitating\u00a0the\u00a0study\u00a0of\u00a0the\u00a0\n\nepidemiology\u00a0 of\u00a0 leptospirosis.\u00a0 \u00a0 Genetic\u00a0 taxonomy\u00a0\n\ninvolves\u00a0 DNA/DNA\u00a0 hybridisation\u00a0 and\u00a0 GC\u00a0 content\u00a0 the\u00a0\n\nguanine\u2010plus\u2010cytosine\u00a0 mol\u00a0 percentages\u00a0 (G+C\u00a0 mol\u00a0 %)\u00a0\n\ncontent\u00a0of\u00a0DNA.\u00a0The\u00a0genotypic\u00a0classification\u00a0based\u00a0on\u00a0\n\nDNA\u00a0 hybridization\u00a0 defined\u00a0 21\u00a0 genome\u00a0 species\u00a0 of\u00a0\n\nLeptospira\u00a0 that\u00a0 include\u00a0 29\u00a0 serogroups\u00a0 and\u00a0 269\u00a0\n\nserovars.\u00a0 Yasuda\u00a0 et\u00a0 al.,\u00a0 1987\u00a0 proposed\u00a0 a\u00a0 new\u00a0\n\nclassification\u00a0 of\u00a0 Leptospira\u00a0 based\u00a0 on\u00a0 \u00a0 DNA\u00a0 homology\u00a0\n\nstudy\u00a0on\u00a046\u00a0pathogenic\u00a0and\u00a0non\u00a0pathogenic\u00a0serovars.\u00a0\n\nWoodward\u00a0 and\u00a0 Redstone,\u00a0 1994\u00a0 developed\u00a0 a\u00a0\n\npolymerase\u00a0 chain\u00a0 reaction\u00a0 (PCR)\u00a0 combined\u00a0 with\u00a0\n\nrestriction\u00a0 fragment\u00a0 length\u00a0 polymorphism\u00a0 which\u00a0 can\u00a0\n\nbe\u00a0used\u00a0for\u00a0differentiation\u00a0of\u00a0leptospira\u00a0serovars.\u00a0\u00a0It\u00a0is\u00a0\n\nsuggested\u00a0 that\u00a0 the\u00a0 PCR\u00a0 combined\u00a0 with\u00a0 restriction\u00a0\n\nfragment\u00a0length\u00a0polymorphism\u00a0is\u00a0useful\u00a0tool\u00a0for\u00a0rapid\u00a0\n\ndetection\u00a0 and\u00a0 preliminary\u00a0 differentiation\u00a0 of\u00a0\n\nLeptospires\u00a0(Levett\u00a0et\u00a0al.,\u00a02001).\u00a0\n\nAlthough\u00a0 over\u00a0 269\u00a0 serovars\u00a0 of\u00a0 Leptospira\u00a0 have\u00a0 been\u00a0\n\ndescribed,\u00a0 all\u00a0 members\u00a0 of\u00a0 the\u00a0 genus\u00a0 have\u00a0 similar\u00a0\n\nmorphology.\u00a0Leptospira\u00a0are\u00a0spiral\u2010shaped\u00a0bacteria\u00a0that\u00a0\n\nare\u00a06\u201020\u00a0\u03bcm\u00a0long\u00a0and\u00a00.1\u00a0\u03bcm\u00a0in\u00a0diameter.\u00a0\u00a0One\u00a0or\u00a0both\u00a0\n\nends\u00a0 of\u00a0 the\u00a0 spirochete\u00a0 are\u00a0 usually\u00a0 hooked.\u00a0 Because\u00a0\n\nthey\u00a0are\u00a0so\u00a0thin,\u00a0 live\u00a0Leptospira\u00a0are\u00a0best\u00a0observed\u00a0by\u00a0\n\ndark\u00a0field\u00a0microscopy.\u00a0The\u00a0bacteria\u00a0have\u00a0a\u00a0number\u00a0of\u00a0\n\nfreedom\u00a0degrees;\u00a0when\u00a0ready\u00a0to\u00a0proliferate\u00a0via\u00a0binary\u00a0\n\nfission,\u00a0the\u00a0bacterium\u00a0noticeably\u00a0bends\u00a0in\u00a0the\u00a0place\u00a0of\u00a0\n\nthe\u00a0future\u00a0split\u00a0(Levett\u00a0et\u00a0al.,\u00a02005).\u00a0Leptospira\u00a0have\u00a0a\u00a0\n\nGram\u2010negative\u2010like\u00a0 cell\u00a0 envelope\u00a0 consisting\u00a0 of\u00a0 a\u00a0\n\ncytoplasmic\u00a0 and\u00a0 outer\u00a0 membrane.\u00a0 However,\u00a0 the\u00a0\n\npeptidoglycan\u00a0layer\u00a0is\u00a0associated\u00a0with\u00a0the\u00a0cytoplasmic\u00a0\n\nrather\u00a0than\u00a0the\u00a0outer\u00a0membrane,\u00a0an\u00a0arrangement\u00a0that\u00a0\n\nis\u00a0unique\u00a0to\u00a0spirochetes.\u00a0The\u00a0two\u00a0flagella\u00a0of\u00a0Leptospira\u00a0\n\nextend\u00a0from\u00a0the\u00a0cytoplasmic\u00a0membrane\u00a0at\u00a0the\u00a0ends\u00a0of\u00a0\n\nthe\u00a0 bacteria\u00a0 into\u00a0 the\u00a0 periplasmic\u00a0 space\u00a0 and\u00a0 are\u00a0\n\nnecessary\u00a0 for\u00a0 the\u00a0 motility\u00a0 of\u00a0 Leptospira.\u00a0 The\u00a0 outer\u00a0\n\nmembrane\u00a0 contains\u00a0 a\u00a0 variety\u00a0 of\u00a0 lipoproteins\u00a0 and\u00a0\n\ntransmembrane\u00a0 outer\u00a0 membrane\u00a0 proteins.", "start_char_idx": 7036, "end_char_idx": 9537, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "cbfdeecb-1e03-4fbd-8a0d-aa1e197186cd": {"__data__": {"id_": "cbfdeecb-1e03-4fbd-8a0d-aa1e197186cd", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c9c3f336-86a8-4cb9-8430-516d6ea109c0", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "2acb9221ffa94d337f1ea00456a42c9f609f7325bdedb7f0ebb0b43b00472abc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "467fc5e9-e831-49da-a2ed-a8132987bdb4", "node_type": "1", "metadata": {}, "hash": "d23f18f9e93ea9c663563631884477e48a42d32bf380425891268f779e84e117", "class_name": "RelatedNodeInfo"}}, "text": "As\u00a0\n\nexpected,\u00a0 the\u00a0 protein\u00a0 composition\u00a0 of\u00a0 the\u00a0 outer\u00a0\n\nmembrane\u00a0differs\u00a0when\u00a0comparing\u00a0Leptospira\u00a0growing\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n24\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nstep\u00a0at\u00a0720C\u00a0for\u00a01\u00a0min\u00a0and\u00a0a\u00a0final\u00a0elongation\u00a0at\u00a0720C\u00a0for\u00a0\n\n5\u00a0min.\u00a0Finally\u00a0annealing\u00a0temperature\u00a0was\u00a0standardized\u00a0\n\nto\u00a0600C\u00a0for\u00a01\u00a0min\u00a0(Fig.\u00a01a).\u00a0\n\n\u00a0The\u00a0 PCR\u00a0 standardization\u00a0 using\u00a0 primers\u00a0 P30/31\u00a0 was\u00a0\n\ninitiated\u00a0in\u00a0a\u00a025\u00a0ml\u00a0reaction\u00a0volume\u00a0containing\u00a010x\u00a0Taq\u00a0\n\nbuffer\u00a0 with\u00a0 KCl\u00a0 (100mM\u00a0 Tris\u00a0 HCl,\u00a0 50mM\u00a0 KCl,\u00a0 0.8%\n\nNonidet\u00a0P40),\u00a01.5mM\u00a0MgCl2,\u00a02.5mM\u00a0of\u00a0each\u00a0dNTPs,\u00a02U\u00a0\n\nof\u00a0 Taq\u00a0 polymerase,\u00a0 25pmoles\u00a0 of\u00a0 each\u00a0 primer\u00a0 and\u00a0\n\n0.25ml\u00a0of\u00a0DNA\u00a0(40ng/ml)\u00a0extract\u00a0from\u00a0L.\u00a0javanica.\u00a0The\u00a0\n\ntest\u00a0 was\u00a0 done\u00a0 for\u00a0 several\u00a0 combinations\u00a0 annealing\u00a0\n\ntemperatures\u00a0from\u00a0550C\u00a0to\u00a0650C.\u00a0The\u00a0reaction\u00a0started\u00a0\n\nwith\u00a0an\u00a0initial\u00a0denaturation\u00a0temperature\u00a0of\u00a0950C\u00a0for\u00a05\u00a0\n\nmin\u00a0followed\u00a035\u00a0cycles\u00a0of\u00a0denaturing\u00a0at\u00a0950C\u00a0for\u00a01\u00a0min,\u00a0\n\nannealing\u00a0at\u00a0600C\u00a0for\u00a01\u00a0min\u00a0at\u00a0G\u00a0\u00b1\u00a050C,\u00a0elongation\u00a0step\u00a0\n\nat\u00a0720C\u00a0for\u00a045\u00a0sec\u00a0and\u00a0a\u00a0final\u00a0elongation\u00a0at\u00a0720C\u00a0for\u00a05\u00a0\n\nmin.\u00a0 Finally\u00a0 annealing\u00a0 temperature\u00a0 was\u00a0 standardized\u00a0\n\nto\u00a0 600C\u00a0 for\u00a0 1\u00a0 min\u00a0 (Fig.\u00a0 1b).\u00a0 \u00a0 Optimized\u00a0 annealing\u00a0\n\ntemperature\u00a0details\u00a0shown\u00a0in\u00a0table\u00a0(Table\u00a02).\u00a0\n\nAgarose\u00a0 gel\u00a0 electrophoresis.\u00a0 A\u00a0 15\u00a0 ml\u00a0 aliquot\u00a0 of\u00a0 each\u00a0\n\nPCR\u00a0 product\u00a0 was\u00a0 mixed\u00a0 with\u00a0 3\u00b5l\u00a0 of\u00a0 6x\u00a0 loading\u00a0 dye\u00a0\n\n(Fermentas)\u00a0and\u00a0loaded\u00a0on\u00a0to\u00a0the\u00a0wells\u00a0of\u00a02%\u00a0agarose\u00a0\n\ngel\u00a0in\u00a0TAE\u00a0buffer.\u00a0It\u00a0was\u00a0run\u00a0at\u00a080V\u00a0for\u00a0about\u00a045\u00a0min.\u00a0\n\nDNA\u00a0bands\u00a0were\u00a0visualized\u00a0under\u00a0UV\u2010Trans\u00a0illuminator\u00a0\n\nand\u00a0documented\u00a0using\u00a0gel\u00a0documentation\u00a0system\u00a0with\u00a0\n\nQuantity\u00a0 one\u00a0 software\u00a0 (Biorad,\u00a0 U.S.A)\u00a0 and\u00a0 the\u00a0 PCR\u00a0\n\nproduct\u00a0 was\u00a0 eluted\u00a0 using\u00a0 eppendorf\u00a0 QIAquick\u00a0 gel\u00a0\n\nextraction\u00a0kit\u00a0(QIAGEN,\u00a0Germany).\u00a0\n\ncolonies\u00a0were\u00a0suspended\u00a0in\u00a0500\u00a0\u00b5l\u00a0TE\u00a0buffer\u00a0or\u00a050\u00b5l\u00a0of\u00a0\n\nmid\u2010log\u00a0phase\u00a0bacterial\u00a0culture\u00a0was\u00a0added\u00a0to\u00a0450\u00b5l\u00a0of\u00a0\n\nTE\u00a0buffer,\u00a0then\u00a0the\u00a0cell\u00a0suspension\u00a0was\u00a0boiled\u00a0for\u00a010\u00a0\n\nmin\u00a0 followed\u00a0 by\u00a0 quick\u00a0 cooling\u00a0 on\u00a0 ice\u00a0 for\u00a0 5\u00a0 min,\u00a0 and\u00a0\n\ncentrifuged\u00a0 at\u00a0 12,800\u00a0 g\u00a0 for\u00a0 5\u00a0 min.\u00a0 \u00a0 The\u00a0 supernatant\u00a0\n\nwas\u00a0collected\u00a0and\u00a0stored\u00a0at\u00a0\u2010300C\u00a0until\u00a0use.\u00a0\n\nPrimer\u00a0 Design.", "start_char_idx": 9539, "end_char_idx": 11634, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "467fc5e9-e831-49da-a2ed-a8132987bdb4": {"__data__": {"id_": "467fc5e9-e831-49da-a2ed-a8132987bdb4", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "cbfdeecb-1e03-4fbd-8a0d-aa1e197186cd", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "8909623c4e0dd7385b92b0abedba84796cd320bbeab657bf1dd870f697bc38cf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "32550e4c-3ec4-4765-a3f0-702d0e5c8815", "node_type": "1", "metadata": {}, "hash": "eba495ee753b3ca8763d22df4b225ddd03b5eb45d1fd32f4d624cb467761ce70", "class_name": "RelatedNodeInfo"}}, "text": "Primer\u00a0 Design.\u00a0 All\u00a0 available\u00a0 sequences\u00a0 of\u00a0 the\u00a0 target\u00a0\n\ngenes\u00a0 among\u00a0 Leptospira\u00a0 spp.\u00a0 were\u00a0 downloaded\u00a0 from\u00a0\n\nthe\u00a0 GenBank\u00a0 (http://www.ncbi.nlm.nih.gov/GenBank/\n\nGenBankSearch.html).\u00a0PerlPrimer\u00a0software\u00a0by\u00a0Marshall\u00a0\n\net\u00a0al.,\u00a0(2004)\u00a0was\u00a0used\u00a0to\u00a0design\u00a0the\u00a0primers.\u00a0Sequence\u00a0\n\nfor\u00a0 LipL21\u00a0 holding\u00a0 accession\u00a0 number\u00a0 AY688426\u00a0 and\u00a0\n\nlipL41\u00a0 sequence\u00a0 of\u00a0 Leptospira\u00a0 borgpetersenii\u00a0 serovar\u00a0\n\nHardjo\u2010bovis\u00a0 holding\u00a0 accession\u00a0 number\u00a0 CP000348.1\u00a0\n\nwas\u00a0retrieved\u00a0from\u00a0NCBI\u2010Genbank.\u00a0\u00a0It\u00a0was\u00a0then\u00a0used\u00a0to\u00a0\n\nget\u00a0 conserved\u00a0 sequences.\u00a0 The\u00a0 conserved\u00a0 part\u00a0 of\u00a0 the\u00a0\n\nnucleotide\u00a0sequence\u00a0was\u00a0retrieved\u00a0and\u00a0used\u00a0for\u00a0primer\u00a0\n\ndesigning.\u00a0The\u00a0criteria\u00a0that\u00a0was\u00a0set\u00a0for\u00a0primer\u00a0design\u00a0\n\nwas\u00a0primer\u00a0length\u00a0is\u00a020\u00b12\u00a0Target\u00a0temperature\u00a0is\u00a055\u00b11\u00b0\n\nC.\u00a0 \u00a0 The\u00a0 overall\u00a0 theoretical\u00a0 specificities\u00a0 of\u00a0 the\u00a0 newly\u00a0\n\ndesigned\u00a0 primers\u00a0 were\u00a0 checked\u00a0 using\u00a0 BLAST\u00a0 search.\u00a0\u00a0\n\nDetails\u00a0 of\u00a0 the\u00a0 primers\u2019\u00a0 sequences,\u00a0 their\u00a0 expected\u00a0\n\namplicons\u00a0sizes\u00a0after\u00a0PCR,\u00a0etc.\u00a0are\u00a0summarized\u00a0in\u00a0table\u00a0\n\n(Table\u00a01).\u00a0\n\nStandardization\u00a0 of\u00a0 polymerase\u00a0 chain\u00a0 reaction\u00a0 (PCR)\u00a0\n\nprotocol.\u00a0In\u00a0order\u00a0to\u00a0achieve\u00a0the\u00a0best\u00a0sensitivity\u00a0for\u00a0the\u00a0\n\nPCR\u00a0 using\u00a0 primer\u00a0 P28/29\u00a0 several\u00a0 combinations\u00a0 of\u00a0\n\nannealing\u00a0 temperatures\u00a0 from\u00a0 580C\u00a0 to\u00a0 680C\u00a0 were\u00a0\n\ntested.\u00a0The\u00a0following\u00a0conditions\u00a0were\u00a0chosen:\u00a0PCR\u00a0was\u00a0\n\nperformed\u00a0 in\u00a0 25\u00a0 ml\u00a0 reaction\u00a0 volume\u00a0 containing\u00a0 10x\u00a0\n\nTaq\u00a0buffer\u00a0with\u00a0KCl\u00a0(100mM\u00a0Tris\u00a0HCl,\u00a050mM\u00a0KCl,\u00a00.8%\u00a0\n\nNonidet\u00a0P40),\u00a01.5mM\u00a0MgCl2,\u00a02.5mM\u00a0of\u00a0each\u00a0dNTPs,\u00a02U\u00a0\n\nof\u00a0 Taq\u00a0 polymerase,\u00a0 25pmoles\u00a0 of\u00a0 each\u00a0 primer\u00a0 and\u00a0\n\n0.25ml\u00a0 of\u00a0 DNA\u00a0 (40ng/ml)\u00a0 extract\u00a0 from\u00a0 L.\u00a0\n\nicterohaemorrhagiae.\u00a0 PCR\u00a0 chemicals\u00a0 were\u00a0 procured\u00a0\n\nfrom\u00a0Fermantas.\u00a0The\u00a0reactions\u00a0were\u00a0carried\u00a0out\u00a0in\u00a0an\u00a0\n\nEppendorf\u00a0thermocycler.\u00a0Initially,\u00a0PCR\u00a0conditions\u00a0were\u00a0\n\noptimized\u00a0 by\u00a0gradient\u00a0 PCR.\u00a0 The\u00a0 reaction\u00a0 started\u00a0 with\u00a0\n\nan\u00a0 initial\u00a0 polymerase\u00a0 activating\u00a0 temperature\u00a0 of\u00a0 950C\u00a0\n\nfor\u00a05\u00a0min\u00a0followed\u00a035\u00a0cycles\u00a0of\u00a0denaturing\u00a0at\u00a0950C\u00a0for\u00a01\u00a0\n\nmin,\u00a0annealing\u00a0at\u00a0630C\u00a0for\u00a01\u00a0min\u00a0at\u00a0G\u00a0\u00b1\u00a050C,\u00a0elongation\u00a0\n\nPrimer\u00a0 Target\u00a0species/gene\u00a0 Sequence(5\u2019\u20103\u2019)\u00a0 Amplicon\u00a0Length\u00a0\nReference\u00a0(Accession\u00a0\n\nno.)", "start_char_idx": 11619, "end_char_idx": 13664, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "32550e4c-3ec4-4765-a3f0-702d0e5c8815": {"__data__": {"id_": "32550e4c-3ec4-4765-a3f0-702d0e5c8815", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "467fc5e9-e831-49da-a2ed-a8132987bdb4", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "a27cfeacf29795bd2c0017f5d2a8c3804d39003c47c82d3c616f8a3fe49f8c19", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "3cc17280-7f9e-4eb2-8959-dbcdb88150b2", "node_type": "1", "metadata": {}, "hash": "3139c0e2170569ca3de156d0f8f8c1ff3d13bacbcf316dcfeb8df6595e3c085d", "class_name": "RelatedNodeInfo"}}, "text": "P28/F\u00a0 Leptospira/LipL21\u00a0 CCAGCACTGACACCGGACAAA\u00a0 385bp\u00a0 AY688426\u00a0\nP29/R\u00a0 Leptospira/LipL21\u00a0 CCGGAACCAACCGCTTTACAT\u00a0 385bp\u00a0 AY688426\u00a0\nP30/F\u00a0 Leptospira/LipL41\u00a0 GACCTCAGTAAACGCGCCGATAT\u00a0 427bp\u00a0 CP000348.1\u00a0\nP31/R\u00a0 Leptospira/LipL41\u00a0 CAGCGGCTTCGTCCAATCCT\u00a0 427bp\u00a0 CP000348.1\u00a0\n\nTable\u00a01.\u00a0Details\u00a0of\u00a0the\u00a0newly\u00a0designed\u00a0primers\u00a0for\u00a0specific\u00a0identification\u00a0\n\n\u00a0\u00a0 PCR\u00a0reaction\u00a0mixture(25ml)\u00a0\n\n\u00a0\u00a0\n\nP28\u00a0/P29\u00a0\n\n\u00a0\u00a0\n\nP30/P31\u00a0\n\n\u00a0\u00a0\n\n10X\u00a0buffer\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.50ml\u00a0\n\nMgCl2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a01.50ml\u00a0\n\ndNTPs\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.00ml\u00a0\n\nPrimer\u2010Forward\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.50ml\u00a0\n\nPrimer\u2010Reverse\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.50ml\u00a0\n\nTemplate\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.25ml\u00a0\n\nTaq\u00a0Pol\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.00ml\u00a0\n\nNuclease\u00a0\u00a0\u00a0free\u00a0water\u00a0\u00a0\u00a0\u00a0\u00a015.75\u00b5l\u00a0\n\nTable\u00a02.\u00a0Details\u00a0of\u00a0Standardized\u00a0thermal\u00a0cycling\u00a0\ncondition \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n25\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nmin.\u00a0Immediately\u00a0mixture\u00a0\u00a0was\u00a0kept\u00a0over\u00a0ice\u00a0for\u00a02min.\u00a0\u00a0\n\n0.8ml\u00a0of\u00a0LB\u00a0broth\u00a0was\u00a0then\u00a0added\u00a0to\u00a0it\u00a0and\u00a0incubated\u00a0\n\nat\u00a0 37\u00b0C\u00a0 for\u00a0 90\u00a0 min\u00a0 in\u00a0 an\u00a0 orbital\u00a0 shaker.\u00a0 \u00a0 It\u00a0 was\u00a0 then\u00a0\n\ncentrifuged\u00a0at\u00a04000\u00a0rpm\u00a0for\u00a010\u00a0min\u00a0and\u00a0the\u00a0pellet\u00a0was\u00a0\n\nplated\u00a0on\u00a0LB\u00a0agar\u00a0plate\u00a0containing\u00a0ampicillin\u00a0(50mg/ml)\u00a0\n\nand\u00a0 40ml\u00a0 of\u00a0 2%\u00a0 X\u2010gal\u00a0 (5\u2010bromo\u20104\u2010chloro\u20103\u2010indolyl\u2010b\u2010\n\ngalactopyranoside)\u00a0and\u00a08ml\u00a0of\u00a02%\u00a0IPTG\u00a0(Isopropyl\u2010b\u2010D\u2013\n\nthiogalactopyranoside).\u00a0 The\u00a0 plates\u00a0 were\u00a0 then\u00a0\n\nincubated\u00a0over\u00a0night\u00a0at\u00a037\u00b0C.\u00a0\n\nWell\u2010isolated\u00a0 white\u00a0 colonies\u00a0 (Fig.\u00a0 4)\u00a0 were\u00a0 picked\u00a0 and\u00a0\n\ntransferred\u00a0 to\u00a0 5ml\u00a0 of\u00a0 LB\u00a0 broth\u00a0 containing\u00a0 5ml\u00a0 of\u00a0\n\nampicillin\u00a0(50mg/ml).\u00a0\u00a0The\u00a0tubes\u00a0were\u00a0then\u00a0incubated\u00a0\n\nat\u00a0 37\u00b0C\u00a0 in\u00a0 the\u00a0 shaker\u00a0 for\u00a0 overnight.\u00a0 Plasmid\u00a0 DNA\u00a0\n\nisolation\u00a0and\u00a0purification\u00a0was\u00a0done\u00a0using\u00a0QIAprep\u00a0spin\u00a0\n\nminiprep\u00a0kit\u00a0(Qiagen,\u00a0Germany)\u00a0and\u00a0microcentrifuge.\u00a0\n\nCharacterization\u00a0 of\u00a0 Recombinants.\u00a0 The\u00a0 plasmids\u00a0\n\nisolated\u00a0 from\u00a0 the\u00a0 white\u00a0 colonies\u00a0 were\u00a0 further\u00a0\n\ncharacterized\u00a0by\u00a0the\u00a0insert\u00a0release\u00a0after\u00a0digestion\u00a0with\u00a0\n\nEcoRI\u00a0 (G\u00a0 \u02c9AATTC)\u00a0 restriction\u00a0 enzyme.\u00a0 The\u00a0 enzyme\u00a0\n\nEcoRI\u00a0was\u00a0used\u00a0to\u00a0cleave\u00a0and\u00a0release\u00a0the\u00a0insert\u00a0in\u00a0the\u00a0\n\nplasmid\u00a0 (Fig.\u00a0 5a\u00a0 and\u00a0 5b).", "start_char_idx": 13666, "end_char_idx": 15834, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "3cc17280-7f9e-4eb2-8959-dbcdb88150b2": {"__data__": {"id_": "3cc17280-7f9e-4eb2-8959-dbcdb88150b2", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "32550e4c-3ec4-4765-a3f0-702d0e5c8815", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "2b4082d82cbb16a6c6c7b8ab7931a8ddf7d99a8cad4ac2c8b08798f31336b7ee", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "835495a5-1c8d-4e9e-8b6b-e4b1614482c0", "node_type": "1", "metadata": {}, "hash": "8a606834d3b61d3a6d896a9084acef7946c3ea29ef5d805b404c0c8869dc4c1e", "class_name": "RelatedNodeInfo"}}, "text": "5a\u00a0 and\u00a0 5b).\u00a0 The\u00a0 reaction\u00a0 mixtures\u00a0 were\u00a0\n\nprepared\u00a0as\u00a0per\u00a0the\u00a0table\u00a0below\u00a0and\u00a0were\u00a0subjected\u00a0to\u00a0\n\ndigestion\u00a0at\u00a037\u00b0C\u00a0in\u00a0a\u00a0water\u00a0bath\u00a0for\u00a01h.\u00a0\u00a0Details\u00a0of\u00a0the\u00a0\n\nreaction\u00a0mixture\u00a0are\u00a0summarized\u00a0in\u00a0table\u00a0(Table\u00a05).\u00a0\n\nPCR\u00a0 for\u00a0 Confirmation.\u00a0 The\u00a0 PCR\u00a0 were\u00a0 carried\u00a0 out\u00a0 for\u00a0\n\nconfirmation\u00a0of\u00a0the\u00a0recombinants\u00a0using\u00a0primer\u00a0P28/29\u00a0\n\nand\u00a0 P30/31\u00a0 under\u00a0 the\u00a0 standardized\u00a0 conditions\u00a0 as\u00a0\n\ndescribed\u00a0 previously\u00a0 using\u00a0 the\u00a0 plasmid\u00a0 DNA\u00a0 as\u00a0\n\ntemplate.\u00a0It\u00a0was\u00a0performed\u00a0in\u00a0a\u00a025\u00a0ml\u00a0reaction\u00a0volume\u00a0\n\ncontaining\u00a0 10x\u00a0 buffer\u00a0 (100mM\u00a0 Tris\u00a0 HCl,\u00a0 50mM\u00a0 KCl,\u00a0\n\n0.8%Nonidet\u00a0 P40),\u00a0 1.5mM\u00a0 MgCl2,\u00a0 2.5mM\u00a0 of\u00a0 each\u00a0\n\ndNTPs,\u00a02U\u00a0of\u00a0Taq\u00a0polymerase,\u00a025pmoles\u00a0each\u00a0primer\u00a0\n\nand\u00a01ml\u00a0of\u00a0template\u00a0(1:10\u00a0diluted).\u00a0\u00a0\n\n\u00a0\u00a05ml\u00a0 aliquot\u00a0 of\u00a0 PCR\u00a0 products\u00a0 mixed\u00a0 with\u00a0 1\u00b5l\u00a0 of\u00a0 6x\u00a0\n\nloading\u00a0dye\u00a0(Fermentas)\u00a0and\u00a0loaded\u00a0on\u00a0to\u00a0the\u00a0wells\u00a0of\u00a0\n\n2.5%\u00a0agarose\u00a0gel.\u00a0It\u00a0was\u00a0run\u00a0at\u00a080V\u00a0for\u00a0about\u00a045\u00a0min.\u00a0\n\nDNA\u00a0bands\u00a0were\u00a0visualized\u00a0under\u00a0UV\u2010Transilluminator\u00a0\n\nand\u00a0 documented\u00a0 using\u00a0 Biorad\u00a0 gel\u00a0 documentation\u00a0\n\nsystem\u00a0with\u00a0Quantity\u00a0one\u00a0software\u00a0(Fig.\u00a06a\u00a0and\u00a06b).\u00a0\u00a0\n\nCharacterization\u00a0 of\u00a0 amplicons.\u00a0 The\u00a0 PCR\u00a0 product\u00a0 of\u00a0\n\nprimer\u00a0 P28/29\u00a0 and\u00a0 primer\u00a0 P30/31\u00a0 were\u00a0 subjected\u00a0 to\u00a0\n\nRE\u00a0 digestion.\u00a0 The\u00a0 enzymes\u00a0 RsaI(GT\u02c9AC)\u00a0 and\u00a0 HindIII\n\n(A\u02c9AGCTT)\u00a0 were\u00a0 used\u00a0 to\u00a0 characterize\u00a0 PCR\u00a0 product\u00a0 of\u00a0\n\nP28/29\u00a0 (Fig.\u00a0 2a)\u00a0 and\u00a0 enzymes\u00a0 ClaI(AT\u02c9CGAT),\u00a0 TaqI\n\n(T\u02c9CGA)\u00a0 and\u00a0 RsaI(GT\u02c9AC)\u00a0 were\u00a0 used\u00a0 to\u00a0 characterize\u00a0\n\nPCR\u00a0product\u00a0of\u00a0P30/31\u00a0(Fig.\u00a02b).\u00a0 \u00a0The\u00a0reactions\u00a0were\u00a0\n\nset\u00a0as\u00a0under\u00a0and\u00a0incubated\u00a0at\u00a037\u00b0C\u00a0in\u00a0a\u00a0water\u00a0bath\u00a0for\u00a0\n\n4h.\u00a0 Restriction\u00a0 enzymes\u00a0 and\u00a0 its\u00a0 corresponding\u00a0 buffer\u00a0\n\nwere\u00a0 procured\u00a0 from\u00a0 Fermentas.\u00a0 \u00a0 ClaI\u00a0 and\u00a0 its\u00a0\n\ncorresponding\u00a0 buffer\u00a0 were\u00a0 procured\u00a0 from\u00a0 Promega.\u00a0\u00a0\n\nDetails\u00a0 of\u00a0 the\u00a0 reaction\u00a0 mixture\u00a0 are\u00a0 summarized\u00a0 in\u00a0\n\nTable\u00a03.\u00a0\u00a0\n\nA\u00a015\u00a0ml\u00a0aliquot\u00a0of\u00a0each\u00a0digested\u00a0products\u00a0were\u00a0mixed\u00a0\n\nwith\u00a03\u00b5l\u00a0of\u00a06x\u00a0loading\u00a0dye\u00a0(Fermentas)\u00a0and\u00a0loaded\u00a0on\u00a0\n\nto\u00a0the\u00a0wells\u00a0of\u00a02%\u00a0agarose\u00a0gel\u00a0in\u00a0TAE\u00a0buffer.\u00a0It\u00a0was\u00a0run\u00a0\n\nat\u00a0 80V\u00a0 for\u00a0 about\u00a0 45\u00a0 min.\u00a0 DNA\u00a0 bands\u00a0 were\u00a0 visualized\u00a0\n\nunder\u00a0UV\u2010Transi\u00a0lluminator\u00a0and\u00a0documented\u00a0using\u00a0gel\u00a0\n\ndocumentation\u00a0 system\u00a0 with\u00a0 Quantity\u00a0 one\u00a0 software\n\n(Biorad,\u00a0U.S.A)\u00a0(Fig.\u00a02a\u00a0and\u00a02b).\u00a0\n\nSpecificity\u00a0 the\u00a0 PCR\u00a0 Assay.", "start_char_idx": 15821, "end_char_idx": 17965, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "835495a5-1c8d-4e9e-8b6b-e4b1614482c0": {"__data__": {"id_": "835495a5-1c8d-4e9e-8b6b-e4b1614482c0", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "3cc17280-7f9e-4eb2-8959-dbcdb88150b2", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "4677da8cd1514d35592a91e37d8057cc4b06f411e47950759a41f734db41db72", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "27e3cf05-20f3-4a01-b1e0-36c31750b03b", "node_type": "1", "metadata": {}, "hash": "bc0a2edb39917b2e50de5aeda3e757c3a7db258017bb3bd1828f534b65c34068", "class_name": "RelatedNodeInfo"}}, "text": "Specificity\u00a0 the\u00a0 PCR\u00a0 Assay.\u00a0 The\u00a0 specificity\u00a0 of\u00a0 both\u00a0 the\u00a0\n\nassay\u00a0developed\u00a0was\u00a0tested\u00a0by\u00a0taking\u00a010\u201020ng\u00a0of\u00a0each\u00a0\n\ntemplate\u00a0DNA\u00a0isolated\u00a0from\u00a0the\u00a0different\u00a0serovars\u00a0and\u00a0\n\nthe\u00a0 PCR\u00a0 was\u00a0 carried\u00a0 out\u00a0 under\u00a0 the\u00a0 standardized\u00a0\n\nconditions\u00a0as\u00a0shown\u00a0in\u00a0table\u00a0(Table\u00a04).\u00a0\n\nA\u00a010ml\u00a0aliquot\u00a0of\u00a0each\u00a0PCR\u00a0product\u00a0mixed\u00a0with\u00a02ml\u00a0of\u00a0\n\n6x\u00a0loading\u00a0dye\u00a0(Fermentas)\u00a0was\u00a0run\u00a0on\u00a0a\u00a02.5%\u00a0agarose\u00a0\n\nelectrophoretic\u00a0gel\u00a0in\u00a0TAE\u00a0buffer\u00a0and\u00a0DNA\u00a0bands\u00a0were\u00a0\n\nvisualized\u00a0under\u00a0UV\u2010Transilluminator\u00a0and\u00a0documented\u00a0\n\nusing\u00a0 gel\u00a0 documentation\u00a0 system\u00a0 with\u00a0 Quantity\u00a0 one\u00a0\n\nsoftware\u00a0(Biorad,\u00a0U.S.A)\u00a0(Fig.\u00a03a\u00a0and\u00a03b).\u00a0\n\nTransformation.\u00a0 Ligation\u00a0 mixture\u00a0 was\u00a0 prepared\u00a0 by\u00a0\n\nadding\u00a01ml\u00a0of\u00a0pGEMT\u00a0Easy\u00a0(50ng)\u00a0vector\u00a0at\u00a0the\u00a0rate\u00a0of\u00a0\n\nand\u00a01ml\u00a0of\u00a0T4\u00a0DNA\u00a0ligase\u00a0(3U/ml)\u00a0to\u00a05ml\u00a0of\u00a02x\u00a0buffer.\u00a0\n\n5ml\u00a0 of\u00a0 DNA\u00a0 (50ng/ml)\u00a0 was\u00a0 added\u00a0 to\u00a0 this\u00a0 mixture\u00a0 for\u00a0\n\nligation.\u00a0Ligation\u00a0was\u00a0done\u00a0at\u00a04\u00b0C\u00a0for\u00a0overnight.\u00a0\n\nLigation\u00a0 mixture\u00a0 was\u00a0 added\u00a0 to\u00a0 ice\u00a0 cold\u00a0 200ml\u00a0 of\u00a0\n\ncompetent\u00a0cells\u00a0and\u00a0tapped\u00a0gently,\u00a0it\u00a0was\u00a0incubated\u00a0on\u00a0\n\nice\u00a0for\u00a030\u00a0min.,\u00a0then\u00a0Heat\u00a0shock\u00a0was\u00a0given\u00a0at\u00a042\u00b0C\u00a0for\u00a01\u00a0\n\nReaction\u00a0\nMixture\u00a0\n\nLipL21\u00a0 LipL41\u00a0\nRsaI\u00a0 HindIII\u00a0 ClaI\u00a0 TaqI\u00a0 RsaI\u00a0\n\nBuffer\u00a0\n\nEnzyme\u00a0\n\nDNA\u00a0\n\nWater\u00a0\n\nBSA\u00a0\n\n2.0\u00a0ml\u00a0\n\n1.0\u00a0ml\u00a0\n\n8.0\u00a0\u00a0ml\u00a0\n\n11.0\u00a0ml\u00a0\n\n\u2010\u00a0\n\n2.0\u00a0ml\u00a0\n\n1.0\u00a0ml\u00a0\n\n8.0\u00a0ml\u00a0\n\n11.0\u00a0ml\u00a0\n\n\u2010\u2010\u2010\u2010\u2010\u2010\u00a0\n\n2.0ml\u00a0\n\n1.0ml\u00a0\n\n5.0ml\u00a0\n\n10.0ml\u00a0\n\n2.0ml\u00a0\n\n2.0ml\u00a0\n\n1.0ml\u00a0\n\n5.0ml\u00a0\n\n12.0ml\u00a0\n\n\u2010\u00a0\n\n2.0ml\u00a0\n\n1.0ml\u00a0\n\n5.0ml\u00a0\n\n12.0ml\u00a0\n\n\u2010\u00a0\n\nTable\u00a03.\u00a0Details\u00a0of\u00a0the\u00a0Restriction\u00a0Enzyme\u00a0\ndigestion\u00a0reaction\u00a0of\u00a0LipL21\u00a0and\u00a0LipL41 \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n26\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nout\u00a0 reactions\u00a0 at\u00a0 various\u00a0 annealing\u00a0 temperatures\u00a0\n\nranging\u00a0from\u00a058\u00b0C\u00a0to\u00a068\u00b0C\u00a0on\u00a0L.\u00a0 icterohaemorrhagiae\u00a0\n\nserovar.\u00a0This\u00a0showed\u00a0non\u2010specific\u00a0amplification\u00a0at\u00a058\u00b0\n\nC,\u00a0 58.7\u00b0C,\u00a0 60.8\u00b0C\u00a0 and\u00a0 63.5\u00b0C.\u00a0 So\u00a0 PCR\u00a0 reaction\u00a0 using\u00a0\n\nP30/31\u00a0was\u00a0carried\u00a0out\u00a0on\u00a0L.javanica\u00a0template\u00a0at\u00a0two\u00a0\n\ndifferent\u00a0MgCl2\u00a0concentration\u00a0i.e.\u00a0(0.5,\u00a01.0\u00b5l).", "start_char_idx": 17936, "end_char_idx": 19850, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "27e3cf05-20f3-4a01-b1e0-36c31750b03b": {"__data__": {"id_": "27e3cf05-20f3-4a01-b1e0-36c31750b03b", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "835495a5-1c8d-4e9e-8b6b-e4b1614482c0", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "8fcfc652c6cb03def4d43459a37fa5b808e3dca6cc4660212626774236cd8a51", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1666ebd1-4825-4f41-8aa3-556dd0a3d5d9", "node_type": "1", "metadata": {}, "hash": "f0611407c354d86079a1b839dd1fdc0a160a4c753ee5ddfd4c3b561e22304fcf", "class_name": "RelatedNodeInfo"}}, "text": "No\u00a0bands\u00a0\n\ncould\u00a0be\u00a0detected\u00a0for\u00a0PCR\u00a0amplification\u00a0at\u00a00.5\u00a0\u00b5l\u00a0MgCl2\u00a0\nbut\u00a0 specific\u00a0 band\u00a0 of\u00a0 430bp\u00a0 was\u00a0 observed\u00a0 at\u00a0 1.0\u00a0 \u00b5l\u00a0\n\nMgCl2.\u00a0 \u00a0 Standardization\u00a0 was\u00a0 done\u00a0 by\u00a0 carrying\u00a0 out\u00a0\n\nreactions\u00a0at\u00a0various\u00a0annealing\u00a0temperatures\u00a0from\u00a055\u00b0\u00a0\n\nto\u00a0 65\u00b0C\u00a0 for\u00a0 1\u00a0 min.\u00a0 Good\u00a0 amplification\u00a0 bands\u00a0 were\u00a0\n\nobserved\u00a0 for\u00a0 PCR\u00a0 reactions\u00a0 carried\u00a0 out\u00a0 at\u00a0 annealing\u00a0\n\ntemperatures\u00a0 60.5\u00b0C,\u00a0 61.8\u00b0C,\u00a0 63.1\u00b0C,\u00a0 64.2\u00b0C,\u00a0 65.0\u00b0C\u00a0\n\nand\u00a065.5\u00b0C\u00a0(Fig.\u00a01b).\u00a0\n\nThe\u00a0427bp\u00a0amplicons\u00a0thus\u00a0amplified\u00a0was\u00a0subjected\u00a0to\u00a0\n\nRE.\u00a0 The\u00a0 restriction\u00a0 enzyme\u00a0 pattern\u00a0 of\u00a0 this\u00a0 amplicon\u00a0\n\nconfirms\u00a0 it\u00a0 to\u00a0 be\u00a0 a\u00a0 lipL41\u00a0 partial\u00a0 gene\u00a0 of\u00a0 leptospira\u00a0\n\ngenus\u00a0(Fig.\u00a02b).\u00a0\n\nThe\u00a0 specificity\u00a0 of\u00a0 this\u00a0 primer\u00a0 was\u00a0 tested\u00a0 on\u00a0 nine\u00a0\n\ndifferent\u00a0serovars\u00a0under\u00a0optimized\u00a0conditions.\u00a0The\u00a0PCR\u00a0\n\nassay\u00a0showed\u00a0nonspecific\u00a0amplification\u00a0along\u00a0with\u00a0the\u00a0\n\nspecific\u00a0 band.\u00a0 Primer\u00a0 degradation\u00a0 was\u00a0 suspected\u00a0 and\u00a0\n\nthe\u00a0 PCR\u00a0 reactions\u00a0 at\u00a0 the\u00a0 optimized\u00a0 conditions\u00a0 were\u00a0\n\nrepeated\u00a0with\u00a0fresh\u00a0primer\u00a0set\u00a0P30/31.\u00a0PCR\u00a0reactions\u00a0\n\nwere\u00a0 again\u00a0 carried\u00a0 out\u00a0 on\u00a0 nine\u00a0 serovars\u00a0 at\u00a0 MgCl2\u00a0\n\nResults\u00a0and\u00a0Discussion\u00a0\nSpecificity\u00a0 of\u00a0 Leptospira\u00a0 spp.\u00a0 Primers.\u00a0 Initially,\u00a0 PCR\u00a0\n\nusing\u00a0 P28/29\u00a0 was\u00a0 standardized\u00a0 by\u00a0 carrying\u00a0 out\u00a0\n\nreactions\u00a0 at\u00a0 various\u00a0 annealing\u00a0 temperatures\u00a0 ranging\u00a0\n\nfrom\u00a058\u00b0C\u00a0to\u00a068\u00b0C\u00a0on\u00a0L.\u00a0 icterohaemorrhagiae\u00a0serovar\u00a0\n\nwhich\u00a0showed\u00a0amplification\u00a0at\u00a058\u00b0C,\u00a058.7\u00b0C\u00a0and\u00a060.8\u00b0C\u00a0\n\n(Fig.1a).\u00a0 A\u00a0 single\u00a0 band\u00a0 of\u00a0 approximately\u00a0 385bp\u00a0 was\u00a0\n\nobserved\u00a0at\u00a060\u00b0C\u00a0annealing\u00a0temperature.\u00a0A\u00a0set\u00a0of\u00a0PCR\u00a0\n\nreactions\u00a0 were\u00a0 done\u00a0 by\u00a0 varying\u00a0 the\u00a0 template\u00a0\n\nconcentration\u00a0from\u00a05ng\u00a0to\u00a060ngand\u00a0could\u00a0amplify\u00a0the\u00a0\n\nspecific\u00a0 band\u00a0 at\u00a0 all\u00a0 the\u00a0 template\u00a0 concentrations.\u00a0 The\u00a0\n\nPCR\u00a0 products\u00a0 were\u00a0 pooled\u00a0 and\u00a0 characterized\u00a0 by\u00a0\n\nsubjecting\u00a0 to\u00a0 RE\u00a0 digestion\u00a0 using\u00a0 RsaI\u00a0 and\u00a0 HindIII\u00a0\n\nrestriction\u00a0 enzymes,\u00a0 both\u00a0 of\u00a0 which\u00a0 confirmed\u00a0 the\u00a0\n\nproduct\u00a0 size\u00a0 of\u00a0 P28/29\u00a0 to\u00a0 be\u00a0 385bp\u00a0 (Fig.2a).\u00a0 The\u00a0\n\nspecificity\u00a0of\u00a0this\u00a0primer\u00a0was\u00a0tested\u00a0on\u00a0nine\u00a0different\u00a0\n\nserovars\u00a0namely\u00a0L.\u00a0icterohaemorrhagiae,\u00a0L.\u00a0canicola,\u00a0L.\u00a0\n\npamona,\u00a0 L.\u00a0 autumnalis,\u00a0 L.\u00a0 javanica,\u00a0 L.\u00a0 pyrogenes,\u00a0 L.\u00a0\n\naustralis,\u00a0 L.\u00a0 hardjo\u00a0 and\u00a0 L.\u00a0 inadai\u00a0 under\u00a0 standardized\u00a0\n\ncondition.\u00a0The\u00a0assay\u00a0targeting\u00a0the\u00a0partial\u00a0sequence\u00a0of\u00a0\n\nlipL21\u00a0 was\u00a0 found\u00a0 to\u00a0 be\u00a0 specific\u00a0 for\u00a0 eight\u00a0 pathogenic\u00a0\n\nLeptospires\u00a0out\u00a0of\u00a0nine\u00a0\u00a0Leptospires\u00a0tested\u00a0(Fig.", "start_char_idx": 19851, "end_char_idx": 22136, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1666ebd1-4825-4f41-8aa3-556dd0a3d5d9": {"__data__": {"id_": "1666ebd1-4825-4f41-8aa3-556dd0a3d5d9", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "27e3cf05-20f3-4a01-b1e0-36c31750b03b", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "d17bd47cb71c1e8b3cc1e425d0720239f42cb18cb62d9ff23f5c32b35a05b8f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9772c448-c762-4731-ab90-cdd51823c229", "node_type": "1", "metadata": {}, "hash": "7424c798d01939f569e9fe7434ecb910c696f7ed3fdac48ffd9378ca56b16139", "class_name": "RelatedNodeInfo"}}, "text": "3a)\u00a0\n\nFor\u00a0the\u00a0PCR\u00a0assay\u00a0targeted\u00a0to\u00a0the\u00a0partial\u00a0sequence\u00a0of\u00a0\n\nlipL41,\u00a0 standardization\u00a0 was\u00a0 done\u00a0 initially\u00a0 by\u00a0 carrying\u00a0\n\nFigure\u00a0 1.\u00a0 (a)\u00a0 Specificity\u00a0 and\u00a0 detection\u00a0 of\u00a0 annealing\u00a0\ntemperature\u00a0of\u00a0LipL21\u00a0gene.\u00a0Annealing\u00a0temperature\u00a0\nwas\u00a0 standardized\u00a0 to\u00a0 600C\u00a0 for\u00a0 1\u00a0 min.\u00a0 \u00a0 Lane1\u20106,\u00a0 PCR\u00a0\nproducts\u00a0 using\u00a0 DNA\u00a0 template\u00a0 of\u00a0 L.\u00a0\nicterohaemorrhagiae.\u00a0 \u00a0 Lane\u00a0 1\u20106\u00a0 annealing\u00a0\ntemperature\u00a0 of\u00a0 each\u00a0 lane\u00a0 corresponding\u00a0 to\u00a0 58oC,\u00a0\n58.7oC,\u00a0 60.8oC,\u00a0 63.5oC,\u00a0 66.1oC,\u00a0 68oC,\u00a0 respectively.\u00a0\u00a0\nLane\u00a0 7,\u00a0 100bp\u00a0 DNA\u00a0 ladder\u00a0 (Fermentas).\u00a0 \u00a0 Gel\u00a0 run\u00a0\nperformed\u00a0using\u00a02%\u00a0agarose. \u00a0\n\nFigure\u00a0 1.\u00a0 (b)\u00a0 Specificity\u00a0and\u00a0detection\u00a0of\u00a0annealing\u00a0\ntemperature\u00a0of\u00a0LipL41\u00a0gene.\u00a0Annealing\u00a0temperature\u00a0\nwas\u00a0standardized\u00a0to\u00a0600C\u00a0for\u00a01\u00a0min.\u00a0 \u00a0Lane2\u20107,\u00a0PCR\u00a0\nproducts\u00a0using\u00a0DNA\u00a0template\u00a0of\u00a0L.\u00a0javanica.\u00a0\u00a0Lane\u00a02\u2010\n7\u00a0annealing\u00a0temperature\u00a0of\u00a0each\u00a0lane\u00a0corresponding\u00a0\nto\u00a0 60.5oC,\u00a0 61.8oC,\u00a0 63.1oC,\u00a0 64.2oC,\u00a0 65.0oC,\u00a0 65.6oC,\u00a0\nrespectively.\u00a0 \u00a0 Lane\u00a0 1\u00a0 and\u00a0 8,\u00a0 100bp\u00a0 DNA\u00a0 ladder\u00a0\n(Fermentas).\u00a0\u00a0Gel\u00a0run\u00a0performed\u00a0using\u00a02%\u00a0agarose. \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n27\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n\u00a0\u00a0 Organism\u00a0 PCR\u00a0reaction\u00a0mixture\u00a0(25ml)\u00a0\n\u00a0\u00a0\n\n\u00a0\u00a0\n\nP28\u00a0/29\u00a0\n\n\u00a0\u00a0\n\nP30/31\u00a0\n\n\u00a0\u00a0\n\nL.\u00a0javanica\u00a0\n\nL.\u00a0autumnalis\u00a0\n\nL.\u00a0hardjo\u00a0\n\nL.\u00a0pyrogenes\u00a0\n\nL.\u00a0pamona\u00a0\n\nL.\u00a0australis\u00a0\n\nL.\u00a0icterohaemorrhagiae\u00a0\n\nL.\u00a0inadai\u00a0\n\nL.\u00a0canicola\u00a0\n\n10x\u00a0buffer\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.50ml\u00a0\n\nMgCl2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a01.50ml\u00a0\n\ndNTP\u00a0mix\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.00ml\u00a0\n\nForward\u00a0Primer\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.50ml\u00a0\n\nReverse\u00a0Primer\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.50ml\u00a0\n\nTemplate\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a00.25ml\u00a0\n\nTaq\u00a0Pol\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a02.00ml\u00a0\n\nNuclease\u00a0free\u00a0water\u00a0\u00a0\u00a0\u00a0\u00a015.75ml\u00a0\n\n\u00a0\u00a0 LipL21\u00a0 LipL41\u00a0\n\nBuffer\u00a0\n(10x)\u00a0\n\nEnzyme\u00a0\n\nDNA\u00a0\n\nWater\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a02.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a01.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a08.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a011.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a02.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a02.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a05.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a010.0\u00a0ml\u00a0\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Total\u00a0 \u00a0\u00a0\u00a020.0\u00a0ml\u00a0 \u00a0\u00a0\u00a0\u00a020.0\u00a0ml\u00a0\n\nTable\u00a04.\u00a0Details\u00a0of\u00a0the\u00a0standardized\u00a0conditions\u00a0for\u00a0PCR \u00a0 Table\u00a05.\u00a0Details\u00a0of\u00a0the\u00a0Restriction\u00a0\nEnzyme\u00a0digestion\u00a0reaction\u00a0for\u00a0insert\u00a0\n\nrelease \u00a0\n\nFigure\u00a0 2.", "start_char_idx": 22137, "end_char_idx": 24243, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9772c448-c762-4731-ab90-cdd51823c229": {"__data__": {"id_": "9772c448-c762-4731-ab90-cdd51823c229", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1666ebd1-4825-4f41-8aa3-556dd0a3d5d9", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "d2399b57f6dd57800afc6447504ee81b17194196800d3f091f2cf78c7c5564d8", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7685aaa7-b6dd-4f91-b5ed-fb4da9756531", "node_type": "1", "metadata": {}, "hash": "eb467eafaa73d2e5a475e22919d54bcea3e48fc2c260e738afdebf9fd8d036bf", "class_name": "RelatedNodeInfo"}}, "text": "(a)\u00a0 Characterization\u00a0 of\u00a0 Amplicons\u00a0 using\u00a0\nrestriction\u00a0enzymes\u00a0RsaI\u00a0in\u00a0lane\u00a02\u00a0and\u00a0HindIII\u00a0in\u00a0lane\u00a0\n3\u00a0and\u00a0lane\u00a0DNA\u00a0template\u00a0without\u00a0RE\u00a0enzyme\u00a0in\u00a0lane\u00a0\n1,\u00a0 lane\u00a0 4,\u00a0 100bp\u00a0 Gene\u00a0 Ruler.\u00a0 \u00a0 \u00a0 Gel\u00a0 run\u00a0 performed\u00a0\nusing\u00a02%\u00a0agarose. \u00a0\n\nFigure\u00a0 2.\u00a0 (b)\u00a0 Characterization\u00a0 of\u00a0 Amplicons\u00a0 using\u00a0\nrestriction\u00a0enzymes\u00a0ClaI\u00a0\u00a0in\u00a0lane\u00a03,\u00a0TaqI\u00a0in\u00a0lane\u00a03\u00a0and\u00a0\nRsaI\u00a0in\u00a0lane\u00a05,\u00a0DNA\u00a0template\u00a0with\u00a0out\u00a0RE\u00a0enzyme\u00a0in\u00a0\nlane\u00a0 2,\u00a0 lane\u00a0 1\u00a0 and\u00a0 6,\u00a0 100bp\u00a0 Gene\u00a0 Ruler.\u00a0 \u00a0 Gel\u00a0 run\u00a0\u00a0\nperformed\u00a0using\u00a02%\u00a0agarose. \u00a0\n\nconcentration\u00a0 of\u00a0 1mM.\u00a0 This\u00a0 assay\u00a0 proved\u00a0 to\u00a0 be\u00a0 only\u00a0\n\nspecific\u00a0 to\u00a0 L.\u00a0 javanica\u00a0 serovar\u00a0 out\u00a0 of\u00a0 nine\u00a0 serovars\u00a0\n\ntested\u00a0(Fig.\u00a03b).\u00a0\n\nThe\u00a0 PCR\u00a0 product\u00a0 of\u00a0 both\u00a0 P28/29\u00a0 and\u00a0 P30/31\u00a0 of\u00a0 size\u00a0\n\n385bp\u00a0 and\u00a0 427bp\u00a0 respectively\u00a0 were\u00a0 then\u00a0 individually\u00a0\n\ncloned\u00a0 into\u00a0 pGEMT\u00a0 Easy\u00a0 TA\u00a0 cloning\u00a0 vector\u00a0 and\u00a0\n\ntransformed\u00a0 to\u00a0 JM109\u00a0 E.\u00a0 coli\u00a0 competent\u00a0 cells\u00a0 and\u00a0\n\nplated\u00a0on\u00a0LB\u00a0ampicillin\u00a0plates\u00a0with\u00a0IPTG\u00a0and\u00a0X\u2010gal\u00a0(Fig.\u00a0\n\n4).\u00a0The\u00a0discrete\u00a0five\u00a0white\u00a0colonies\u00a0and\u00a0a\u00a0blue\u00a0colony\u00a0\n\nwere\u00a0then\u00a0picked,\u00a0cultured\u00a0and\u00a0plasmid\u00a0was\u00a0 isolated.\u00a0\n\nThe\u00a0 recombinants\u00a0 were\u00a0 then\u00a0 characterized\u00a0 by\u00a0\n\nreleasing\u00a0 the\u00a0 insert\u00a0 (Fig.\u00a0 5a\u00a0 and\u00a0 5b)\u00a0 using\u00a0 EcoRI\u00a0\n\nrestriction\u00a0 enzyme\u00a0 followed\u00a0 by\u00a0 PCR\u00a0 reaction\u00a0 (Fig.\u00a0 6a\u00a0\n\nand\u00a06b)\u00a0for\u00a0confirmation.\u00a0The\u00a0cloned\u00a0sequences\u00a0were\u00a0\n\nthen\u00a0 sent\u00a0 for\u00a0 sequence\u00a0 analysis\u00a0 (MWG\u00a0 Biotech\u00a0 Pvt.\u00a0\n\nLtd.,\u00a0Bangalore)\u00a0in\u00a0order\u00a0to\u00a0facilitate\u00a0further\u00a0studies.\u00a0\n\nOne\u00a0of\u00a0the\u00a0main\u00a0advantages\u00a0of\u00a0using\u00a0this\u00a0technique\u00a0on\u00a0\n\nvarious\u00a0 biological\u00a0 samples\u00a0 is\u00a0 \u00a0 possibility\u00a0 to\u00a0 detect\u00a0\n\npathogenic\u00a0 Leptospires\u00a0 first\u00a0 by\u00a0 PCR\u00a0 assay\u00a0 and\u00a0\n\nsubsequently\u00a0 confirming\u00a0 it\u00a0 through\u00a0 RE\u00a0 digestion\u00a0 thus\u00a0\n\nimproving\u00a0the\u00a0specificity\u00a0of\u00a0the\u00a0test.\u00a0Further\u00a0sequence\u00a0\n\nanalysis\u00a0may\u00a0reveal\u00a0the\u00a0associated\u00a0serovar\u00a0causing\u00a0the\u00a0\n\ndisease.\u00a0\n\nThe\u00a0 direct\u00a0 method\u00a0 of\u00a0 diagnosis\u00a0 of\u00a0 leptospirosis\u00a0 is\u00a0 by", "start_char_idx": 24245, "end_char_idx": 26051, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7685aaa7-b6dd-4f91-b5ed-fb4da9756531": {"__data__": {"id_": "7685aaa7-b6dd-4f91-b5ed-fb4da9756531", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9772c448-c762-4731-ab90-cdd51823c229", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "c5fff0730a8956d4d521a3bac75b8596769d8f8b36e9411b747d1be09cba4114", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "783cc899-e9f2-4e03-9add-9d16b0ce89ae", "node_type": "1", "metadata": {}, "hash": "bd78a59b9aec82c3561b9e4be4193f79ea1c6da08c268a87a2b1793d15ae7927", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n28\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nculture\u00a0 isolation,\u00a0 but\u00a0 it\u00a0 is\u00a0 time\u00a0 consuming\u00a0 and\u00a0\n\npotentially\u00a0 biohazardous.\u00a0 Another\u00a0 traditional\u00a0 method\u00a0\n\nis\u00a0the\u00a0detection\u00a0of\u00a0antibodies\u00a0(serological\u00a0tests)\u00a0but\u00a0it\u00a0is\u00a0\n\nalso\u00a0time\u00a0consuming\u00a0and\u00a0fails\u00a0to\u00a0identify\u00a0the\u00a0infecting\u00a0\n\nserovar.\u00a0To\u00a0overcome\u00a0the\u00a0limitations\u00a0of\u00a0cultivation\u00a0and\u00a0\n\nserology,\u00a0we\u00a0have\u00a0used\u00a0PCR\u00a0amplification\u00a0of\u00a0leptospiral\u00a0\n\nDNA\u00a0for\u00a0the\u00a0diagnosis\u00a0of\u00a0leptospirosis\u00a0at\u00a0an\u00a0early\u00a0stage\u00a0\n\nof\u00a0illness.\u00a0\n\nIn\u00a0 a\u00a0 previous\u00a0 study\u00a0 by\u00a0 (Merien\u00a0 et\u00a0 al.,\u00a0 1992\u00a0 and\u00a0\n\nGravekamp\u00a0 et\u00a0 al,.\u00a0 1993),\u00a0 the\u00a0 16S\u00a0 rRNA\u00a0 gene\u00a0 target\u00a0\n\ncould\u00a0not\u00a0differentiate\u00a0pathogenic\u00a0and\u00a0nonpathogenic\u00a0\n\nLeptospira.\u00a0 \u00a0 The\u00a0 development\u00a0 of\u00a0 PCR\u00a0 assay\u00a0 targeting\u00a0\n\npartial\u00a0 sequence\u00a0 of\u00a0 lipL21\u00a0 and\u00a0 lipL41\u00a0 gene\u00a0 of\u00a0\n\npathogenic\u00a0Leptospires\u00a0using\u00a0in\u2010house\u00a0designed\u00a0P28/29\u00a0\n\nand\u00a0P30/31\u00a0primers,\u00a0with\u00a0a\u00a0product\u00a0size\u00a0of\u00a0385bp\u00a0and\u00a0\n\n427bp,\u00a0 respectively.\u00a0 These\u00a0 primers\u00a0 were\u00a0 designed\u00a0 by\u00a0\n\nusing\u00a0 Gene\u00a0 Tool\u00a0 Lite\u00a0 software.\u00a0 The\u00a0 gene\u00a0 sequence\u00a0\n\ncoding\u00a0for\u00a0the\u00a0respective\u00a0surface\u00a0protein\u00a0was\u00a0subjected\u00a0\n\nto\u00a0 nucleotide\u00a0 blast\u00a0 to\u00a0 get\u00a0 conserved\u00a0 sequences.\u00a0 The\u00a0\n\nmost\u00a0 conserved\u00a0 sequence\u00a0 was\u00a0 then\u00a0 chosen\u00a0 and\u00a0\n\nprimers\u00a0were\u00a0designed.\u00a0\n\nThe\u00a0 newly\u00a0 developed\u00a0 PCR\u00a0 using\u00a0 in\u2010house\u00a0 designed\u00a0\n\nprimers\u00a0produces\u00a0expected\u00a0\u00a0\u00a0amplicons\u00a0sizes\u00a0i.e.\u00a0385bp\u00a0\n\nfor\u00a0lipL21\u00a0and\u00a0427bp\u00a0for\u00a0lipL41.\u00a0\u00a0Afterwards\u00a0when\u00a0this\u00a0\n\nPCR\u00a0 protocol\u00a0 was\u00a0 applied\u00a0 to\u00a0 examine\u00a0 all\u00a0 the\u00a0 test\u00a0\n\nsamples\u00a0 of\u00a0 this\u00a0 species\u00a0 also\u00a0 produces\u00a0 specific\u00a0\n\namplicons.\u00a0\u00a0No\u00a0amplicons\u00a0of\u00a0specific\u00a0size\u00a0was\u00a0observed\u00a0\n\nin\u00a0 case\u00a0 of\u00a0 non\u2010target\u00a0 Leptospira,\u00a0 or\u00a0 other\u00a0 bacterial\u00a0\n\nFigure\u00a03.\u00a0(a)\u00a0The\u00a0specificity\u00a0of\u00a0this\u00a0primer\u00a0tested\u00a0on\u00a0\nnine\u00a0different\u00a0serovars\u00a0under\u00a0optimized\u00a0conditions\u00a0\nfor\u00a0lipL21.\u00a0\u00a0Lane\u00a03\u201011\u00a0different\u00a0serovars,\u00a0lane\u00a02,\u00a0nega\u2010\ntive\u00a0control\u00a0and\u00a0lane\u00a01,\u00a0100bp\u00a0Gene\u00a0Ruler.\u00a0Gel\u00a0run\u00a0\nperformed\u00a0using\u00a02.5%\u00a0agarose. \u00a0\n\nFigure\u00a03.\u00a0(b)\u00a0The\u00a0specificity\u00a0of\u00a0this\u00a0primer\u00a0tested\u00a0on\u00a0\nnine\u00a0 different\u00a0 serovars\u00a0 under\u00a0 optimized\u00a0 conditions\u00a0\nfor\u00a0lipL41.\u00a0\u00a0Lane\u00a02\u20105\u00a0different\u00a0serovars\u00a0and\u00a0lane\u00a01and\u00a0\n6,\u00a0100bp\u00a0Gene\u00a0Ruler.\u00a0\u00a0Gel\u00a0run\u00a0performed\u00a0using\u00a02.5%\u00a0\nagarose. \u00a0\n\nFigure\u00a04.\u00a0\u00a0\u00a0\nPlate\u00a0showing\u00a0\nrecombinant\u00a0\ncolonies", "start_char_idx": 26056, "end_char_idx": 28264, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "783cc899-e9f2-4e03-9add-9d16b0ce89ae": {"__data__": {"id_": "783cc899-e9f2-4e03-9add-9d16b0ce89ae", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7685aaa7-b6dd-4f91-b5ed-fb4da9756531", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "4457577d74d494ee7df3e65923c857cb5186a3f6cfc150ff31ef9e85ca17a688", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ef2810ad-b87a-4b0b-9a79-28ac21062104", "node_type": "1", "metadata": {}, "hash": "a8d4fce7870d59e8aaeb22f5d81e2dca4cf936652c5e483c0a01d6feb68a91af", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n29\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nspecies.\u00a0\u00a0Besides\u00a0non\u2010specific\u00a0amplicon\u00a0of\u00a0different\u00a0size\u00a0\n\ndid\u00a0not\u00a0appear\u00a0in\u00a0any\u00a0case.\u00a0\n\nIn\u00a0conclusion,\u00a0the\u00a0assay\u00a0targeting\u00a0partial\u00a0sequence\u00a0of\u00a0\n\nLipL41\u00a0gene\u00a0showed\u00a0high\u00a0sensitivity\u00a0and\u00a0specificity\u00a0for\u00a0\n\nL.\u00a0javanica\u00a0out\u00a0of\u00a09\u00a0Leptospires\u00a0tested.\u00a0The\u00a0PCR\u00a0assay\u00a0\n\ntargeting\u00a0 partial\u00a0 sequence\u00a0 of\u00a0 lipL21\u00a0 gene,\u00a0 which\u00a0\n\nshowed\u00a0 good\u00a0 sensitivity\u00a0 and\u00a0 specificity\u00a0 with\u00a0 all\u00a0 the\u00a0\n\nnine\u00a0serovars\u00a0tested\u00a0and\u00a0may\u00a0also\u00a0likely\u00a0to\u00a0detect\u00a0other\u00a0\n\npathogenic\u00a0 serovars\u00a0 as\u00a0 the\u00a0 lipL21\u00a0 sequence\u00a0 targeted\u00a0\n\nunder\u00a0the\u00a0assay\u00a0is\u00a0conserved\u00a0among\u00a0all\u00a0the\u00a0pathogenic\u00a0\n\nLeptospires\u00a0 hence\u00a0 may\u00a0 be\u00a0 valuable\u00a0 tool\u00a0 for\u00a0 early\u00a0\n\ndiagnosis\u00a0 of\u00a0 pathogenic\u00a0 Leptospires\u00a0 directly\u00a0 from\u00a0\n\nbiological\u00a0 samples\u00a0 with\u00a0 clinical\u00a0 suspicion\u00a0 of\u00a0\n\nleptospirosis.\u00a0\n\nFigure\u00a05.\u00a0(a)\u00a0Insert\u00a0release\u00a0of\u00a0lipL21from\u00a0recombinant\u00a0\npGEM\u2010TE\u00a0 vector.\u00a0 \u00a0 Gel\u00a0 run\u00a0 performed\u00a0 using\u00a0 2.5%\u00a0\nagarose. \u00a0\n\nFigure\u00a05.\u00a0(b)\u00a0Insert\u00a0release\u00a0of\u00a0lipL41\u00a0from\u00a0recombi\u2010\nnant\u00a0pGEM\u2010TE\u00a0vector.\u00a0\u00a0Gel\u00a0run\u00a0performed\u00a0using\u00a02.5%\u00a0\nagarose. \u00a0\n\nFigure\u00a0 6.\u00a0 (a)\u00a0 PCR\u00a0 for\u00a0 lipL21\u00a0 gene\u00a0 for\u00a0 confirmation.\u00a0\u00a0\nGel\u00a0run\u00a0performed\u00a0using\u00a02.5%\u00a0agarose. \u00a0\n\nFigure\u00a0 6.\u00a0 (b)\u00a0 PCR\u00a0 for\u00a0 lipL41\u00a0 gene\u00a0 for\u00a0 confirmation.\u00a0\u00a0\nGel\u00a0run\u00a0performed\u00a0using\u00a02.5%\u00a0agarose. \u00a0\n\nReferences\u00a0\nAbdollahpour,\u00a0 G:\u00a0 A\u00a0 review\u00a0 on\u00a0 Leptospirosis.\u00a0\u00a0\n\nProceedings\u00a0of\u00a0Leptospirosis\u00a0Research\u00a0\u00a0\u00a0\u00a0\u00a0Conference,\u00a0\n\nJapan,\u00a0Matsoyamam\u00a0;1990:\u00a034\u201037.\u00a0\n\nBharti,\u00a0AR,\u00a0Nally\u00a0JE,\u00a0Ricaldi\u00a0JN,\u00a0Matthias\u00a0MA,\u00a0Diaz\u00a0MM:\u00a0\n\nLeptospirosis:\u00a0 A\u00a0 zoonotic\u00a0 disease\u00a0 of\u00a0 global\u00a0\n\nimportance.\u00a0Lancet\u00a0Infect\u00a0Dis\u00a02003,\u00a03:757\u2013771.\u00a0\u00a0\n\nGravekamph\u00a0C,\u00a0Kemp\u00a0VAD,\u00a0Franzend\u00a0M,\u00a0Carrington\u00a0D,\u00a0\u00a0\u00a0\n\nSchoone\u00a0GJ,\u00a0\u00a0Eys\u00a0GJJMV,\u00a0\u00a0Everardr\u00a0C0R,\u00a0\u00a0Hartskeeraln\u00a0\n\nRA,\u00a0 Terpstra\u00a0 WJ:\u00a0 \u00a0 Detection\u00a0 of\u00a0 seven\u00a0 species\u00a0 of\u00a0\n\npathogenic\u00a0 Leptospires\u00a0 by\u00a0 PCR\u00a0 using\u00a0 two\u00a0 sets\u00a0 of\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a022\u201030\u00a0\n\n30\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nprimers.\u00a0\u00a0J\u00a0of\u00a0Gen\u00a0Microbiol\u00a0\u00a01993,\u00a0139:\u00a01691\u20101700.", "start_char_idx": 28270, "end_char_idx": 30310, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ef2810ad-b87a-4b0b-9a79-28ac21062104": {"__data__": {"id_": "ef2810ad-b87a-4b0b-9a79-28ac21062104", "embedding": null, "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-55", "node_type": "4", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "5ba0f80f505189e070927911ab84fcbf386364a1ef45fe430b6d4e04a24aa19b", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "783cc899-e9f2-4e03-9add-9d16b0ce89ae", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "07d57b4072f95cd2c60255ab78fe7106cd13a29136915c471cc05803bbc09f17", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "fa0800d9-24a7-42be-a0ee-475e1dd312fc", "node_type": "1", "metadata": {}, "hash": "7defb9fcc53ade2b8fecdd6d28b0f454db77b5ea1ceda06f85d4c3952f63135f", "class_name": "RelatedNodeInfo"}}, "text": "Hoshino\u00a0 K,\u00a0 Yamasaki\u00a0 S,\u00a0 Mukhopadhyaym,\u00a0 AK,\u00a0\n\nChakraborty\u00a0 S,\u00a0 Basu\u00a0 A,\u00a0 Bhattacharya\u00a0 SK,\u00a0 Nair,\u00a0 GB,\u00a0\n\nShimada\u00a0 T:\u00a0 Development\u00a0 and\u00a0 evaluation\u00a0 of\u00a0 a\u00a0\n\nmultiplex\u00a0PCR\u00a0assay\u00a0for\u00a0rapid\u00a0detection\u00a0of\u00a0toxigenic\u00a0\n\nVibrio\u00a0cholerae\u00a0O1\u00a0and\u00a0O139.\u00a0 \u00a0FEMS\u00a0 Immunol\u00a0Med\u00a0\n\nMicrobiol\u00a01998,\u00a020:\u00a0201\u2013207.\u00a0\u00a0\n\nLevett,\u00a0 PN,\u00a0 Songee\u00a0 L,\u00a0 Branch,\u00a0 Carol\u00a0 U,\u00a0 Whittington,\u00a0\n\nCharles\u00a0 N,\u00a0 Edwards,\u00a0 \u00a0 Paxton\u00a0 H:\u00a0 \u00a0 Two\u00a0 Methods\u00a0 for\u00a0\n\nRapid\u00a0 Serological\u00a0 Diagnosis\u00a0 of\u00a0 Acute\u00a0 Leptospirosis.\u00a0\n\nClin\u00a0and\u00a0Diag.\u00a0Lab\u00a0Immunology\u00a02001,\u00a08(2):349\u2010351.\u00a0\n\nLevett\u00a0 PN,\u00a0 Morey\u00a0 RE,\u00a0 Galloway\u00a0 RL,\u00a0 Turner\u00a0 DE,\u00a0\n\nSteigerwalt\u00a0AG,\u00a0Mayer\u00a0LW:\u00a0Detection\u00a0of\u00a0pathogenic\u00a0\n\nLeptospires\u00a0 by\u00a0 real\u2010time\u00a0 quantitative\u00a0 PCR.\u00a0 \u00a0 J\u00a0 Med\u00a0\n\nMicrobiol\u00a02005\u00a054(1):45\u201049.\u00a0\n\nMerien\u00a0 F,\u00a0 Amouriaux\u00a0 P,\u00a0 Perolat\u00a0 P,\u00a0 Baranton\u00a0 G,\u00a0 Saint\u00a0\n\nGirons\u00a0I:\u00a0\u00a0Polymerase\u00a0Chain\u00a0Reaction\u00a0for\u00a0Detection\u00a0of\u00a0\n\nLeptospira\u00a0 spp.\u00a0 in\u00a0 Clinical\u00a0 Samples.\u00a0 J\u00a0 Clin\u00a0 Microbiol\u00a0\n\n1992,\u00a030(9):2219\u20102224.\u00a0\n\nMarshall\u00a0 \u00a0 OJ:\u00a0 Perl\u00a0 Primer:\u00a0 cross\u2010platform,\u00a0 graphical\u00a0\n\nprimer\u00a0design\u00a0for\u00a0standard,\u00a0bisulphite\u00a0and\u00a0real\u2010time\u00a0\n\nPCR.\u00a0Bio\u00a0Inf\u00a0Applications\u00a0Note\u00a02004,\u00a020\u00a0(15):\u00a02471\u2013\n\n2472.\u00a0\n\nSerres\u00a0GD,\u00a0Levesque\u00a0B,\u00a0Higgins\u00a0R,\u00a0Major\u00a0M,\u00a0Laliberre\u00a0D,\u00a0\n\nBouliance\u00a0N,\u00a0Duval\u00a0B:\u00a0Need\u00a0for\u00a0vaccination\u00a0of\u00a0sewer\u00a0\n\nworkers\u00a0against\u00a0leptospirosis\u00a0and\u00a0hepatitis\u00a0A.\u00a0\u00a0Occup\u00a0\n\nand\u00a0Envt.\u00a0Med\u00a01995,52:\u00a0505\u2010507.\u00a0\n\nWoodward\u00a0 \u00a0 M\u00a0 J,\u00a0 Redstone\u00a0 JS\u00a0 :\u00a0 Differentiation\u00a0 of\u00a0\u00a0\n\nleptospira\u00a0serovars\u00a0by\u00a0polymerase\u00a0chain\u00a0reaction\u00a0and\u00a0\n\nrestriction\u00a0 fragment\u00a0 length\u00a0 polymorphism.\u00a0 Vet\u00a0 Rec\u00a0\n\n1993,\u00a0132:\u00a0325\u2010326.\u00a0\n\nYasuda\u00a0PH,\u00a0Arnold\u00a0G,\u00a0Steigerwalt,\u00a0Katherine\u00a0R,\u00a0Sulzer,\u00a0\n\nArnold\u00a0F,\u00a0Kaufmann,\u00a0Faye\u00a0Rogers\u00a0 \u00a0 \u00a0Don,\u00a0J,\u00a0Brenner:\u00a0\n\nDeoxyribonucleic\u00a0 Acid\u00a0 Relatedness\u00a0 between\u00a0\n\nSerogroups\u00a0 and\u00a0 Serovars\u00a0 in\u00a0 the\u00a0 Family\u00a0\n\nLeptospiraceae\u00a0 with\u00a0 Proposals\u00a0 for\u00a0 Seven\u00a0 New\u00a0\n\nLeptospira\u00a0Species.\u00a0\u00a0Int\u00a0\u00a0J\u00a0\u00a0Systc\u00a0Bact\u00a01987,\u00a037(4):\u00a0407\n\n\u2010415.\u00a0\n\nZhang,\u00a0Y.,\u00a0Dai,\u00a0B.\u00a0\u00a0(1992)\u00a0Detection\u00a0of\u00a0Leptospiral\u00a0DNA\u00a0\n\nin\u00a0the\u00a0serum\u00a0of\u00a0175\u00a0patients\u00a0with\u00a0early\u00a0leptospirosis\u00a0\n\nby\u00a0polymerase\u00a0chain\u00a0reaction.\u00a0\u00a0Hua\u00a0Xi\u00a0Yi\u00a0Ke\u00a0Da\u00a0Xue\u00a0\n\nXue\u00a0Bao\u00a023(3):256\u201060.", "start_char_idx": 30313, "end_char_idx": 32323, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "fa0800d9-24a7-42be-a0ee-475e1dd312fc": {"__data__": {"id_": "fa0800d9-24a7-42be-a0ee-475e1dd312fc", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ef2810ad-b87a-4b0b-9a79-28ac21062104", "node_type": "1", "metadata": {"identifier": "njb-55", "author": "Chandan, S; Umesha, S; Bhure, SK; Haraprasad, N; Chandrashekar, S", "title": "Development of PCR assay for targeting partial lipL21 and lipL41 gene of leptospira", "date": "2022-07-11", "file": "njb-55.pdf"}, "hash": "3bec038e939ed5fa56463b02f2b7c498f16d2acaf4189a5d4cbc145784e941d6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "86028e91-691c-4229-bba7-21531ff27b93", "node_type": "1", "metadata": {}, "hash": "7af784e863cc4731e725904048bb38164f4887fb9ea0b72600deee844b2d8e17", "class_name": "RelatedNodeInfo"}}, "text": "3\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n14\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nORIGINAL\u00a0RESEARCH\u00a0ARTICLE\u00a0\n\nAnalysis\u00a0of\u00a0KatG\u00a0Ser315Thr\u00a0Mutation\u00a0in\u00a0Multidrug\u00a0Resistant\u00a0\nMycobacterium\u00a0tuberculosis\u00a0and\u00a0SLC11A1\u00a0Polymorphism\u00a0in\u00a0\nMultidrug\u00a0Resistance\u00a0Tuberculosis\u00a0in\u00a0Central\u00a0Development\u00a0\nRegion\u00a0of\u00a0Nepal\u00a0Using\u00a0PCR\u2010RFLP\u00a0Technique:\u00a0A\u00a0Pilot\u00a0Study\u00a0\n\nRaunak\u00a0Shrestha1,\u00a0Rubin\u00a0Narayan\u00a0Joshi1,\u00a0Kriti\u00a0Joshi1,\u00a0Bal\u00a0Hari\u00a0Poudel2\u00a0and\u00a0Bhupal\u00a0Govinda\u00a0\nShrestha1\u00a0\n\n1\u00a0Department\u00a0of\u00a0Biotechnology,\u00a0Kathmandu\u00a0University,\u00a0Dhulikhel,\u00a0Nepal.\u00a0\n2\u00a0Genetics\u00a0Nepal\u00a0Pvt.\u00a0Ltd.,\u00a0Lalitpur,\u00a0Nepal.\u00a0\n\nAbstract\u00a0\n\nSer315Thr\u00a0mutations\u00a0in\u00a0genes\u00a0encoding\u00a0the\u00a0mycobacteria\u00a0catalase\u2010peroxidase\u00a0(KatG)\u00a0has\u00a0been\u00a0associated\u00a0with\u00a0\n\nthe\u00a0major\u00a0resistance\u00a0to\u00a0isoniazid\u00a0(INH)\u00a0in\u00a0Mycobacterium\u00a0tuberculosis\u00a0(MTB).\u00a0Also\u00a0G/C\u00a0polymorphisms\u00a0in\u00a0INT4\u00a0\n\nregion\u00a0of\u00a0the\u00a0solute\u00a0carrier\u00a0family\u00a011\u00a0member\u00a01\u00a0gene\u00a0(SLC11A1)\u00a0and\u00a0susceptibility\u00a0towards\u00a0tuberculosis\u00a0(TB)\u00a0has\u00a0\n\nbeen\u00a0demonstrated\u00a0worldwide.\u00a024\u00a0drug\u00a0resistant\u00a0MTB\u00a0culture\u00a0positive\u00a0samples\u00a0and\u00a024\u00a0whole\u2010blood\u00a0samples\u00a0\n\nwere\u00a0collected\u00a0from\u00a0different\u00a0TB\u00a0patients\u00a0of\u00a0Central\u00a0Development\u00a0Region\u00a0of\u00a0Nepal\u00a0in\u00a02009.\u00a0A\u00a0Polymerase\u00a0Chain\u00a0\n\nReaction\u00a0(PCR)\u00a0\u2010\u00a0Restriction\u00a0Fragment\u00a0Length\u00a0Polymorphism\u00a0(RFLP)\u00a0assay\u00a0was\u00a0carried\u00a0out\u00a0in\u00a0order\u00a0to\u00a0investigate\u00a0\n\nSer315Thr\u00a0 KatG\u00a0 mutation\u00a0 and\u00a0G/C\u00a0 polymorphism\u00a0 in\u00a0 INT4\u00a0 region.\u00a0 4\u00a0 (16.67%)\u00a0 samples\u00a0 out\u00a0 of\u00a024\u00a0 MTB\u00a0 culture\u00a0\n\nsamples\u00a0demonstrated\u00a0the\u00a0Ser315Thr\u00a0KatG\u00a0mutation\u00a0whereas\u00a0none\u00a0of\u00a0the\u00a024\u00a0whole\u00a0blood\u00a0samples\u00a0were\u00a0found\u00a0\n\nto\u00a0 contain\u00a0 G/C\u00a0 polymorphism\u00a0 in\u00a0 INT4.\u00a0 Though\u00a0 no\u00a0 significant\u00a0 correlation\u00a0 could\u00a0 be\u00a0 found\u00a0 between\u00a0 INT4\u00a0\n\npolymorphism\u00a0 and\u00a0 TB\u00a0 susceptibility,\u00a0 overall\u00a0 scenario\u00a0 of\u00a0 Nepal\u00a0 cannot\u00a0 be\u00a0 drawn\u00a0 from\u00a0 this\u00a0 data.\u00a0 Molecular\u00a0\n\ndiagnostic\u00a0technique\u00a0such\u00a0as\u00a0PCR\u2010RFLP\u00a0can\u00a0be\u00a0used\u00a0 in\u00a0a\u00a0robust\u00a0scale\u00a0to\u00a0carry\u00a0out\u00a0base\u00a0 line\u00a0studies\u00a0 in\u00a0the\u00a0TB\u00a0\n\npopulation\u00a0of\u00a0Nepal.\u00a0\n\n\u00a0\nKey\u00a0words:\u00a0Multi\u2010drug\u00a0resistance,\u00a0Tuberculosis,\u00a0PCR,\u00a0RFLP\u00a0\n\n\u00a0\nCorrespondence\u00a0Author:\u00a0\nE\u2010mail:\u00a0sraunak@gmail.com\u00a0\n\nIntroduction\u00a0\nDuring\u00a0the\u00a0last\u00a0decades,\u00a0incidence\u00a0of\u00a0tuberculosis\u00a0(TB)\u00a0\nhas\u00a0increased\u00a0in\u00a0many\u00a0countries\u00a0and\u00a0more\u00a0people\u00a0have\u00a0\nthe\u00a0disease\u00a0now\u00a0than\u00a0at\u00a0any\u00a0other\u00a0time\u00a0in\u00a0history.", "start_char_idx": 45, "end_char_idx": 2224, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "86028e91-691c-4229-bba7-21531ff27b93": {"__data__": {"id_": "86028e91-691c-4229-bba7-21531ff27b93", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "fa0800d9-24a7-42be-a0ee-475e1dd312fc", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "0799ddcefcb8746455851a49764e2b89811a7833079b18c22153707b699265b3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6f68a801-b3fe-455a-ab24-aacaf82a7d59", "node_type": "1", "metadata": {}, "hash": "ff9c55e2d9cd69073d9c34f714178b23aafd01222213400da066e296668cad3a", "class_name": "RelatedNodeInfo"}}, "text": "It\u00a0is\u00a0\nestimated\u00a0that\u00a0TB\u00a0still\u00a0kills\u00a0more\u00a0people\u00a0globally\u00a0than\u00a0\nany\u00a0 other\u00a0 infection\u00a0 (WHO,\u00a0 2009).\u00a0 The\u00a0 situation\u00a0 has\u00a0\nworsened\u00a0 by\u00a0 the\u00a0 emergence\u00a0 of\u00a0 antibiotic\u2010resistant\u00a0\nstrains\u00a0of\u00a0Mycobacterium\u00a0tuberculosis\u00a0(MTB)\u00a0(Musser,\u00a0\n1995).\u00a0The\u00a0strains\u00a0resistant\u00a0to\u00a0the\u00a0two\u00a0most\u00a0important\u00a0\nanti\u2010TB\u00a0 drugs,\u00a0 rifampin\u00a0 (RIF)\u00a0 and\u00a0 isoniazid\u00a0 (INH)\u00a0 are\u00a0\ncommonly\u00a0defined\u00a0as\u00a0multidrug\u00a0resistant\u00a0(MDR).\u00a0Drug\u00a0\nresistance\u00a0 in\u00a0M.\u00a0tuberculosis\u00a0 is\u00a0attributed\u00a0primarily\u00a0to\u00a0\nthe\u00a0 accumulation\u00a0 of\u00a0 mutations\u00a0 in\u00a0 the\u00a0 drug\u00a0 target\u00a0\ngenes;\u00a0 these\u00a0 mutations\u00a0 either\u00a0 leading\u00a0 to\u00a0 an\u00a0 altered\u00a0\n\ntarget\u00a0or\u00a0to\u00a0an\u00a0alternation\u00a0in\u00a0effective\u00a0titration\u00a0of\u00a0the\u00a0\ndrug\u00a0(Rattan\u00a0et\u00a0al.,\u00a01998).\u00a0\u00a0\n\nIn\u00a0 2008,\u00a0 there\u00a0 were\u00a0 an\u00a0 estimated\u00a0 8.9\u20139.9\u00a0 million\u00a0\n\nincident\u00a0cases\u00a0of\u00a0TB,\u00a09.6\u201313.3\u00a0million\u00a0prevalent\u00a0cases\u00a0\n\nof\u00a0 TB\u00a0 (WHO,\u00a0 2009)\u00a0 and\u00a0 390,000\u2013510,000\u00a0 cases\u00a0 of\u00a0\n\nMDRTB\u00a0 (WHO,\u00a0 2010).\u00a0 Among\u00a0 the\u00a0 incident\u00a0 TB\u00a0 cases\u00a0\n\nglobally,\u00a0 3.6%\u00a0 (95%\u00a0 confidence\u00a0 interval\u00a0 (CI):\u00a0 3.0\u20134.4)\u00a0\n\nare\u00a0estimated\u00a0to\u00a0have\u00a0MDR\u2010TB.\u00a0Almost\u00a050%\u00a0of\u00a0MDR\u2010TB\u00a0\n\ncases\u00a0worldwide\u00a0are\u00a0estimated\u00a0to\u00a0occur\u00a0 in\u00a0China\u00a0and\u00a0\n\nIndia.\u00a0In\u00a02008,\u00a0MDR\u2010TB\u00a0caused\u00a0an\u00a0estimated\u00a0death\u00a0of\u00a0\n\n150,000\u00a0people\u00a0across\u00a0the\u00a0globe\u00a0(WHO,\u00a02010).\u00a0\u00a0\n\nWHO\u00a0 estimated\u00a0 the\u00a0 prevalence\u00a0 of\u00a0 all\u00a0 types\u00a0 of\u00a0\n\ntuberculosis\u00a0 cases\u00a0 for\u00a0 Nepal\u00a0 to\u00a0 be\u00a0 67,546\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n15\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\n(240/100,000).\u00a0The\u00a0proportion\u00a0of\u00a0MDR\u2010TB\u00a0in\u00a0Nepal\u00a0was\u00a0\n\n2.9%\u00a0 (95%\u00a0 CI:\u00a0 1.8\u20104.3)\u00a0 among\u00a0 new\u00a0 cases\u00a0 and\u00a0 11.7%\u00a0\n\n(95%\u00a0 CI:\u00a0 7.2\u201017.7)\u00a0 among\u00a0 retreatment\u00a0 cases\u00a0 (NTC,\u00a0\n\n2009).\u00a0\n\nIn\u00a0 2009,\u00a0 with\u00a0 the\u00a0 assistance\u00a0 of\u00a0 WHO,\u00a0 National\u00a0 TB\u00a0\n\nCenter\u00a0in\u00a0collaboration\u00a0with\u00a0National\u00a0Anti\u2010Tuberculosis\u00a0\n\nAssociation\u00a0 (NATA)\u00a0 and\u00a0 German\u00a0 Nepal\u00a0 Tuberculosis\u00a0\n\nProject\u00a0 (GENTUP)\u00a0 had\u00a0 conducted\u00a0 a\u00a0 surveillance\u00a0 of\u00a0\n\nextremely\u00a0drug\u2010resistant\u00a0tuberculosis\u00a0(XDR\u2010TB)\u00a0among\u00a0\n\nthe\u00a0 registered\u00a0 MDR\u00a0 TB\u00a0 patients.\u00a0 The\u00a0 study\u00a0 showed\u00a0 a\u00a0\n\nprevalence\u00a0 of\u00a0 5%\u00a0 of\u00a0 XDR\u2010TB\u00a0 cases\u00a0 among\u00a0 MDR\u00a0 TB\u00a0\n\ncases\u00a0registered\u00a0(NTC,\u00a02009).\u00a0\n\nIsoniazid\u00a0 (INH)\u00a0 is\u00a0 one\u00a0 of\u00a0 the\u00a0 important\u00a0 first\u2010line\u00a0\n\ntuberculosis\u00a0 drugs.", "start_char_idx": 2225, "end_char_idx": 4331, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6f68a801-b3fe-455a-ab24-aacaf82a7d59": {"__data__": {"id_": "6f68a801-b3fe-455a-ab24-aacaf82a7d59", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "86028e91-691c-4229-bba7-21531ff27b93", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "4acb8f3e4045ef494cac52c4f1d895729d270a5795035f881055b723c4407e6f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "6ac9b9bc-828e-4d38-b4ef-e563eb3c9d97", "node_type": "1", "metadata": {}, "hash": "6e2f1b72656b5dd2f2ef1aad3c68d3ef58d040e5ee57e7268716d0b0b96c8f86", "class_name": "RelatedNodeInfo"}}, "text": "MTB\u00a0 is\u00a0 highly\u00a0 susceptible\u00a0 to\u00a0 INH,\u00a0\n\nwith\u00a0a\u00a0Minimum\u00a0Inhibitory\u00a0Concentration\u00a0(MIC)\u00a0of\u00a00.03\n\n\u20100.06\u00a0mg\u00a0ml\u20101.\u00a0INH\u00a0inhibits\u00a0the\u00a0biosynthesis\u00a0of\u00a0cell\u00a0wall\u00a0\n\nmycolic\u00a0 acids\u00a0 (long\u00a0 chain\u00a0 \u03b1\u2010branched\u00a0 \u00df\u2010hydroxylated\u00a0\n\nfatty\u00a0 acids),\u00a0 thereby\u00a0 making\u00a0 the\u00a0 mycobacteria\u00a0\n\nsusceptible\u00a0 to\u00a0 reactive\u00a0 oxygen\u00a0 radicals\u00a0 and\u00a0 other\u00a0\n\nenvironmental\u00a0factors\u00a0(Rattan\u00a0et\u00a0al.,\u00a01998).\u00a0It\u00a0is\u00a0a\u00a0pro\u2010\n\ndrug\u00a0 that\u00a0 requires\u00a0 activation\u00a0 by\u00a0 the\u00a0 MTB\u00a0 catalase\u2010\n\nperoxidase\u00a0enzyme\u00a0(KatG)\u00a0to\u00a0its\u00a0active\u00a0form.\u00a0Mutation\u00a0\n\nof\u00a0 the\u00a0 KatG\u00a0 gene,\u00a0 which\u00a0 leads\u00a0 to\u00a0 loss\u00a0 of\u00a0 or\u00a0 reduced\u00a0\n\ncatalase\u2010peroxidase\u00a0 activity,\u00a0 is\u00a0 a\u00a0 major\u00a0 mechanism\u00a0 of\u00a0\n\nINH\u00a0 resistance\u00a0 in\u00a0 MTB\u00a0 (Musser,\u00a0 1995).\u00a0 Although\u00a0\n\nvarious\u00a0 mutations\u00a0 in\u00a0 the\u00a0 KatG\u00a0 gene\u00a0 have\u00a0 been\u00a0\n\nreported\u00a0 in\u00a0 INH\u2010resistant\u00a0 isolates,\u00a0 the\u00a0 most\u00a0 common\u00a0\n\nmutation\u00a0 is\u00a0the\u00a0Ser315Thr\u00a0mutation,\u00a0which\u00a0 is\u00a0present\u00a0\n\nin\u00a0approximately\u00a050\u201390%\u00a0of\u00a0all\u00a0INH\u2010resistant\u00a0isolates,\u00a0is\u00a0\n\nassociated\u00a0 with\u00a0 relatively\u00a0 high\u2010level\u00a0 resistance\u00a0to\u00a0 INH\u00a0\n\n(Mokrousov\u00a0et\u00a0al.,\u00a02002).\u00a0\u00a0\n\nMore\u00a0 than\u00a0 one\u2010third\u00a0 of\u00a0 the\u00a0 global\u00a0 population\u00a0 is\u00a0\ninfected\u00a0 with\u00a0 MTB.\u00a0 However,\u00a0 only\u00a0 10%\u00a0 develop\u00a0 the\u00a0\nclinical\u00a0 disease.\u00a0 Many\u00a0 factors\u00a0 contribute\u00a0 to\u00a0 the\u00a0\nimmune\u00a0 response\u00a0 against\u00a0 tuberculosis.\u00a0 The\u00a0 solute\u00a0\ncarrier\u00a0 family\u00a0 11\u00a0 member\u00a0 1\u00a0 gene\u00a0 (SLC11A1,\u00a0 formerly\u00a0\n\nknown\u00a0 as\u00a0 NRAMP1:\u00a0 natural\u00a0 resistance\u00a0 associated\u00a0\nmacrophage\u00a0protein\u00a01)\u00a0 is\u00a0one\u00a0of\u00a0the\u00a0candidate\u00a0genes\u00a0\nfor\u00a0susceptibility\u00a0to\u00a0human\u00a0tuberculosis\u00a0(Takahashi\u00a0et\u00a0\nal.,\u00a02008).\u00a0The\u00a0gene\u00a0is\u00a0located\u00a0on\u00a0human\u00a0chromosome\u00a0\n2q35\u00a0and\u00a0has\u00a015\u00a0exons\u00a0spanning\u00a0about\u00a014\u00a0kb\u00a0(Marquet\u00a0\net\u00a0 al.,\u00a0 2000).\u00a0 The\u00a0 gene\u00a0 encodes\u00a0 a\u00a0 transmembrane\u00a0\nprotein\u00a0 expressed\u00a0 exclusively\u00a0 in\u00a0 macrophages/\nmonocytes\u00a0 and\u00a0 polymorphonuclear\u00a0 leukocytes.\u00a0 The\u00a0\nprotein\u00a0acts\u00a0as\u00a0a\u00a0transporter\u00a0for\u00a0divalent\u00a0cations\u00a0Fe2+,\u00a0\nZn2+\u00a0 and\u00a0 Mn2+\u00a0 and\u00a0 has\u00a0 pleiotropic\u00a0 effects\u00a0 on\u00a0\nmacrophage\u00a0activation\u00a0(Goswami\u00a0et\u00a0al.,\u00a02001).\u00a0\u00a0Studies\u00a0\nhave\u00a0 demonstrated\u00a0 an\u00a0 association\u00a0 between\u00a0 three\u00a0\ndifferent\u00a0 polymorphisms\u00a0 (INT4,\u00a0 D543N\u00a0 and\u00a0 3\u2019UTR)\u00a0 in\u00a0\nSLC11A1\u00a0and\u00a0pulmonary\u00a0TB\u00a0(Taype\u00a0et\u00a0al.,\u00a02006).\u00a0\n\nIn\u00a0the\u00a0present\u00a0study,\u00a0Ser315Thr\u00a0KatG\u00a0mutation\u00a0 in\u00a0the\u00a0\nMDR\u2010TB\u00a0 culture\u00a0 samples\u00a0 and\u00a0 G/C\u00a0 polymorphisms\u00a0 in\u00a0\nINT4\u00a0 region\u00a0 of\u00a0 SLC11A1\u00a0 of\u00a0 TB\u00a0 patients\u00a0 were\u00a0 studied\u00a0\nusing\u00a0 Polymerase\u00a0 Chain\u00a0 Reaction\u00a0 (PCR)\u00a0 \u2010\u00a0 Restriction\u00a0\nFragment\u00a0Length\u00a0Polymorphism\u00a0(RFLP)\u00a0technique.\u00a0\n\nMaterials\u00a0and\u00a0Methods\u00a0\nStudy\u00a0samples.", "start_char_idx": 4333, "end_char_idx": 6691, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "6ac9b9bc-828e-4d38-b4ef-e563eb3c9d97": {"__data__": {"id_": "6ac9b9bc-828e-4d38-b4ef-e563eb3c9d97", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6f68a801-b3fe-455a-ab24-aacaf82a7d59", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "3c35d782f8e4de259d3117c9c569ad5e3aa373f4be6d29ebc05d74795132aba3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91e74032-f81c-407a-87a2-59442036e6d3", "node_type": "1", "metadata": {}, "hash": "f93b35f2fcd12e9d02df522b94ce38b111cd1975c9f6b7387313080066974acb", "class_name": "RelatedNodeInfo"}}, "text": "Materials\u00a0and\u00a0Methods\u00a0\nStudy\u00a0samples.\u00a024\u00a0MTB\u00a0Culture\u00a0(all\u00a0AFB\u00a0stain\u00a0positive)\u00a0\n\nsamples\u00a0were\u00a0obtained\u00a0from\u00a0GENTUP,\u00a0Kathmandu.\u00a0The\u00a0\n\ncultured\u00a0 samples\u00a0 comprised\u00a0 of\u00a0 both\u00a0 pulmonary\u00a0 and\u00a0\n\nextra\u2010pulmonary\u00a0origin.\u00a0Drug\u00a0Susceptibility\u00a0Testing\u00a0was\u00a0\n\nalso\u00a0carried\u00a0out\u00a0 in\u00a0GENTUP\u00a0and\u00a0all\u00a0of\u00a0the\u00a024\u00a0samples\u00a0\n\nwere\u00a0 graded\u00a0 as\u00a0 MDR\u2010TB.\u00a0 24\u00a0 whole\u00a0 blood\u00a0 samples\u00a0 of\u00a0\n\nMDR\u2010TB\u00a0 patients\u00a0 were\u00a0 also\u00a0 obtained\u00a0 for\u00a0 SLC11A1\u00a0\n\nanalysis.\u00a0The\u00a0samples\u00a0were\u00a0collected\u00a0from\u00a0the\u00a0Central\u00a0\n\nDevelopment\u00a0 Region\u00a0 of\u00a0 Nepal.\u00a0 However,\u00a0 we\u00a0 were\u00a0\n\nunable\u00a0 to\u00a0 obtain\u00a0 the\u00a0 clinical\u00a0 history\u00a0 of\u00a0 the\u00a0 test\u00a0\n\nsubjects.\u00a0Further\u00a0molecular\u00a0analysis\u00a0was\u00a0carried\u00a0out\u00a0in\u00a0\n\na\u00a0BSL\u2010III\u00a0laboratory\u00a0at\u00a0Genetics\u00a0Nepal\u00a0Pvt.\u00a0Ltd.,\u00a0Lalitpur,\u00a0\n\nNepal.\u00a0\n\nGenomic\u00a0 DNA\u00a0 isolation\u00a0 of\u00a0 M.\u00a0 tuberculosis.\u00a0 DNA\u00a0\n\nisolation\u00a0from\u00a0cultured\u00a0samples\u00a0was\u00a0done\u00a0using\u00a0SORPO\u00a0\n\nclean\u2122\u00a0 Genomic\u00a0 DNA\u00a0 extraction\u00a0 kit.\u00a0 A\u00a0 loop\u00a0 full\u00a0 of\u00a0\n\nbacterial\u00a0 colonies\u00a0 from\u00a0 the\u00a0 culture\u00a0 samples\u00a0 were\u00a0\n\nsuspended\u00a0 in\u00a0 200\u00b5l\u00a0 of\u00a0 sterile\u00a0 water.\u00a0 A\u00a0 spin\u00a0 column\u00a0\n\nextraction\u00a0 was\u00a0 carried\u00a0 out\u00a0 as\u00a0 prescribed\u00a0 by\u00a0 the\u00a0\n\nmanufacturer.\u00a0\n\nHuman\u00a0 genomic\u00a0 DNA\u00a0 isolation.\u00a0 The\u00a0 whole\u2010blood\u00a0\n\nsamples\u00a0 from\u00a0 human\u00a0 test\u00a0 subjects\u00a0 were\u00a0 collected\u00a0 in\u00a0\n\nEDTA\u00a0 coated\u00a0 vial\u00a0 and\u00a0 immediately\u00a0 centrifuged\u00a0 at\u00a0\n\n5000rpm/5min.\u00a0 Plasma\u00a0 was\u00a0 separated\u00a0 and\u00a0 200\u00b5l\u00a0 of\u00a0\n\nFigure\u00a01:\u00a0Prevalence\u00a0of\u00a0MDR\u2010TB\u00a0in\u00a0Nepal.\u00a0\u00a0\n\n(Source:\u00a0NTC,\u00a02009)\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n16\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nwhole\u2010blood\u00a0 was\u00a0 taken\u00a0 as\u00a0 sample\u00a0 for\u00a0 further\u00a0\n\nprocessing.\u00a0SORPO\u00a0clean\u2122\u00a0Genomic\u00a0DNA\u00a0extraction\u00a0kit\u00a0\n\nwas\u00a0used\u00a0for\u00a0genomic\u00a0DNA\u00a0isolation.\u00a0All\u00a0the\u00a0other\u00a0steps\u00a0\n\nwere\u00a0same\u00a0as\u00a0described\u00a0above.\u00a0\n\nGenomic\u00a0 DNA\u00a0 visualization:\u00a0 The\u00a0 extracted\u00a0 DNA\u00a0 was\u00a0\n\nvisualized\u00a0in\u00a0a\u00a00.8%\u00a0agarose\u00a0(Amresco\u00ae)\u00a0gel.", "start_char_idx": 6654, "end_char_idx": 8453, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91e74032-f81c-407a-87a2-59442036e6d3": {"__data__": {"id_": "91e74032-f81c-407a-87a2-59442036e6d3", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "6ac9b9bc-828e-4d38-b4ef-e563eb3c9d97", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "43abadb80f2f1f54c797ca75832667035b041d4576fc7723148851c4912e1011", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7cf4b5f8-6fd2-4b2c-acfa-966b7b0a9ae0", "node_type": "1", "metadata": {}, "hash": "9a7668a3a21ab03f0d29f3eec4a3dd40a8a430622c9f2dcbd767b2a5792dfd3a", "class_name": "RelatedNodeInfo"}}, "text": "PCR\u2010RFLP\u00a0 analysis:\u00a0 Amplification\u00a0 of\u00a0 the\u00a0 200\u00a0 bp\u00a0\n\nfragment\u00a0with\u00a0KatG\u00a0codon\u00a0315\u00a0(the\u00a0fragment\u00a0 in\u00a0KatG\u00a0\n\nfrom\u00a0 nucleotide\u00a0 positions\u00a0 904\u00a0 to\u00a0 1103;\u00a0 http://\n\ngenolist.pasteur.fr/TubercuList)\u00a0 was\u00a0 performed\u00a0 in\u00a0\n\nTPersonal\u00a0 Thermocycler\u00a0 (Biometra\u00ae)\u00a0 with\u00a0 primers\u00a0\n\nkatG1F\u00a0and\u00a0katG4RB\u00a0(Mokrousov\u00a0et\u00a0al.,\u00a02002)\u00a0(Table\u00a01)\u00a0\n\nin\u00a0 25\u00b5l\u00a0 of\u00a0 a\u00a0 PCR\u00a0 mixture\u00a0 (0.4\u00a0 \u03bcM\u00a0 of\u00a0 each\u00a0 primers,\u00a0\n\n2.5mM\u00a0 of\u00a0 dNTP\u00a0 mix,\u00a0 1U\u00a0 of\u00a0 recombinant\u00a0 Taq\u00a0 DNA\u00a0\n\npolymerase\u00a0(Fermentas\u00ae)\u00a0and\u00a02.5\u00a0mM\u00a0of\u00a0MgCl2)\u00a0under\u00a0\n\nthe\u00a0 following\u00a0 conditions:\u00a0 initial\u00a0 denaturation\u00a0 at\u00a0 94\u2070C\u00a0\n\nfor\u00a05\u00a0min;\u00a030\u00a0cycles\u00a0of\u00a094\u2070C\u00a0for\u00a01\u00a0min,\u00a056\u2070C\u00a0for\u00a045\u00a0sec\u00a0\n\nand\u00a072\u2070C\u00a0for\u00a045\u00a0sec;\u00a0and\u00a0a\u00a0final\u00a0elongation\u00a0at\u00a072\u2070C\u00a0for\u00a05\u00a0\n\nmin.\u00a0 The\u00a0 amplified\u00a0 fragment\u00a0 was\u00a0 assessed\u00a0 by\u00a0\n\nelectrophoresis\u00a0 in\u00a0a\u00a02%\u00a0agarose\u00a0gel\u00a0and\u00a0cleaved\u00a0with\u00a0\n\nrestriction\u00a0 enzyme\u00a0 MspI\u00a0 (HpaII)\u00a0 (Fermentas\u00ae)\u00a0 as\u00a0 per\u00a0\n\nthe\u00a0 instructions\u00a0 of\u00a0 the\u00a0 manufacturer.\u00a0 The\u00a0 restriction\u00a0\n\nfragments\u00a0 obtained\u00a0 were\u00a0 electrophoresed\u00a0 in\u00a0 a\u00a0 2%\u00a0\n\nagarose\u00a0gel\u00a0and\u00a0were\u00a0visualized\u00a0under\u00a0ultra\u2010violet\u00a0(UV)\u00a0\n\nlight\u00a0on\u00a0a\u00a0transilluminator\u00a0(Biometra\u00ae).\u00a0M.\u00a0tuberculosis\u00a0\n\nH37Rv\u00a0was\u00a0taken\u00a0as\u00a0the\u00a0reference\u00a0strain\u00a0for\u00a0this\u00a0study.\u00a0\n\nPCR\u2010RFLP\u00a0assay\u00a0was\u00a0designed\u00a0to\u00a0detect\u00a0the\u00a0KatG\u00a0codon\u00a0\n\nmutation\u00a0AGC(Ser)\u00a0\u00e0\u00a0ACC(Thr),\u00a0which\u00a0leads\u00a0to\u00a0the\u00a0INH\u00a0\n\nresistant\u00a0 phenotype.\u00a0 This\u00a0 mutation\u00a0 creates\u00a0 an\u00a0\n\nadditional\u00a0MspI\u00a0site\u00a0(CCGG)\u00a0and\u00a0thus\u00a0can\u00a0be\u00a0detected\u00a0\n\nby\u00a0use\u00a0of\u00a0this\u00a0restriction\u00a0endonuclease.\u00a0As\u00a0a\u00a0result,\u00a0the\u00a0\n\nlongest\u00a0 fragment\u00a0 size\u00a0 in\u00a0 the\u00a0 wild\u00a0 type\u00a0 KatG\u00a0 product\u00a0\n\nwould\u00a0 be\u00a0 153\u00a0 bp\u00a0 and\u00a0 in\u00a0 Ser315Thr\u00a0 KatG\u00a0 mutant\u00a0 the\u00a0\n\nlongest\u00a0 fragment\u00a0 would\u00a0 be\u00a0 132\u00a0 bp\u00a0 which\u00a0 could\u00a0 be\u00a0\n\neasily\u00a0 resolved\u00a0 in\u00a0 a\u00a0 2%\u00a0 agarose\u00a0 gel\u00a0 while\u00a0 the\u00a0 shorter\u00a0\n\nbands\u00a0 (21\u00a0 bp\u00a0 \u2010\u00a0 8\u00a0 bp)\u00a0 cannot\u00a0 be\u00a0 resolved\u00a0 in\u00a0 the\u00a0 gel\u00a0\n\n(Figure\u00a02).\u00a0\u00a0\n\nSimilarly,\u00a0514\u00a0bp\u00a0of\u00a0INT4\u00a0region\u00a0of\u00a0SLC11A1\u00a0gene\u00a0was\u00a0\namplified\u00a0using\u00a0primers\u00a0INT4F\u00a0and\u00a0INT4R\u00a0(Taype\u00a0et\u00a0al.,\u00a0\n2006)\u00a0(Table\u00a01).", "start_char_idx": 8456, "end_char_idx": 10324, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7cf4b5f8-6fd2-4b2c-acfa-966b7b0a9ae0": {"__data__": {"id_": "7cf4b5f8-6fd2-4b2c-acfa-966b7b0a9ae0", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91e74032-f81c-407a-87a2-59442036e6d3", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "44377a121cc533ca8610aa1da5b715f60c577dc9fa7ece8646ee981646f522bf", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "75825f6c-474c-45f0-a863-3dcc9110cd86", "node_type": "1", "metadata": {}, "hash": "071f442136c0f6c2fcbd972363aef60e30203b6495585f7984317eb6c1f11c39", "class_name": "RelatedNodeInfo"}}, "text": "A\u00a025\u00b5l\u00a0of\u00a0a\u00a0PCR\u00a0mixture\u00a0(0.4\u00a0\u03bcM\u00a0of\u00a0\neach\u00a0primers,\u00a02.5mM\u00a0of\u00a0dNTP\u00a0mix,\u00a01U\u00a0of\u00a0recombinant\u00a0\nTaq\u00a0 DNA\u00a0 polymerase\u00a0 and\u00a0 2.5\u00a0 mM\u00a0 of\u00a0 MgCl2)\u00a0 was\u00a0\nprepared\u00a0and\u00a0the\u00a0reaction\u00a0was\u00a0carried\u00a0out\u00a0under\u00a0the\u00a0\nfollowing\u00a0 condition:\u00a0 initial\u00a0 denaturation\u00a0 at\u00a0 95\u2070C\u00a0 for\u00a0 5\u00a0\nmin;\u00a030\u00a0cycles\u00a0of\u00a094\u2070C\u00a0for\u00a01\u00a0min,\u00a061\u00b0C\u00a0for\u00a01\u00a0min\u00a0and\u00a0\n72\u2070C\u00a0for\u00a01\u00a0min;\u00a0and\u00a0a\u00a0final\u00a0elongation\u00a0at\u00a072\u2070C\u00a0for\u00a04\u00a0min.\u00a0\nThe\u00a0 amplified\u00a0 fragment\u00a0 was\u00a0 assessed\u00a0 by\u00a0\nelectrophoresis\u00a0in\u00a0a\u00a02%\u00a0agarose\u00a0gel.\u00a0To\u00a0analyze\u00a0the\u00a0G/C\u00a0\n\npolymorphisms\u00a0 in\u00a0 INT4,\u00a0 the\u00a0 amplified\u00a0 products\u00a0 were\u00a0\ncleaved\u00a0 with\u00a0 restriction\u00a0 enzyme\u00a0 ApaI\u00a0 (Fermentas\u00ae)\u00a0 as\u00a0\nper\u00a0 the\u00a0 instructions\u00a0 of\u00a0 the\u00a0 manufacturer.\u00a0 The\u00a0\nrestriction\u00a0 fragments\u00a0 obtained\u00a0 were\u00a0 electrophoresed\u00a0\nin\u00a0a\u00a02%\u00a0agarose\u00a0gel\u00a0and\u00a0were\u00a0visualized\u00a0under\u00a0UV\u00a0light\u00a0\non\u00a0a\u00a0transilluminator.\u00a0\u00a0\n\nResults:\u00a0\nGenomic\u00a0 DNA\u00a0 visualization:\u00a0 All\u00a0 the\u00a0 extracted\u00a0 DNA\u00a0\n\nsamples\u00a0 displayed\u00a0 a\u00a0 positive\u00a0 band\u00a0 when\u00a0 they\u00a0 were\u00a0\n\nelectrophoresed\u00a0in\u00a00.8%\u00a0agarose\u00a0gel.\u00a0(Figure\u00a03\u00a0and\u00a04)\u00a0\n\nPCR\u2010RFLP\u00a0analysis:\u00a0All\u00a0the\u00a024\u00a0MTB\u00a0DNA\u00a0samples\u00a0were\u00a0\n\nobserved\u00a0to\u00a0have\u00a0a\u00a0positive\u00a0amplification\u00a0for\u00a0KatG\u00a0gene\u00a0\n\nfragment.\u00a0An\u00a0amplicon\u00a0of\u00a0200\u00a0bp\u00a0size\u00a0were\u00a0observed\u00a0in\u00a0\n\na\u00a02%\u00a0agarose\u00a0gel\u00a0(Figure\u00a05).\u00a0\u00a0In\u00a0the\u00a0electrophoresis\u00a0of\u00a0\n\nthe\u00a0 KatG\u00a0 amplified\u00a0 product\u00a0 digested\u00a0 with\u00a0 MspI,\u00a0 4\u00a0\n\n(samples\u00a0 015,\u00a0 016,\u00a0 020\u00a0 and\u00a0 024)\u00a0 out\u00a0 of\u00a0 24\u00a0 samples\u00a0\n\nwere\u00a0 obtained\u00a0 at\u00a0 132\u00a0 bp\u00a0 region\u00a0 indicating\u00a0 the\u00a0\n\noccurrence\u00a0of\u00a0Ser315Thr\u00a0mutation\u00a0whereas\u00a0rest\u00a0of\u00a0the\u00a0\n\n20\u00a0samples\u00a0were\u00a0obtained\u00a0at\u00a0153\u00a0bp\u00a0region\u00a0indication\u00a0\n\nno\u00a0 Ser315Thr\u00a0 mutation\u00a0 (Figure\u00a0 6).\u00a0 Gel\u00a0 picture\u00a0 of\u00a0\n\nsamples\u00a0015\u00a0and\u00a0016\u00a0are\u00a0not\u00a0shown.\u00a0\n\nAll\u00a0the\u00a024\u00a0human\u00a0DNA\u00a0samples\u00a0from\u00a0whole\u00a0blood\u00a0had\u00a0a\u00a0\npositive\u00a0amplification\u00a0at\u00a0514\u00a0bp\u00a0region\u00a0for\u00a0INT4\u00a0(Figure\u00a0\n7).\u00a0 When\u00a0 these\u00a0 amplicons\u00a0 were\u00a0 subjected\u00a0 to\u00a0 ApaI\u00a0\ndigestion,\u00a0all\u00a0of\u00a0the\u00a024\u00a0samples\u00a0were\u00a0observed\u00a0at\u00a0514\u00a0\nbp\u00a0 region\u00a0 indicating\u00a0 no\u00a0 G/C\u00a0 polymorphisms\u00a0 in\u00a0 INT4\u00a0\nregion\u00a0of\u00a0SLC11A1\u00a0(Figure\u00a08).\u00a0\u00a0\n\nDiscussion\u00a0\nSouth\u00a0Asia\u00a0holds\u00a0around\u00a040%\u00a0of\u00a0the\u00a0global\u00a0TB\u00a0burden\u00a0\nand\u00a0 Nepal\u00a0 has\u00a0 no\u00a0 different\u00a0 scenario.\u00a0 The\u00a0 Millennium\u00a0\nDevelopment\u00a0 Goal\u00a0 (MDG)\u00a0 aims\u00a0 to\u00a0 eliminate\u00a0 TB\u00a0 as\u00a0 a\u00a0\npublic\u00a0health\u00a0problem\u00a0(1\u00a0case\u00a0per\u00a0million\u00a0population)\u00a0\nby\u00a02050.\u00a0Nepal\u00a0has\u00a0made\u00a0satisfactory\u00a0progress\u00a0towards\u00a0\nMDG.", "start_char_idx": 10327, "end_char_idx": 12506, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "75825f6c-474c-45f0-a863-3dcc9110cd86": {"__data__": {"id_": "75825f6c-474c-45f0-a863-3dcc9110cd86", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7cf4b5f8-6fd2-4b2c-acfa-966b7b0a9ae0", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "a7cf8110ef533aa8d4fa4e397386fcdc1d6b79259ebc26ac83c562d9c4f4b9ca", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "9fde521d-3df0-4d2a-a99c-a45f08fb4341", "node_type": "1", "metadata": {}, "hash": "0d68a387378796223524763507f9a6f37b3eee40a9fdfcbddff27f40923f100a", "class_name": "RelatedNodeInfo"}}, "text": "However,\u00a0the\u00a0challenges\u00a0to\u00a0address\u00a0the\u00a0growing\u00a0\nburden\u00a0of\u00a0TB\u00a0and\u00a0MDR\u2010TB\u00a0population\u00a0are\u00a0yet\u00a0far\u00a0away.\u00a0\nNepal\u00a0still\u00a0lacks\u00a0the\u00a0health\u00a0care\u00a0facilities\u00a0and\u00a0access\u00a0to\u00a0\nmodern\u00a0 diagnostic\u00a0 technologies\u00a0 is\u00a0 very\u00a0 poor\u00a0 in\u00a0 the\u00a0\narea\u00a0(Basnet\u00a0et\u00a0al.,\u00a02009).\u00a0Although\u00a0Directly\u00a0Observed\u00a0\nTreatment,\u00a0 Short\u2010course\u00a0 (DOTS)\u00a0 strategy\u00a0 has\u00a0\nsignificantly\u00a0contributed\u00a0towards\u00a0the\u00a0treatment\u00a0of\u00a0TB,\u00a0\na\u00a0 lack\u00a0 of\u00a0 rapid\u00a0 and\u00a0 sensitive\u00a0 rapid\u00a0 and\u00a0 sensitive\u00a0\nmethods\u00a0 of\u00a0 detection\u00a0 is\u00a0 a\u00a0 major\u00a0 hindrance\u00a0 to\u00a0 the\u00a0\nongoing\u00a0battle\u00a0against\u00a0the\u00a0disease.\u00a0\n\nAFB\u00a0 microscopy\u00a0 is\u00a0 the\u00a0 primary\u00a0 screening\u00a0 tool\u00a0 for\u00a0 TB\u00a0\n\nand\u00a0 culture\u00a0 of\u00a0 mycobacteria\u00a0 is\u00a0 still\u00a0 regarded\u00a0 as\u00a0 the\u00a0\n\n\u201cgold\u00a0 standard\u201d\u00a0 in\u00a0 TB\u00a0 diagnosis.\u00a0 But\u00a0 these\u00a0 days\u00a0 new\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n17\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nTarget\u00a0Gene\u00a0Region\u00a0 Primer\u00a0 Sequence\u00a0\n\nKatG\u00a0\nkatG1F\u00a0 5'\u2010AGCTCGTATGGCACCGGAAC\u20103\u2019\u00a0\n\nkatG4RB\u00a0 5'\u2010AACGGGTCCGGGATGGTG\u20103'\u00a0\n\nINT4\u00a0\nINT4F\u00a0 5'\u2010GTCTGCCATCTCTACTACCCTAAGGTG\u20103\u2019\u00a0\n\nINT4R\u00a0 5'\u2010CATGTCCCTCTAGGTATGTGCTATCAG\u20103'\u00a0\n\nTable\u00a01.\u00a0Primer\u00a0sequence\u00a0for\u00a0KatG\u00a0and\u00a0INT4\u00a0\n\nFigure\u00a0 2.\u00a0 Schematic\u00a0 illustration\u00a0 of\u00a0 the\u00a0 KatG\u00a0 200\u2010bp\u00a0 fragment\u00a0 amplified\u00a0 with\u00a0 primers\u00a0 katG1F\u00a0 and\u00a0 katG4RB.\u00a0 The\u00a0\nvertical\u00a0 line\u00a0 represents\u00a0 the\u00a0 MspI\u00a0 restriction\u00a0 site\u00a0 (CCGG).\u00a0 In\u00a0 the\u00a0 wild\u2010type\u00a0 KatG\u00a0 PCR\u00a0 product\u00a0 there\u00a0 is\u00a0 no\u00a0 MspI\u00a0\nrestriction\u00a0site\u00a0at\u00a0315th\u00a0Codon\u00a0(AGC)\u00a0and\u00a0the\u00a0 longest\u00a0fragment\u00a0 is\u00a0of\u00a0153\u00a0bp.\u00a0But\u00a0 in\u00a0the\u00a0Mutant\u00a0KatG\u00a0PCR\u00a0product\u00a0\nthere\u00a0is\u00a0an\u00a0addition\u00a0of\u00a0MspI\u00a0restriction\u00a0site\u00a0at\u00a0315th\u00a0codon\u00a0(ACC)\u00a0and\u00a0it\u00a0gives\u00a0132\u00a0bp\u00a0band\u00a0as\u00a0the\u00a0longest\u00a0fragment. \u00a0\n\nFigure\u00a0 3.\u00a0 Visualization\u00a0 of\u00a0 the\u00a0 extracted\u00a0 MTB\u00a0 DNA\u00a0 from\u00a0\ncultured\u00a0sample\u00a0 in\u00a0a\u00a00.8%\u00a0agarose\u00a0gel.\u00a0020\u2010024\u00a0are\u00a0the\u00a0\nDNA\u00a0 samples,\u00a0 PC\u00a0 is\u00a0 the\u00a0 Positive\u00a0 Control\u00a0 and\u00a0 NC\u00a0 is\u00a0 the\u00a0\nNegative\u00a0Control.\u00a0The\u00a0bands\u00a0are\u00a0very\u00a0close\u00a0to\u00a0the\u00a0loading\u00a0\nwell\u00a0indicating\u00a0the\u00a0larger\u00a0fragment\u00a0of\u00a0genomic\u00a0DNA. \u00a0\n\nFigure\u00a04.\u00a0Visualization\u00a0of\u00a0the\u00a0extracted\u00a0DNA\u00a0sample\u00a0from\u00a0\nHuman\u00a0whole\u00a0blood\u00a0in\u00a0a\u00a00.8%\u00a0agarose\u00a0gel.\u00a0001\u2010005\u00a0are\u00a0\nthe\u00a0 DNA\u00a0 samples,\u00a0 PC\u00a0 is\u00a0 the\u00a0 Positive\u00a0 Control\u00a0 and\u00a0 NC\u00a0 is\u00a0\nthe\u00a0 Negative\u00a0 Control.\u00a0 The\u00a0 bands\u00a0 are\u00a0 very\u00a0 close\u00a0 to\u00a0 the\u00a0\nloading\u00a0 well\u00a0 indicating\u00a0 the\u00a0 larger\u00a0 fragment\u00a0 of\u00a0 genomic\u00a0\nDNA.", "start_char_idx": 12507, "end_char_idx": 14717, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "9fde521d-3df0-4d2a-a99c-a45f08fb4341": {"__data__": {"id_": "9fde521d-3df0-4d2a-a99c-a45f08fb4341", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "75825f6c-474c-45f0-a863-3dcc9110cd86", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "1169fa3acb4c69b0526fbea8149522aa3a3abfd22e232d0ebcfefddb1322ad13", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "93798574-ff46-4d6e-9673-983d7445bea4", "node_type": "1", "metadata": {}, "hash": "b2cff785f260228b93201fee5c418eca97abdcfe6af39624ab71fb660993c994", "class_name": "RelatedNodeInfo"}}, "text": "Nepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n18\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nFigure\u00a05.\u00a0PCR\u00a0product\u00a0of\u00a0KatG\u00a0gene\u00a0fragment\u00a0visualized\u00a0\nin\u00a0a\u00a02%\u00a0agarose\u00a0gel.\u00a0020\u2010024\u00a0are\u00a0the\u00a0MTB\u00a0samples,\u00a0PC\u00a0is\u00a0\nthe\u00a0Positive\u00a0Control\u00a0and\u00a0NTC\u00a0is\u00a0the\u00a0No\u00a0Template\u00a0Con\u2010\ntrol.\u00a0All\u00a0the\u00a0samples\u00a0have\u00a0positive\u00a0band\u00a0amplification\u00a0at\u00a0\n200\u00a0bp\u00a0region. \u00a0\n\nFigure\u00a06.MspI\u00a0digested\u00a0product\u00a0of\u00a0KatG\u00a0gene\u00a0fragment\u00a0\nvisualized\u00a0 in\u00a0 a\u00a0 2%\u00a0 agarose\u00a0 gel.\u00a0 020\u2010024\u00a0 are\u00a0 the\u00a0 MTB\u00a0\nsamples,\u00a0PC\u00a0is\u00a0the\u00a0Positive\u00a0Control\u00a0and\u00a0NTC\u00a0is\u00a0the\u00a0No\u00a0\nTemplate\u00a0 Control.\u00a0 Samples\u00a0 021,\u00a0 022,\u00a0 and\u00a0 023\u00a0 has\u00a0 a\u00a0\nband\u00a0 size\u00a0 of\u00a0 153\u00a0 bp\u00a0 indicating\u00a0 no\u00a0 mutation\u00a0 at\u00a0 315th\u00a0\ncodon\u00a0 position\u00a0 where\u00a0 as\u00a0 sample\u00a0 020\u00a0 and\u00a0 024\u00a0 has\u00a0 a\u00a0\nband\u00a0sized\u00a0132\u00a0bp\u00a0indicating\u00a0Ser\u00e0Thr\u00a0mutation\u00a0at\u00a0315th\u00a0\ncodon\u00a0position\u00a0of\u00a0KatG\u00a0gene. \u00a0\n\nsensitivity\u00a0and\u00a0specificity.\u00a0\n\nThe\u00a0present\u00a0study\u00a0 looks\u00a0at\u00a0the\u00a0resistance\u00a0 in\u00a0the\u00a0KatG\u00a0\nSer315Thr\u00a0 towards\u00a0 a\u00a0 popular\u00a0 anti\u2010TB\u00a0 drug\u00a0 isoniazid.\u00a0\nThis\u00a0 is\u00a0 just\u00a0 one\u00a0 among\u00a0 many\u00a0 possible\u00a0 mutations\u00a0 in\u00a0\nKatG\u00a0accounting\u00a0for\u00a0INH\u00a0resistance.\u00a04\u00a0(16.67%)\u00a0samples\u00a0\n\nmolecular\u00a0 diagnostic\u00a0 tools\u00a0 are\u00a0 promoted\u00a0 globally\u00a0\n\nmostly\u00a0in\u00a0the\u00a0detection\u00a0of\u00a0MTB,\u00a0drug\u00a0monitoring\u00a0and\u00a0in\u00a0\n\nMDR\u2010TB\u00a0diagnosis\u00a0(Pai\u00a0et\u00a0al.,\u00a02006).\u00a0This\u00a0is\u00a0due\u00a0to\u00a0many\u00a0\n\nadvantages\u00a0 of\u00a0 molecular\u00a0 genomic\u00a0 tools\u00a0 over\u00a0\n\nconventional\u00a0diagnostic\u00a0methods\u00a0that\u00a0 lack\u00a0the\u00a0speed,\u00a0\n\nFigure\u00a07.\u00a0PCR\u00a0product\u00a0of\u00a0INT4\u00a0gene\u00a0fragment\u00a0visualized\u00a0\nin\u00a0 a\u00a0 2%\u00a0 agarose\u00a0 gel.\u00a0 001\u2010005\u00a0 are\u00a0 the\u00a0 human\u00a0 DNA\u00a0\nsamples,\u00a0PC\u00a0 is\u00a0the\u00a0Positive\u00a0Control\u00a0and\u00a0NTC\u00a0 is\u00a0the\u00a0No\u00a0\nTemplate\u00a0 Control.\u00a0 All\u00a0 the\u00a0 samples\u00a0 has\u00a0 positive\u00a0 band\u00a0\namplification\u00a0at\u00a0514\u00a0bp\u00a0region \u00a0\n\nFigure\u00a08.\u00a0ApaI\u00a0digested\u00a0product\u00a0of\u00a0INT4\u00a0gene\u00a0fragment\u00a0\nvisualized\u00a0in\u00a0a\u00a02%\u00a0agarose\u00a0gel.\u00a0001\u2010002\u00a0are\u00a0the\u00a0Human\u00a0\nDNA\u00a0samples,\u00a0PC\u00a0is\u00a0the\u00a0Positive\u00a0Control\u00a0and\u00a0NTC\u00a0is\u00a0the\u00a0\nNo\u00a0Template\u00a0Control.\u00a0All\u00a0the\u00a0samples\u00a0were\u00a0observed\u00a0at\u00a0\n514\u00a0bp\u00a0region\u00a0indicating\u00a0no\u00a0G/C\u00a0polymorphisms\u00a0in\u00a0INT4\u00a0\nregion\u00a0of\u00a0SLC11A1\u00a0gene \u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n19\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nout\u00a0 of\u00a0 24\u00a0 MTB\u00a0 culture\u00a0 samples\u00a0 demonstrated\u00a0\nSer315Thr\u00a0KatG\u00a0mutation\u00a0(i.e.\u00a0these\u00a0samples\u00a0were\u00a0INH\u00a0\nresistant).", "start_char_idx": 14723, "end_char_idx": 16897, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "93798574-ff46-4d6e-9673-983d7445bea4": {"__data__": {"id_": "93798574-ff46-4d6e-9673-983d7445bea4", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "9fde521d-3df0-4d2a-a99c-a45f08fb4341", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "dd4bdd5110d3ee4d38a85c9212332e6af2d253645c8b5e436cd682ae75497349", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "496fb350-c4b1-4c19-8673-abf5fc2f200c", "node_type": "1", "metadata": {}, "hash": "e9521cdd69ade20c5810bf9e94e032cec5d9bd37ec20a2f59a60cd4883b66d6c", "class_name": "RelatedNodeInfo"}}, "text": "these\u00a0samples\u00a0were\u00a0INH\u00a0\nresistant).\u00a0 It\u00a0 indicates\u00a0 that\u00a0 there\u00a0 could\u00a0 possibly\u00a0 be\u00a0\nsignificantly\u00a0 high\u00a0 anti\u2010TB\u00a0 drug\u00a0 resistant\u00a0 population\u00a0 if\u00a0\nscreened\u00a0in\u00a0a\u00a0larger\u00a0population.\u00a0One\u00a0of\u00a0the\u00a0drawbacks\u00a0\nof\u00a0 this\u00a0 study\u00a0 is\u00a0 that\u00a0 other\u00a0 mutation\u00a0 in\u00a0 various\u00a0 genes\u00a0\n(InhA,\u00a0 OxyR,\u00a0 AhpC)\u00a0 that\u00a0 contributes\u00a0 to\u00a0 the\u00a0 INH\u00a0\nresistance\u00a0 were\u00a0 not\u00a0 studied.\u00a0 Studies\u00a0 have\u00a0 reported\u00a0\nthat\u00a0even\u00a0if\u00a0there\u00a0is\u00a0Ser315Ther\u00a0KatG\u00a0mutation,\u00a0it\u00a0does\u00a0\nnot\u00a0necessarily\u00a0account\u00a0to\u00a0INH\u00a0resistance\u00a0and\u00a0have\u00a0an\u00a0\noverall\u00a0high\u00a0level\u00a0of\u00a0catalase\u00a0activity\u00a0(Guo\u00a0et\u00a0al.,\u00a02006).\u00a0\nBoth\u00a0 the\u00a0 catalase\u00a0 activity\u00a0 and\u00a0 drug\u00a0 sensitivity\u00a0\ninformation\u00a0could\u00a0not\u00a0be\u00a0obtained.\u00a0\u00a0\n\nThe\u00a0association\u00a0between\u00a0SLC11A1\u00a0polymorphisms\u00a0and\u00a0\n\nsusceptibility\u00a0 to\u00a0 tuberculosis\u00a0 has\u00a0 been\u00a0 described\u00a0 in\u00a0\n\nmany\u00a0 studies,\u00a0 which\u00a0 showed\u00a0 positive\u00a0 association\u00a0 in\u00a0\n\nsome,\u00a0while\u00a0no\u00a0association\u00a0in\u00a0others\u00a0(Takahashi\u00a0et\u00a0al.,\u00a0\n\n2008).\u00a0 A\u00a0 study\u00a0 showed\u00a0 that\u00a0 NRAMP1\u00a0 polymorphisms\u00a0\n\nmay\u00a0be\u00a0associated\u00a0with\u00a0progression\u00a0to\u00a0severe\u00a0forms\u00a0of\u00a0\n\npulmonary\u00a0tuberculosis\u00a0rather\u00a0than\u00a0with\u00a0susceptibility\u00a0\n\nto\u00a0M.\u00a0tuberculosis\u00a0infection\u00a0(Zhang\u00a0et\u00a0al.,\u00a02005).\u00a0All\u00a0the\u00a0\n\nINT4\u00a0 amplicons\u00a0 when\u00a0 subjected\u00a0 to\u00a0 ApaI\u00a0 digestion,\u00a0\n\nbands\u00a0of\u00a0514\u00a0bp\u00a0were\u00a0observed.\u00a0If\u00a0the\u00a0INT4\u00a0region\u00a0had\u00a0\n\nG/C\u00a0polymorphisms\u00a0then\u00a0restriction\u00a0site(s)\u00a0would\u00a0have\u00a0\n\nbeen\u00a0additionally\u00a0created\u00a0and\u00a0the\u00a0RFLP\u00a0bands\u00a0of\u00a0<500\u00a0\n\nbp\u00a0size\u00a0would\u00a0have\u00a0been\u00a0observed.\u00a0\u00a0This\u00a0indicates\u00a0that\u00a0\n\nno\u00a0 G/C\u00a0 polymorphism\u00a0 was\u00a0 present\u00a0 in\u00a0 the\u00a0 amplified\u00a0\n\nFigure\u00a09.", "start_char_idx": 16862, "end_char_idx": 18317, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "496fb350-c4b1-4c19-8673-abf5fc2f200c": {"__data__": {"id_": "496fb350-c4b1-4c19-8673-abf5fc2f200c", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "93798574-ff46-4d6e-9673-983d7445bea4", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "96a277d87b915cbda2ae3dae4bd32f4fe9664e462d2e137d79e77ab3e81fc176", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8c936f50-7218-4a40-a350-5497d0e197d1", "node_type": "1", "metadata": {}, "hash": "2e5c803a8d6f2646090edbc13a7950d3f2f7d27259c323ca299eea4c54771ab3", "class_name": "RelatedNodeInfo"}}, "text": "Percentage\u00a0Distribution\u00a0of\u00a0KatG\u00a0Ser315Thr\u00a0Mu\u2010\ntation \u00a0\n\nSample\u00a0ID\u00a0 AFB\u00a0Stain\u00a0 MTB\u00a0Culture\u00a0 MDR\u2010TB\u00a0 KatG\u00a0PCR\u00a0ampli\u2010\ncon\u00a0size\u00a0(bp)\u00a0\n\nMspI\u00a0digested\u00a0\nproduct\u00a0size\u00a0\n\n(bp)\u00a0\n\nSer315Thr\u00a0KatG\u00a0\nmutation\u00a0\n\nMTB\u2010001\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010002\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010003\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010004\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010005\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010006\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010007\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010008\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010009\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010010\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010011\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010012\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010013\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010014\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010015\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 132\u00a0 POSITIVE\u00a0\n\nMTB\u2010016\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 132\u00a0 POSITIVE\u00a0\n\nMTB\u2010017\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010018\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010019\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010020\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 132\u00a0 POSITIVE\u00a0\n\nMTB\u2010021\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010022\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010023\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 153\u00a0 NEGATIVE\u00a0\n\nMTB\u2010024\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 POSITIVE\u00a0 200\u00a0 132\u00a0 POSITIVE\u00a0\n\nTable\u00a02.\u00a0Relationship\u00a0between\u00a0AFB\u00a0Stain,\u00a0MTB\u00a0Culture\u00a0and\u00a0PCR\u2010RFLP\u00a0Results\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n20\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nregion\u00a0of\u00a0INT4.\u00a0The\u00a0present\u00a0study\u00a0shows\u00a0no\u00a0significant\u00a0\n\nassociation\u00a0between\u00a0G/C\u00a0polymorphism\u00a0INT4\u00a0region\u00a0of\u00a0\n\nSLC11A1\u00a0with\u00a0TB.\u00a0With\u00a0the\u00a0limited\u00a0number\u00a0of\u00a0samples\u00a0\n\nand\u00a0failure\u00a0to\u00a0study\u00a0D543N\u00a0and\u00a03\u2019UTR\u00a0region,\u00a0the\u00a0role\u00a0\n\nof\u00a0 SLC11A1\u00a0 in\u00a0 susceptibility\u00a0 towards\u00a0 TB\u00a0 cannot\u00a0 over\u2010\n\nruled.", "start_char_idx": 18318, "end_char_idx": 20453, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8c936f50-7218-4a40-a350-5497d0e197d1": {"__data__": {"id_": "8c936f50-7218-4a40-a350-5497d0e197d1", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "496fb350-c4b1-4c19-8673-abf5fc2f200c", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "0af0a624a58df2b8f58af309069a4c7f8abd08857100d170d1c71e797b94c855", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "23c19f04-0898-4766-8470-6d3d466cf18c", "node_type": "1", "metadata": {}, "hash": "cc2cf541c82d08f0093dcee37c5a41f5a7f5b8edcd1b50d9fbda45a883ac1e08", "class_name": "RelatedNodeInfo"}}, "text": "As\u00a0 WHO\u00a0 claims\u00a0 one\u2010third\u00a0 of\u00a0 the\u00a0 world\u2019s\u00a0\n\npopulation\u00a0 to\u00a0 be\u00a0 infected\u00a0 with\u00a0 latent\u00a0 form\u00a0 of\u00a0 TB,\u00a0\n\nSLC11A1\u00a0 polymorphisms\u00a0 can\u00a0 be\u00a0 accredited\u00a0 to\u00a0 be\u00a0 a\u00a0\n\nhelping\u00a0factor\u00a0for\u00a0TB\u00a0susceptibility\u00a0or\u00a0progression.\u00a0\n\nThis\u00a0study\u00a0has\u00a0considered\u00a0M.\u00a0tuberculosis\u00a0H37Rv\u00a0as\u00a0a\u00a0\n\nreference\u00a0 strain\u00a0 but\u00a0 other\u00a0 clinical\u00a0 strain\u00a0 could\u00a0 also\u00a0\n\nhave\u00a0been\u00a0prevalent.\u00a0This\u00a0fact\u00a0remained\u00a0as\u00a0a\u00a0challenge\u00a0\n\nSampleID\u00a0 MDR\u2010TB\u00a0 INT4\u00a0PCR\u00a0amplicon\u00a0size\u00a0\n(bp)\u00a0\n\nApaI\u00a0digested\u00a0product\u00a0of\u00a0\nINT4\u00a0(bp)\u00a0\n\nG/C\u00a0Polymorphism\u00a0in\u00a0INT4\u00a0\n\nSLC\u2010001\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010002\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010003\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010004\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010005\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010006\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010007\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010008\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010009\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010010\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010011\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010012\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010013\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010014\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010015\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010016\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010017\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010018\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010019\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010020\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010021\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010022\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010023\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nSLC\u2010024\u00a0 POSITIVE\u00a0 514\u00a0 514\u00a0 NEGATIVE\u00a0\n\nTable\u00a03.\u00a0Relationship\u00a0MDR\u2010TB\u00a0and\u00a0G/C\u00a0polymorphism\u00a0in\u00a0INT4\u00a0region\u00a0of\u00a0SLC11A1\u00a0\n\nto\u00a0 our\u00a0 study\u00a0 as\u00a0 no\u00a0 base\u2010line\u00a0 study\u00a0 on\u00a0 MTB\u00a0 strain\u00a0\n\ngenotyping\u00a0 has\u00a0 been\u00a0 conducted\u00a0 for\u00a0 Nepalese\u00a0 MTB\u00a0\n\nisolates\u00a0till\u00a0date.\u00a0\n\nConclusion\u00a0\nThe\u00a0 findings\u00a0 in\u00a0 this\u00a0 study\u00a0 suggests\u00a0 that\u00a0 still\u00a0 more\u00a0\npeople\u00a0could\u00a0be\u00a0properly\u00a0diagnosed\u00a0for\u00a0the\u00a0actual\u00a0drug\u00a0\nresistance\u00a0 mutations\u00a0 utilizing\u00a0 molecular\u00a0 genomic\u00a0\ndiagnostic\u00a0tools.\u00a0Further\u00a0study\u00a0with\u00a0a\u00a0greater\u00a0number\u00a0\nof\u00a0 MDR\u2010TB\u00a0 patients\u00a0 is\u00a0 needed\u00a0 to\u00a0 get\u00a0 the\u00a0 actual\u00a0\nscenario\u00a0of\u00a0TB\u00a0population\u00a0of\u00a0Nepal.\u00a0\u00a0\n\nReference\u00a0\nWorld\u00a0Health\u00a0Organization.\u00a0Global\u00a0Tuberculosis\u00a0\nControl.\u00a0A\u00a0short\u00a0update\u00a0to\u00a0the\u00a02009\u00a0report.\u00a02009\u00a0\n\n\u00a0\nWorld\u00a0Health\u00a0Organization.", "start_char_idx": 20455, "end_char_idx": 22609, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "23c19f04-0898-4766-8470-6d3d466cf18c": {"__data__": {"id_": "23c19f04-0898-4766-8470-6d3d466cf18c", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "8c936f50-7218-4a40-a350-5497d0e197d1", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "4a972a9d1d7273c98bafa2ae567eb4d124c83f14cff4d7b73f2e25d334938aac", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a4ff725f-fe58-4822-8b21-9bf77540b72b", "node_type": "1", "metadata": {}, "hash": "0f2e20d34c1ac15aa49fea8e5ad0fb489f5c2bf9b99c569cf00fb60eaf3ad5d6", "class_name": "RelatedNodeInfo"}}, "text": "2009\u00a0\n\n\u00a0\nWorld\u00a0Health\u00a0Organization.\u00a0Multidrug\u00a0and\u00a0extensively\u00a0\ndrug\u2010resistant\u00a0TB\u00a0(M/XDR\u2010TB)\u00a02010\u00a0Global\u00a0Report\u00a0on\u00a0\nSurveillance\u00a0and\u00a0Response.\u00a02010\u00a0\n\n\u00a0\nNational\u00a0 Tuberculosis\u00a0 Center.\u00a0 National\u00a0 Tuberculosis\u00a0\n\nControl\u00a0 Programme\u00a0 Nepal\u00a0 \u2010\u00a0 Annual\u00a0 Report\u00a0 FY\u00a0\n\n2065/66\u00a0(2008/2009).\u00a02009\u00a0\n\nMusser\u00a0 JM.\u00a0 Antimicrobial\u00a0 agent\u00a0 resistance\u00a0 in\u00a0\n\nmycobacteria:\u00a0 molecular\u00a0 genetic\u00a0 insights.\u00a0 Clin\u00a0\n\nMicrobiol\u00a0Rev.\u00a01995\u00a0Oct;8(4):496\u2010514.\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a014\u201021\u00a0\n\n21\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nRattan\u00a0 A,\u00a0 Kalia\u00a0 A,\u00a0 Ahmad\u00a0 N.\u00a0 Multidrug\u2010resistant\u00a0\n\nMycobacterium\u00a0 tuberculosis:\u00a0 molecular\u00a0\n\nperspectives.\u00a0 Emerg\u00a0 Infect\u00a0 Dis.\u00a0 1998\u00a0 Apr\u2010Jun;4\n\n(2):195\u2010209.\u00a0\n\nMokrousov\u00a0 I,\u00a0 Narvskaya\u00a0 O,\u00a0 Otten\u00a0 T,\u00a0 Limeschenko\u00a0 E,\u00a0\n\nSteklova\u00a0 L,\u00a0 Vyshnevskiy\u00a0 B.\u00a0 High\u00a0 prevalence\u00a0 of\u00a0 KatG\u00a0\n\nSer315Thr\u00a0 substitution\u00a0 among\u00a0 isoniazid\u2010resistant\u00a0\n\nMycobacterium\u00a0 tuberculosis\u00a0 clinical\u00a0 isolates\u00a0 from\u00a0\n\nnorthwestern\u00a0 Russia,\u00a0 1996\u00a0 to\u00a0 2001.\u00a0 Antimicrob\u00a0\n\nAgents\u00a0Chemother.\u00a02002\u00a0May;46(5):1417\u201024.\u00a0\n\nTakahashi\u00a0 K,\u00a0 Hasegawa\u00a0 Y,\u00a0 Abe\u00a0 T,\u00a0 Yamamoto\u00a0 T,\u00a0\n\nNakashima\u00a0 K,\u00a0 Imaizumi\u00a0 K,\u00a0 Shimokata\u00a0 K.\u00a0 SLC11A1\u00a0\n\n(formerly\u00a0 NRAMP1)\u00a0 polymorphisms\u00a0 associated\u00a0 with\u00a0\n\nmultidrug\u2010resistant\u00a0 tuberculosis.\u00a0 Tuberculosis\u00a0\n\n(Edinb).\u00a02008\u00a0Jan;88(1):52\u20107.\u00a0\n\nMarquet\u00a0S,\u00a0Lepage\u00a0P,\u00a0Hudson\u00a0TJ,\u00a0Musser\u00a0JM,\u00a0Schurr\u00a0E.\u00a0\n\nComplete\u00a0 nucleotide\u00a0 sequence\u00a0 and\u00a0 genomic\u00a0\n\nstructure\u00a0 of\u00a0 the\u00a0 human\u00a0 NRAMP1\u00a0 gene\u00a0 region\u00a0 on\u00a0\n\nchromosome\u00a0 region\u00a0 2q35.\u00a0 Mamm\u00a0 Genome\u00a0 2000,\u00a0\n\n11:755\u2010762.\u00a0\n\nGoswami\u00a0T,\u00a0Bhattacharjee\u00a0A,\u00a0Babal\u00a0P,\u00a0Searle\u00a0S,\u00a0Moore\u00a0\n\nE,\u00a0 Li\u00a0 M,\u00a0 Blackwell\u00a0 JM.\u00a0 Natural\u2010resistanceassociated\u00a0\n\nmacrophage\u00a0 protein\u00a0 1\u00a0 is\u00a0 an\u00a0 H+/bivalent\u00a0 cation\u00a0\n\nantiporter.\u00a0Biochem\u00a0J\u00a02001;\u00a0354:\u00a0511\u2013519.\u00a0\n\nTaype\u00a0CA,\u00a0Castro\u00a0JC,\u00a0Accinelli\u00a0RA,\u00a0Herrera\u2010Velit\u00a0P,\u00a0Shaw\u00a0\n\nMA,\u00a0 Espinoza\u00a0 JR.\u00a0 Association\u00a0 between\u00a0 SLC11A1\u00a0\n\npolymorphisms\u00a0and\u00a0susceptibility\u00a0to\u00a0different\u00a0clinical\u00a0\n\nforms\u00a0 of\u00a0 tuberculosis\u00a0 in\u00a0 the\u00a0 Peruvian\u00a0 population.\u00a0\n\nInfect\u00a0Genet\u00a0Evol.\u00a02006\u00a0Sep;6(5):361\u20107.\u00a0\n\nPai\u00a0 M,\u00a0 Kalantri\u00a0 S,\u00a0 Dheda\u00a0 K.\u00a0 New\u00a0 tools\u00a0 and\u00a0 emerging\u00a0\n\ntechnologies\u00a0for\u00a0the\u00a0diagnosis\u00a0of\u00a0tuberculosis:\u00a0part\u00a0II.", "start_char_idx": 22574, "end_char_idx": 24696, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a4ff725f-fe58-4822-8b21-9bf77540b72b": {"__data__": {"id_": "a4ff725f-fe58-4822-8b21-9bf77540b72b", "embedding": null, "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-56", "node_type": "4", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "6fdb5a37a00bd3e8b55ed73cf364a32b0a8c9a2505ce36ba6815f710c6f7eaa6", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "23c19f04-0898-4766-8470-6d3d466cf18c", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "a5649b1c04c93586a76e85fe4668fa044f91d1e77c479c72e884093745f21a5d", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "a68d3a0c-8cb6-48c1-b273-ace6242c339a", "node_type": "1", "metadata": {}, "hash": "d8ffbfe4b68074f6bb515c3e35438024e8862af717dfbbd2ee7cef28b6fc502e", "class_name": "RelatedNodeInfo"}}, "text": "Active\u00a0 tuberculosis\u00a0 and\u00a0 drug\u00a0 resistance.\u00a0 Expert\u00a0 Rev\u00a0\n\nMol\u00a0Diagn.\u00a02006\u00a0May;6(3):423\u201032.\u00a0\n\nBasnet\u00a0R,\u00a0Hinderaker\u00a0SG,\u00a0Enarson\u00a0D,\u00a0Malla\u00a0P,\u00a0M\u00f8rkve\u00a0\n\nO.\u00a0 Delay\u00a0 in\u00a0 the\u00a0 diagnosis\u00a0 of\u00a0 tuberculosis\u00a0 in\u00a0 Nepal.\u00a0\n\nBMC\u00a0Public\u00a0Health.\u00a02009\u00a0Jul\u00a014;9:236.\u00a0\n\nGuo\u00a0H,\u00a0Seet\u00a0Q,\u00a0Denkin\u00a0S,\u00a0Parsons\u00a0L,\u00a0Zhang\u00a0Y.\u00a0Molecular\u00a0\n\ncharacterization\u00a0of\u00a0isoniazid\u2010resistant\u00a0clinical\u00a0isolates\u00a0\n\nof\u00a0Mycobacterium\u00a0tuberculosis\u00a0from\u00a0the\u00a0USA.\u00a0J\u00a0Med\u00a0\n\nMicrobiol.\u00a02006\u00a0Nov;55(Pt\u00a011):1527\u201031.\u00a0\n\nZhang\u00a0W,\u00a0Shao\u00a0L,\u00a0Weng\u00a0X,\u00a0Hu\u00a0Z,\u00a0Jin\u00a0A,\u00a0Chen\u00a0S,\u00a0Pang\u00a0M,\u00a0\nChen\u00a0 ZW.\u00a0 Variants\u00a0 of\u00a0 the\u00a0 natural\u00a0 resistance\u2010\nassociated\u00a0 macrophage\u00a0 protein\u00a0 1\u00a0 gene\u00a0 (NRAMP1)\u00a0\nare\u00a0 associated\u00a0 with\u00a0 severe\u00a0 forms\u00a0 of\u00a0 pulmonary\u00a0\ntuberculosis.\u00a0Clin\u00a0Infect\u00a0Dis.\u00a02005\u00a0May\u00a01;40(9):1232\u2010\n6.\u00a0Epub\u00a02005\u00a0Mar\u00a023.", "start_char_idx": 24699, "end_char_idx": 25445, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "a68d3a0c-8cb6-48c1-b273-ace6242c339a": {"__data__": {"id_": "a68d3a0c-8cb6-48c1-b273-ace6242c339a", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a4ff725f-fe58-4822-8b21-9bf77540b72b", "node_type": "1", "metadata": {"identifier": "njb-56", "author": "Shrestha, Raunak; Joshi, Rubin Narayan; Joshi, Kriti; Poudel, Bal Hari; Shrestha, Bhupal Govinda", "title": "Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study", "date": "2022-07-11", "file": "njb-56.pdf"}, "hash": "748b875da136810672a2a7f358916b2f857aecc75b30597a79c58c432a9017d9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2459357a-9167-41bf-89ce-13868ae6800e", "node_type": "1", "metadata": {}, "hash": "c132b1ce35e6a79c2191ea4cc4f6f1d28d001237ae92337d4abe3a719a642172", "class_name": "RelatedNodeInfo"}}, "text": "2\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a09\u201013\u00a0\n\n9\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nORIGINAL\u00a0RESEARCH\u00a0ARTICLE\u00a0\n\nAn\u00a0Evaluation\u00a0of\u00a0the\u00a0Fungi\u00a0Isolated\u00a0from\u00a0Sub\u2010\nepidermal\u00a0Region\u00a0of\u00a0Post\u2010harvested\u00a0Stored\u00a0Wheat\u00a0\n\nGrains\u00a0\nShiju\u00a0Mathew1,\u00a0George\u00a0Thomas2,\u00a0Tufail\u00a0Ahmad3\u00a0\n\n1\u00a0Ministry\u00a0of\u00a0Higher\u00a0Education,\u00a0Aksum\u00a0University,\u00a0Aksum\u00a0(Ethiopia)\u00a0\u00a02\u00a0Department\u00a0of\u00a0Molecular\u00a0Biology\u00a0&\u00a0Genetic\u00a0Engineering,\u00a0Allahabad\u00a0\nAgricultural\u00a0Institute\u00a0\u2010Deemed\u00a0University,\u00a0Allahabad\u00a0211007\u00a0(India)\u00a0\u00a03\u00a0\u00a0Department\u00a0of\u00a0Agriculture\u00a0Process\u00a0and\u00a0Food\u00a0Engineering,\u00a0Allahabad\u00a0\n\nAgricultural\u00a0Institute\u00a0\u2013\u00a0Deemed\u00a0University,\u00a0Allahabad\u00a0211007\u00a0(India)\u00a0\n\nAbstract\u00a0\n\nThe\u00a0 criteria\u00a0 of\u00a0 wheat\u00a0 quality\u00a0 are\u00a0 as\u00a0 varied\u00a0 as\u00a0 their\u00a0 different\u00a0 uses.\u00a0 Wheat,\u00a0 which\u00a0 is\u00a0 suitable\u00a0 for\u00a0 a\u00a0\nparticular\u00a0use\u00a0/\u00a0product,\u00a0may\u00a0have\u00a0certain\u00a0characters\u00a0that\u00a0make\u00a0it\u00a0entirely\u00a0unsatisfactory\u00a0for\u00a0other\u00a0\npurposes.\u00a0 The\u00a0 storage\u00a0 fungi\u00a0 damage\u00a0 the\u00a0 grains\u00a0 in\u00a0 several\u00a0 ways;\u00a0 they\u00a0 reduce\u00a0 the\u00a0 germinability,\u00a0\nproduce\u00a0undesirable\u00a0odor\u00a0and\u00a0kernel\u00a0discoloration,\u00a0decrease\u00a0the\u00a0food\u00a0value\u00a0and\u00a0also\u00a0produce\u00a0toxins\u00a0\ninjurious\u00a0 to\u00a0 the\u00a0 health\u00a0 of\u00a0 consumers.\u00a0 The\u00a0 sub\u2010epidermal\u00a0 mycoflora\u00a0 of\u00a0 stored\u00a0 wheat\u00a0 grains\u00a0\npredominantly\u00a0consisted\u00a0of\u00a0ubiquitous\u00a0mould\u00a0genera\u00a0Aspergillus,\u00a0Alternaria\u00a0and\u00a0Penicillium\u00a0possibly\u00a0\nbecause\u00a0 of\u00a0 their\u00a0 omnipresence,\u00a0 capacity\u00a0 to\u00a0 grow\u00a0 on\u00a0 all\u00a0 possible\u00a0 substrates\u00a0 and\u00a0 a\u00a0 wide\u00a0 range\u00a0 of\u00a0\ntemperature\u00a0 and\u00a0 humidity.\u00a0 The\u00a0 most\u00a0 frequent\u00a0 species\u00a0 observed\u00a0 in\u00a0 the\u00a0 stored\u00a0 wheat\u00a0 grains\u00a0 of\u00a0\nAspergillus\u00a0 were\u00a0 A.\u00a0 niger\u00a0 and\u00a0 A.\u00a0 fumigates,\u00a0 Alternaria\u00a0 alternata\u00a0 and\u00a0 Pencillium\u00a0 citrinum.\u00a0 Among\u00a0\nthese\u00a0the\u00a0frequency\u00a0of\u00a0Alternaria\u00a0alternata\u00a0was\u00a0highest\u00a0which\u00a0has\u00a0the\u00a0capacity\u00a0to\u00a0produce\u00a0mycotoxin\u00a0\nwhich\u00a0can\u00a0contaminate\u00a0and\u00a0cause\u00a0spoilage.\u00a0The\u00a0grain\u00a0losses\u00a0found\u00a0in\u00a0quantity\u00a0and\u00a0quality;\u00a0can\u00a0be\u00a0in\u00a0\nthe\u00a0 form\u00a0 of\u00a0 depletion\u00a0 in\u00a0 seed\u00a0 viability,\u00a0 hardness,\u00a0 color,\u00a0 size\u00a0 and\u00a0 shape,\u00a0 grain\u00a0 weight\u00a0 and\u00a0 various\u00a0\nbiochemical\u00a0parameters\u00a0viz.,\u00a0protein,\u00a0carbohydrate\u00a0and\u00a0vitamins\u00a0under\u00a0post\u00a0harvest\u00a0storages.\u00a0\u00a0\n\u00a0\nKey\u00a0words:\u00a0wheat,\u00a0post\u2010harvest,\u00a0storage,\u00a0sub\u2010epidermal\u00a0fungi\u00a0and\u00a0spoilage.\u00a0\n\n\u00a0\nCorrespondence\u00a0Author:\u00a0\nE\u2010mail:\u00a0shijumathew_biotech@yahoo.com;\u00a0Contact\u00a0no.:\u00a0(+251)\u00a0920126043\u00a0\n\nIntroduction\u00a0\nGrain\u00a0 production\u00a0 in\u00a0 any\u00a0 country\u00a0 varies\u00a0 from\u00a0 year\u00a0 to\u00a0\n\nyear\u00a0 and\u00a0 hence\u00a0 the\u00a0 grains\u00a0 should\u00a0 be\u00a0 stored\u00a0\n\nstrategically\u00a0 from\u00a0 years\u00a0 of\u00a0 overproduction\u00a0 for\u00a0 use\u00a0 in\u00a0\n\nyear\u00a0of\u00a0under\u00a0production.", "start_char_idx": 45, "end_char_idx": 2478, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2459357a-9167-41bf-89ce-13868ae6800e": {"__data__": {"id_": "2459357a-9167-41bf-89ce-13868ae6800e", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "a68d3a0c-8cb6-48c1-b273-ace6242c339a", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "347a0d5fdb50a0cc313942e85e64f471417dee03987df3c21f27301effa4b759", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "bd3cd14c-c906-4b73-8a28-89aea230249d", "node_type": "1", "metadata": {}, "hash": "eb6449af3fe9c1b02b605528bf7c52b90a8ba7022dc0c5ddcdccd96e594624ec", "class_name": "RelatedNodeInfo"}}, "text": "Grain\u00a0quality\u00a0after\u00a0harvest\u00a0is\u00a0\n\ninfluenced\u00a0 by\u00a0 a\u00a0 wide\u00a0 variety\u00a0 of\u00a0 biotic\u00a0 and\u00a0 abiotic\u00a0\n\nfactors\u00a0 and\u00a0 has\u00a0 been\u00a0 studied\u00a0 as\u00a0 a\u00a0 stored\u00a0 grain\u00a0\n\necosystem.\u00a0 Spoilage\u00a0 of\u00a0 stored\u00a0 grain\u00a0 by\u00a0 fungi\u00a0 is\u00a0\n\ndetermined\u00a0 by\u00a0 a\u00a0 range\u00a0 of\u00a0 factors\u00a0 which\u00a0 can\u00a0 be\u00a0\n\nclassified\u00a0 into\u00a0 four\u00a0 main\u00a0 groups\u00a0 including\u00a0 (a)\u00a0 intrinsic\u00a0\n\nnutritional\u00a0 factors,\u00a0 (b)\u00a0 extrinsic\u00a0 factors\u00a0 (c)\u00a0 processing\u00a0\n\nfactors\u00a0 and\u00a0 (d)\u00a0 implicit\u00a0 microbial\u00a0 factors.\u00a0 The\u00a0 factors\u00a0\n\nproduce\u00a0 fungal\u00a0 colonization\u00a0 within\u00a0 the\u00a0 stored\u00a0 grains\u00a0\n\n(Wallace\u00a0and\u00a0Sinha,\u00a01981;\u00a0Sinha,\u00a01995).\u00a0In\u00a01970s,\u00a0it\u00a0was\u00a0\n\nconsidered\u00a0for\u00a0the\u00a0first\u00a0time\u00a0that\u00a0the\u00a0stored\u00a0grain\u00a0as\u00a0a\u00a0\n\nmanmade\u00a0ecosystem\u00a0which\u00a0needed\u00a0to\u00a0be\u00a0examined\u00a0in\u00a0\n\na\u00a0 more\u00a0 holistic\u00a0 and\u00a0 ecological\u00a0 manner\u00a0 to\u00a0 enable\u00a0 a\u00a0\n\nproper\u00a0 understanding\u00a0 of\u00a0 the\u00a0 processes\u00a0 occurring\u00a0 and\u00a0\n\nto\u00a0 improve\u00a0 post\u2010harvest\u00a0 management\u00a0 of\u00a0 stored\u00a0 food\u00a0\n\ncommodities\u00a0of\u00a0all\u00a0types.\u00a0The\u00a0post\u00a0harvest\u00a0losses\u00a0at\u00a0the\u00a0\n\nfarm\u00a0 level\u00a0 have\u00a0 been\u00a0 estimated\u00a0 to\u00a0 be\u00a0 3.28\u00a0 kg/q\u00a0 in\u00a0\n\nwheat.\u00a0The\u00a0post\u00a0harvest\u00a0loss\u00a0of\u00a0wheat\u00a0grain\u00a0has\u00a0been\u00a0\n\nfound\u00a0 to\u00a0 be\u00a0 highest\u00a0 during\u00a0 storage\u00a0 (Magan\u00a0 et\u00a0 al.,\u00a0\n\n2003).\u00a0Stored\u00a0grains\u00a0can\u00a0have\u00a0 losses\u00a0 in\u00a0both\u00a0quantity\u00a0\n\nand\u00a0quality.\u00a0Losses\u00a0occur\u00a0when\u00a0the\u00a0grain\u00a0is\u00a0attacked\u00a0by\u00a0\n\nmicroorganisms\u00a0and\u00a0other\u00a0organisms\u00a0including\u00a0insects,\u00a0\n\nmites,\u00a0rodents\u00a0and\u00a0birds\u00a0(Neetirajan\u00a0et\u00a0al.,\u00a02007).\u00a0\n\nThe\u00a0 wheat\u00a0 grains\u00a0 come\u00a0 in\u00a0 association\u00a0 with\u00a0 the\u00a0 fungi\u00a0\n\nfrom\u00a0the\u00a0time\u00a0of\u00a0grain\u00a0maturity\u00a0and\u00a0also\u00a0at\u00a0the\u00a0time\u00a0of\u00a0\n\nstorage.\u00a0 Some\u00a0 of\u00a0 these\u00a0 fungi\u00a0 are\u00a0 in\u00a0 intimate\u00a0\n\nassociation\u00a0 and\u00a0 are\u00a0 present\u00a0 as\u00a0 dormant\u00a0 mycelium\u00a0\n\nunder\u00a0the\u00a0pericarp\u00a0or\u00a0dormant\u00a0spores\u00a0on\u00a0the\u00a0surface\u00a0of\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a09\u201013\u00a0\n\n10\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nthe\u00a0 kernel.\u00a0 However,\u00a0 there\u00a0 are\u00a0 a\u00a0 number\u00a0 of\u00a0 fungi\u00a0\n\nwhich\u00a0 are\u00a0 only\u00a0 superficially\u00a0 associated\u00a0 with\u00a0 stored\u00a0\n\ngrains.\u00a0 The\u00a0 mycelium\u00a0 was\u00a0 usually\u00a0 septate,\u00a0 thin\u00a0 and\u00a0\n\nbranching\u00a0 repeatedly\u00a0 to\u00a0 form\u00a0 a\u00a0 network\u00a0 on\u00a0 the\u00a0 sub\u2010\n\nepidermal\u00a0surface\u00a0of\u00a0stored\u00a0wheat\u00a0grains.\u00a0Sometimes,\u00a0\n\ndue\u00a0 to\u00a0 more\u00a0 frequent\u00a0 formation\u00a0 of\u00a0 transverse\u00a0 walls,\u00a0\n\nbeaded\u00a0cells\u00a0were\u00a0seen.\u00a0The\u00a0sub\u2010epidermal\u00a0mycelium\u00a0\n\nwas\u00a0observed\u00a0even\u00a0in\u00a0apparently,\u00a0healthy,\u00a0undamaged\u00a0\n\ngrains\u00a0 examined\u00a0 superficially\u00a0 with\u00a0 hand\u00a0 lens.\u00a0 The\u00a0\n\nassociation\u00a0of\u00a0fungi\u00a0with\u00a0cereal\u00a0grains\u00a0starts\u00a0from\u00a0the\u00a0\n\nfield\u00a0itself.", "start_char_idx": 2479, "end_char_idx": 4822, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "bd3cd14c-c906-4b73-8a28-89aea230249d": {"__data__": {"id_": "bd3cd14c-c906-4b73-8a28-89aea230249d", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2459357a-9167-41bf-89ce-13868ae6800e", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "3d9b0450a5badc1a4cd001a849c5bf5028b01a24af4e515c7d51e5b705be6451", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5ebed3e2-cc95-4026-ae76-48cd6ae814bd", "node_type": "1", "metadata": {}, "hash": "6231e67f45a70aff2b94503144d32d98108436a6570a04f733c3df511f0b10cc", "class_name": "RelatedNodeInfo"}}, "text": "Shortly\u00a0after\u00a0the\u00a0grain\u00a0reaches\u00a0to\u00a0maximum\u00a0\n\nsize,\u00a0 the\u00a0 lemma\u00a0 and\u00a0 palea\u00a0 protecting\u00a0 it\u00a0 are\u00a0 pushed\u00a0\n\napart\u00a0 exposing\u00a0 the\u00a0 grain\u00a0 to\u00a0 infection\u00a0 by\u00a0 fungi\u00a0\n\n(Machacek\u00a0 and\u00a0 Greaney,\u00a0 1938)\u00a0 and\u00a0 their\u00a0 extensive\u00a0\n\nstudies\u00a0has\u00a0been\u00a0carried\u00a0out\u00a0in\u00a0the\u00a0laboratory\u00a0on\u00a0these\u00a0\n\naspects\u00a0 (Sankaran\u00a0 et\u00a0 al.,\u00a0 1975;\u00a0 Sankaran,\u00a0 1976;\u00a0\n\nSankaran\u00a0 et\u00a0 al.,\u00a0 1976).\u00a0 An\u00a0 extensive\u00a0 microflora\u00a0 has\u00a0\n\nbeen\u00a0found\u00a0to\u00a0be\u00a0associated\u00a0with\u00a0stored\u00a0wheat\u00a0grains\u00a0\n\n(Duggeli,\u00a01904;\u00a0Kent\u2010\u00a0Jones\u00a0and\u00a0Amos,\u00a01930;\u00a0James\u00a0et\u00a0\n\nal.,\u00a0 1946;\u00a0 Christensen,\u00a0 1956;\u00a0 Poisson\u00a0 and\u00a0 Guilbot,\u00a0\n\n1956;\u00a0 Inagaski\u00a0and\u00a0 Ikeda,\u00a01959;\u00a0Field\u00a0and\u00a0King,\u00a01962;\u00a0\n\nBrook\u00a0 and\u00a0 White,\u00a0 1966;\u00a0 Graves\u00a0 et\u00a0 al.,\u00a0 1967;\u00a0 Pelhate,\u00a0\n\n1968;\u00a0 Hesseltine,\u00a0 1968;\u00a0 Wallace,\u00a0 1973).\u00a0 Earlier\u00a0 in\u00a0 the\u00a0\n\nlaboratory\u00a0 a\u00a0 number\u00a0 of\u00a0 cereals\u00a0 have\u00a0 been\u00a0 screened\u00a0\n\nwith\u00a0respect\u00a0to\u00a0microflora\u00a0associated\u00a0in\u00a0storage\u00a0grains\u00a0\n\n(Basu,\u00a0 1974;\u00a0 Mehrothra,\u00a0 1974;\u00a0 Palni,\u00a0 1975;\u00a0 Jayas,\u00a0\n\n1995).\u00a0Fungal\u00a0activity\u00a0can\u00a0cause\u00a0undesirable\u00a0effects\u00a0in\u00a0\n\ngrains\u00a0 including\u00a0 discolouration,\u00a0 contribute\u00a0 to\u00a0 heating\u00a0\n\nand\u00a0 losses\u00a0 in\u00a0 nutritional\u00a0 value,\u00a0 produce\u00a0 off\u2010odours,\u00a0\n\nlosses\u00a0 in\u00a0 germinability,\u00a0 deterioration\u00a0 in\u00a0 baking\u00a0 and\u00a0\n\nmilling\u00a0 quality,\u00a0 and\u00a0 can\u00a0 result\u00a0 in\u00a0 contamination\u00a0 by\u00a0\n\nmycotoxins\u00a0(Hocking,\u00a02003;\u00a0Magan\u00a0et\u00a0al.,\u00a02003).\u00a0\n\nMaterials\u00a0and\u00a0Methods\u00a0\nThe\u00a0 investigation\u00a0 was\u00a0 done\u00a0 at\u00a0 Allahabad\u00a0 Agricultural\u00a0\n\nInstitute\u2010Demmed\u00a0University,\u00a0Allahabad\u00a0in\u00a0India.\u00a0.\u00a0The\u00a0\n\nwork\u00a0 was\u00a0 an\u00a0 attempt\u00a0 to\u00a0 correlate\u00a0 the\u00a0 sub\u2010epidermal\u00a0\n\nfungal\u00a0 infestation\u00a0 and\u00a0 quality\u00a0 of\u00a0 wheat\u00a0 grain\u00a0 under\u00a0\n\nstorage.\u00a0The\u00a0wheat\u00a0samples\u00a0were\u00a0collected\u00a0separately\u00a0\n\nin\u00a0 3\u00a0 replicates\u00a0 for\u00a0 each\u00a0 of\u00a0 the\u00a0 wheat\u00a0 (Triticium\u00a0\n\naestivum\u00a0L.)\u00a0varieties\u00a0viz.,\u00a0U.P.\u00a0262\u00a0and\u00a0H.D.\u00a01982\u00a0from\u00a0\n\nF.C.I.\u00a0 godown,\u00a0 Naini,\u00a0 Allahabad\u00a0 district,\u00a0 whole\u00a0 sale\u00a0\n\ndealers\u00a0from\u00a0Naini\u00a0and\u00a0Muttiganj\u00a0markets\u00a0of\u00a0Allahabad\u00a0\n\nand\u00a0from\u00a0the\u00a0local\u00a0farmers.\u00a0The\u00a0samples\u00a0were\u00a0brought\u00a0\n\nto\u00a0the\u00a0 laboratory\u00a0under\u00a0aseptic\u00a0condition\u00a0where\u00a0they\u00a0\n\nwere\u00a0screened\u00a0(before\u00a0washing\u00a0and\u00a0after\u00a0washing\u00a0with\u00a0\n\nwater)\u00a0for\u00a0their\u00a0associated\u00a0sub\u2010epidermal\u00a0fungal\u00a0flora.\u00a0\n\nOut\u00a0of\u00a0these,\u00a0some\u00a0samples\u00a0were\u00a0heavily\u00a0infested\u00a0with\u00a0\n\nfungi,\u00a0some\u00a0were\u00a0slightly\u00a0infested\u00a0and\u00a0some\u00a0were\u00a0not\u00a0\n\nat\u00a0 all\u00a0 infested.\u00a0 Twenty\u00a0 samples\u00a0 of\u00a0 wheat\u00a0 grains\u00a0\n\nbelonging\u00a0to\u00a0two\u00a0different\u00a0varieties\u00a0were\u00a0screened\u00a0for\u00a0\n\nthe\u00a0presence\u00a0of\u00a0sub\u2010epidermal\u00a0mycelium.", "start_char_idx": 4823, "end_char_idx": 7139, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5ebed3e2-cc95-4026-ae76-48cd6ae814bd": {"__data__": {"id_": "5ebed3e2-cc95-4026-ae76-48cd6ae814bd", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "bd3cd14c-c906-4b73-8a28-89aea230249d", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "5026b543c6959bd2e5714f61ec56b240c666fe1bef6e822373a84acccdc26815", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "671537fc-3ae8-4b6d-a16a-782d9df44c5a", "node_type": "1", "metadata": {}, "hash": "1b8ae434b54fec0520f9efdaac088a3e973a326249551838591ee3bbefe5df80", "class_name": "RelatedNodeInfo"}}, "text": "Five\u00a0different\u00a0\n\nsample\u00a0 collection\u00a0 sites\u00a0 were\u00a0 selected\u00a0 and\u00a0 12\u00a0 month\u00a0\n\nstored\u00a0 (moisture\u00a0 content\u00a0 8\u201016%)\u00a0 samples\u00a0 were\u00a0\n\nscreened\u00a0 within\u00a0 a\u00a0 week\u00a0 of\u00a0 their\u00a0 collection.\u00a0 One\u00a0\n\nhundered\u00a0 grains\u00a0 from\u00a0 each\u00a0 samples\u00a0 were\u00a0 taken\u00a0\n\nrandomly,\u00a0they\u00a0were\u00a0examined\u00a0for\u00a0the\u00a0presence\u00a0of\u00a0sub\n\n\u2010epidermal\u00a0mycelium\u00a0within\u00a0the\u00a0grain\u00a0by\u00a0the\u00a0method\u00a0of\u00a0\n\nHyde\u00a0and\u00a0Galleymore\u00a0(1951)\u00a0as\u00a0described\u00a0below:\u00a0\u00a0\n\n1.\u00a0 The\u00a0 grain\u00a0 without\u00a0 cracks\u00a0 and\u00a0 holes\u00a0 on\u00a0 their\u00a0\n\nepidermis\u00a0were\u00a0soaked\u00a0in\u00a0water\u00a0for\u00a0a\u00a0short\u00a0period\u00a0\n\nand\u00a0 then\u00a0 the\u00a0 epidermis\u00a0 was\u00a0 peeled\u00a0 off\u00a0 with\u00a0\n\nforceps.\u00a0\n\n2.\u00a0 The\u00a0stored\u00a0grains\u00a0were\u00a0also\u00a0examined\u00a0superficially\u00a0\n\nwith\u00a0a\u00a0hand\u00a0lens\u00a0(10\u00a0X).\u00a0\n\n3.\u00a0 The\u00a0 peeled\u00a0 epidermis\u00a0 contains\u00a0 sub\u2010epidermal\u00a0\n\nmycelium.\u00a0\n\n4.\u00a0 The\u00a0 epidermis\u00a0 was\u00a0 then\u00a0 placed\u00a0 in\u00a0 aniline\u00a0 blue\u00a0\n\n(0.2%\u00a0 in\u00a0 66%\u00a0 lactic\u00a0 acid)\u00a0 and\u00a0 warmed\u00a0 for\u00a0 5\u00a0 \u2013\u00a0 10\u00a0\n\nminutes\u00a0which\u00a0stained\u00a0the\u00a0mycelium\u00a0alone\u00a0and\u00a0the\u00a0\n\nepidermal\u00a0cells\u00a0were\u00a0left\u00a0unstained.\u00a0\n\nThe\u00a0 sub\u2010epidermal\u00a0 fungi\u00a0 was\u00a0 brought\u00a0 into\u00a0 culture\u00a0 by\u00a0\n\nfirst\u00a0 surface\u00a0 disinfecting\u00a0 the\u00a0 soaked\u00a0 grains\u00a0 by\u00a0\n\nimmersion\u00a0 for\u00a0 two\u00a0 minutes\u00a0 in\u00a0 0.2%\u00a0 sodium\u00a0\n\nhypochlorite\u00a0 and\u00a0 washing\u00a0 in\u00a0 two\u00a0 changes\u00a0 of\u00a0 sterile\u00a0\n\nwater.\u00a0Then\u00a0the\u00a0peeling\u00a0of\u00a0the\u00a0epidermis\u00a0were\u00a0taken\u00a0\n\nout\u00a0 and\u00a0 transferred\u00a0 to\u00a0 five\u00a0 different\u00a0 culture\u00a0 medium\u00a0\n\nviz.,\u00a0 Czapek\u2019s\u00a0 solution\u00a0 agar,\u00a0 Czapek\u2019s\u00a0 osmophilic\u00a0\n\nsolution\u00a0 agar,\u00a0 Malt\u00a0 extract\u00a0 agar,\u00a0 Wheat\u00a0 extract\u00a0 agar\u00a0\n\nand\u00a0oat\u00a0meal\u00a0agar\u00a0with\u00a0the\u00a0following\u00a0composition:\u00a0\n\n1.\u00a0 Czapek\u2019s\u00a0 solution\u00a0 agar\u00a0 media,\u00a0 Oat\u00a0 meal\u00a0 Agar\u00a0\n\nMedia\u00a0and\u00a0Malt\u00a0extract\u00a0agar:\u00a0The\u00a0composition\u00a0of\u00a0\n\nthese\u00a0media\u00a0has\u00a0been\u00a0mentioned\u00a0above.\u00a0\n\n2.\u00a0 Czapek\u2019s\u00a0 osmophilic\u00a0 solution\u00a0 agar:\u00a0 Same\u00a0 as\u00a0\n\nCzapek\u2019s\u00a0 solution\u00a0 agar\u00a0 media\u00a0 except\u00a0 for\u00a0 sucrose\u00a0\n\nwhich\u00a0is\u00a020%\u00a0instead\u00a0of\u00a03%.\u00a0\n\n3.\u00a0 Wheat\u00a0extract\u00a0agar:\u00a0\u00a0Wheat\u00a0extract\u00a020g;\u00a0Agar,\u00a030g;\u00a0\n\nYeast\u00a0extract,\u00a00.5g;\u00a0Distilled\u00a0water,\u00a01000ml.\u00a0\u00a0\n\n\u00a0The\u00a0culture\u00a0was\u00a0incubated\u00a0at\u00a025\u00b1\u00a02\u00a00C\u00a0for\u00a07\u00a0days.\u00a0\n\nEach\u00a0set\u00a0of\u00a0experiment\u00a0had\u00a0a\u00a0control\u00a0to\u00a0differentiate\u00a0\n\nthe\u00a0 laboratory\u00a0 contaminants\u00a0 from\u00a0 the\u00a0 microflora\u00a0\n\nactually\u00a0 associated\u00a0 with\u00a0 the\u00a0 sample.\u00a0 The\u00a0 fungal\u00a0\n\ncolonies\u00a0were\u00a0isolated\u00a0and\u00a0 identified\u00a0with\u00a0the\u00a0help\u00a0of\u00a0\n\nauthentic\u00a0 literature.\u00a0 The\u00a0 frequency\u00a0 of\u00a0 occurrence\u00a0 of\u00a0\n\ndifferent\u00a0 fungi\u00a0 isolated\u00a0 from\u00a0 wheat\u00a0 samples\u00a0 was\u00a0\n\ncalculated\u00a0by\u00a0the\u00a0following\u00a0formula;\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 7140, "end_char_idx": 9522, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "671537fc-3ae8-4b6d-a16a-782d9df44c5a": {"__data__": {"id_": "671537fc-3ae8-4b6d-a16a-782d9df44c5a", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5ebed3e2-cc95-4026-ae76-48cd6ae814bd", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "a2527f8d4a0c271ae31130365589d39a2e8f43a7a6c408966f8bc60c765bc6c9", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4e4574c7-e7c8-4155-9c31-0146a5d0fdac", "node_type": "1", "metadata": {}, "hash": "e9b7a966e2c4f2ae2cff24e9f3b2f76d009bc33942db859c0519f4ad73ac63a6", "class_name": "RelatedNodeInfo"}}, "text": "1\u00a0:\u00a09\u201013\u00a0\n\n11\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nThe\u00a0 various\u00a0 media\u00a0 used\u00a0 for\u00a0 the\u00a0 identification\u00a0 of\u00a0\n\nsubepidermal\u00a0 fungi\u00a0 with\u00a0 different\u00a0 composition\u00a0 of\u00a0\n\nCzepek\u2019s\u00a0 solution\u00a0 agar\u00a0 {NaNO3\u00a0 (3.0\u00a0 g);\u00a0 K2PO4\u00a0 (1.0\u00a0 g);\u00a0\n\nKCL\u00a0(0.5\u00a0g);\u00a0MgSO4\u00a0.7H2O\u00a0(0.5\u00a0g);\u00a0FeSO4\u00a0.7H2O\u00a0(0.01\u00a0g);\u00a0\n\nSucrose\u00a0(30.0\u00a0g);\u00a0Agar\u00a0(20.0\u00a0g);\u00a0Distilled\u00a0water\u00a0(1000.0\u00a0\n\nml);\u00a0 PH\u00a0 6.5)},\u00a0 Czepek\u2019s\u00a0 yeast\u00a0 agar\u00a0 {\u00a0 K2HPO4\u00a0 (1.0\u00a0 gm);\u00a0\n\nCzepek\u2019s\u00a0concentrate\u00a0(10.0\u00a0gm);\u00a0Yeast\u00a0extract\u00a0(5.0\u00a0gm);\u00a0\n\nSucrose\u00a0 (30.0\u00a0 gm);\u00a0 Agar\u00a0 (20.0\u00a0 gm);\u00a0 Distilled\u00a0 water\u00a0\n\n(1000.0\u00a0ml);\u00a0PH\u00a06.5},\u00a0malt\u00a0extract\u00a0agar\u00a0{Malt\u00a0\u2013\u00a0Extract\u00a0\n\n(20.0\u00a0g);\u00a0Peptone\u00a0(1.0\u00a0g);\u00a0Dextrose\u00a0(20.0\u00a0g);\u00a0Agar\u00a0(20.0\u00a0\n\ng);\u00a0 Distilled\u00a0 water\u00a0 (1000.0\u00a0ml)\u00a0 PH\u00a0 6.5},\u00a0Oat\u00a0 meal\u00a0 agar\u00a0\n\n{Oat\u00a0meal\u00a0(20.0\u00a0g);\u00a0Yeast\u00a0extract\u00a0\u00a0(0.5\u00a0g);\u00a0Agar\u00a0(20.0\u00a0g);\u00a0\n\nDistilled\u00a0 water\u00a0 (1000.0\u00a0 ml);\u00a0 PH\u00a0 6.5},\u00a0 Potato\u2010dextrose\u00a0\n\nagar\u00a0{Potato\u00a0Dextrose\u00a0(20.0\u00a0g);\u00a0Agar\u00a0(20.0\u00a0g);\u00a0Distilled\u00a0\n\nwater\u00a0 (1000.0\u00a0 ml);\u00a0 PH\u00a0 6.5},\u00a0 Peptone\u00a0 agar\u00a0 {Peptone\u00a0\n\n(10.0\u00a0 g);\u00a0 Dextrose\u00a0 (20.0\u00a0 g);\u00a0 Agar\u00a0 (20.0\u00a0 g);\u00a0 Distilled\u00a0\n\nwater\u00a0 (1000.0\u00a0 ml);\u00a0 PH\u00a0 6.5}\u00a0 and\u00a0 Synthetic\u00a0 mucor\u00a0 agar\n\n{\u00a0Dextrose\u00a0(20.0\u00a0g);\u00a0Asparagine\u00a0(2.0\u00a0g);\u00a0KH2PO4\u00a0(0.5\u00a0g);\u00a0\n\nMgSO4\u00a0 .7H2O\u00a0 (0.25\u00a0 g);\u00a0 Thiamine\u00a0 chloride\u00a0 (0.5\u00a0 g);\u00a0\n\nDistilled\u00a0water\u00a0(1000.0\u00a0ml);\u00a0PH\u00a06.5}.\u00a0These\u00a0media\u00a0were\u00a0\n\nsterilized\u00a0at\u00a01210C\u00a0and\u00a015\u00a0lbs\u00a0p.s.i.\u00a0for\u00a020\u00a0minutes.\u00a0\n\nResults\u00a0and\u00a0Discussion\u00a0\nThe\u00a0result\u00a0of\u00a0sub\u2010epidermal\u00a0fungi\u00a0in\u00a0the\u00a0stored\u00a0wheat\u00a0\n\ngrain\u00a0 varieties\u00a0 U.P.\u00a0 232\u00a0 and\u00a0 H.D.1982\u00a0 collected\u00a0 from\u00a0\n\ndifferent\u00a0sites\u00a0of\u00a0Allahabad\u00a0are\u00a0given\u00a0in\u00a0Figure\u00a01.\u00a0Sub\u2010\n\nepidermal\u00a0mycelium\u00a0was\u00a0seen\u00a0in\u00a0all\u00a0samples\u00a0screened.\u00a0\n\nThese\u00a0mycelia\u00a0were\u00a0usually\u00a0septate,\u00a0thin\u00a0and\u00a0branching\u00a0\n\nrepeatedly\u00a0to\u00a0form\u00a0a\u00a0network.\u00a0Sometimes,\u00a0due\u00a0to\u00a0more\u00a0\n\nfrequent\u00a0 formation\u00a0 of\u00a0 transverse\u00a0 walls,\u00a0 beaded\u00a0 cells\u00a0\n\nwere\u00a0seen.\u00a0Four\u00a0species\u00a0of\u00a0fungi\u00a0as\u00a0shown\u00a0 in\u00a0Table\u00a01\u00a0\n\nwere\u00a0 isolated\u00a0 from\u00a0 sub\u2010epidermal\u00a0 region\u00a0 of\u00a0 wheat\u00a0\n\ngrains\u00a0and\u00a0the\u00a0microscopic\u00a0view\u00a0 is\u00a0shown\u00a0 in\u00a0Figure\u00a02.", "start_char_idx": 9523, "end_char_idx": 11452, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4e4574c7-e7c8-4155-9c31-0146a5d0fdac": {"__data__": {"id_": "4e4574c7-e7c8-4155-9c31-0146a5d0fdac", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "671537fc-3ae8-4b6d-a16a-782d9df44c5a", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "f89508b891c09b79dc0c940e4f91e0fc38a4730b69b0406bf4e6dfbe6dc524d6", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "70c3e191-4f4b-42b1-9246-8f1e25ff084b", "node_type": "1", "metadata": {}, "hash": "8f62108b6d75b0d6918f74f0ad871d47adee7510b77abc4edb47c2b0d3cfb426", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 most\u00a0 common\u00a0 storage\u00a0 fungi\u00a0 observed\u00a0 in\u00a0 the\u00a0\n\nstored\u00a0 wheat\u00a0 grain\u00a0 samples\u00a0 was\u00a0 Alternaria\u00a0 alternata\u00a0\n\n(14.6%).\u00a0Species\u00a0of\u00a0Aspergillus\u00a0were\u00a0also\u00a0encountered\u00a0\n\nwith\u00a0A.\u00a0fumigatus\u00a0(10.5%),\u00a0and\u00a0A.\u00a0niger\u00a0(8.3%)\u00a0followed\u00a0\n\nby\u00a0 Penicillium\u00a0 citrinum\u00a0 (3.8%).\u00a0 The\u00a0 sub\u2010epidermal\u00a0\n\nmycelium\u00a0 was\u00a0 observed\u00a0 even\u00a0 in\u00a0 apparently,\u00a0 healthy,\u00a0\n\nundamaged\u00a0 grains\u00a0 examined\u00a0 superficially\u00a0 with\u00a0 hand\u00a0\n\nlens\u00a0 prior\u00a0 to\u00a0 peeling.\u00a0 Sporophores\u00a0 were\u00a0 sometimes\u00a0\n\nseen\u00a0emerging\u00a0from\u00a0the\u00a0groove\u00a0region\u00a0or\u00a0from\u00a0cracks\u00a0\n\nand\u00a0holes\u00a0on\u00a0the\u00a0smooth\u00a0surface\u00a0of\u00a0the\u00a0stored\u00a0wheat\u00a0\n\ngrain.\u00a0\u00a0\n\nThe\u00a0 entry\u00a0 of\u00a0 the\u00a0 fungus\u00a0 in\u00a0 stored\u00a0 grains\u00a0 could\u00a0 be\u00a0\n\nthrough\u00a0the\u00a0epidermis\u00a0as\u00a0the\u00a0sub\u2010epidermal\u00a0mycelium\u00a0\n\nwas\u00a0 observed\u00a0 even\u00a0 in\u00a0 apparently\u00a0 healthy\u00a0 grains\u00a0\n\nexamined\u00a0 superficially\u00a0 with\u00a0 a\u00a0 hand\u00a0 lens\u00a0 and\u00a0 prior\u00a0 to\u00a0\n\npeeling.\u00a0However,\u00a0sporophores\u00a0were\u00a0sometimes\u00a0seen\u00a0\n\nemerging\u00a0in\u00a0the\u00a0groove\u00a0region\u00a0or\u00a0from\u00a0the\u00a0cracks\u00a0and\u00a0\n\nholes\u00a0 in\u00a0 the\u00a0 smooth\u00a0 surface.\u00a0 Simmonds\u00a0 (1968)\u00a0\n\nobserved\u00a0that\u00a0infection\u00a0takes\u00a0place\u00a0through\u00a0the\u00a0cracks\u00a0\n\nin\u00a0the\u00a0epidermis\u00a0or\u00a0the\u00a0opening\u00a0over\u00a0the\u00a0embryo\u00a0since\u00a0\n\nabundant\u00a0fungal\u00a0fruiting\u00a0was\u00a0evident\u00a0where\u00a0cracks\u00a0had\u00a0\n\noccurred.\u00a0Welling\u00a0(1968)\u00a0found\u00a0that\u00a0Aspergillus\u00a0species\u00a0\n\ninfected\u00a0 seeds\u00a0 independent\u00a0 of\u00a0 the\u00a0 amount\u00a0 of\u00a0 injury.\u00a0\n\nThe\u00a0 mature\u00a0 stored\u00a0 wheat\u00a0 grains\u00a0 were\u00a0 found\u00a0 to\u00a0 have\u00a0\n\nmore\u00a0 injury.\u00a0 Hyde\u00a0 and\u00a0 Galleymore\u00a0 (1951)\u00a0 observed\u00a0\n\nthat\u00a0 mature\u00a0 grains\u00a0 contain\u00a0 more\u00a0 internal\u00a0 mycelia.\u00a0\n\nFlannigan\u00a0 (1974)\u00a0 also\u00a0 found\u00a0 Alternaria\u00a0 alternata\u00a0 in\u00a0\n\n78.5%\u00a0 of\u00a0 the\u00a0 wheat\u00a0 grains\u00a0 established\u00a0 fungi\u00a0 with\u00a0\n\nAlternaria\u00a0 alternata\u00a0 was\u00a0 among\u00a0 the\u00a0 ones\u00a0 which\u00a0\n\nappeared\u00a0later.\u00a0\n\nReports\u00a0from\u00a0various\u00a0countries\u00a0show\u00a0that\u00a0this\u00a0species\u00a0\n\nis\u00a0 found\u00a0 to\u00a0 be\u00a0 the\u00a0 most\u00a0 common\u00a0 post\u00a0 harvest\u00a0 fungi.\u00a0\n\nSaponaro\u00a0and\u00a0Madaluni\u00a0(1960)\u00a0reported\u00a0the\u00a0presence\u00a0\n\nof\u00a0Aspergillus\u00a0 in\u00a0stored\u00a0wheat\u00a0grains\u00a0 in\u00a0 Italy,\u00a0Wallace\u00a0\n\nand\u00a0 Sinha\u00a0 (1962)\u00a0 in\u00a0 Canada,\u00a0 Kurata\u00a0 et\u00a0 al.\u00a0 (1968),\u00a0\n\nTsunado\u00a0(1970)\u00a0and\u00a0Tsuruta\u00a0(1970)\u00a0in\u00a0Japan.\u00a0However,\u00a0\n\nJames\u00a0and\u00a0Smith,\u00a01948\u00a0from\u00a0Canada\u00a0reported\u00a0A.\u00a0niger\u00a0\n\nstood\u00a0second\u00a0with\u00a0respect\u00a0to\u00a0frequency\u00a0of\u00a0occurrence\u00a0\n\nof\u00a0fungi\u00a0in\u00a0stored\u00a0wheat\u00a0grains.\u00a0\u00a0Conversely\u00a0Aspergillus\u00a0\n\nand\u00a0Penicillium\u00a0are\u00a0more\u00a0often\u00a0considered\u00a0as\u00a0\u2018storage\u00a0\n\nfungi\u2019.", "start_char_idx": 11455, "end_char_idx": 13657, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "70c3e191-4f4b-42b1-9246-8f1e25ff084b": {"__data__": {"id_": "70c3e191-4f4b-42b1-9246-8f1e25ff084b", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4e4574c7-e7c8-4155-9c31-0146a5d0fdac", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "7ab06840400c998f744b19b7561c920d6061a8fa9842931595a6de81afd26cec", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "48cca47d-31a4-4b23-ac2a-9d028547f6d0", "node_type": "1", "metadata": {}, "hash": "0c0f5cae82cf784f91b5b16959750350d1df700b3a6417a2072b92f976141611", "class_name": "RelatedNodeInfo"}}, "text": "They\u00a0 are\u00a0 known\u00a0 to\u00a0 form\u00a0 mycotoxins\u00a0 in\u00a0 stored\u00a0\n\ngrains\u00a0and\u00a0are\u00a0usually\u00a0not\u00a0regarded\u00a0as\u00a0fungi\u00a0that\u00a0can\u00a0\n\nproduce\u00a0 mycotoxins\u00a0 before\u00a0 harvest\u00a0 (Frisvad,\u00a0 1995;\u00a0\n\nWicklow,\u00a01995;\u00a0Hockings,\u00a02003)\u00a0\n\nAcknowledgements\u00a0\nI\u00a0 gratefully\u00a0 acknowledge\u00a0 the\u00a0 cooperation,\u00a0\nuninterrupted\u00a0 guidance,\u00a0 impeccable\u00a0 and\u00a0 valuable\u00a0\nsuggestions\u00a0 rendered\u00a0 to\u00a0 me\u00a0 by\u00a0 Prof.\u00a0 (Dr.)\u00a0 George\u00a0\nThomas\u00a0during\u00a0my\u00a0research\u00a0work.\u00a0\u00a0\n\nS.\u00a0No.\u00a0 Organisms\u00a0 Frequency\u00a0of\u00a0occurrence\u00a0(%)\u00a0\n\n1\u00a0 Alternaria\u00a0alternata\u00a0 14.6\u00a0\n\n2\u00a0 Aspergillus\u00a0fumigatus\u00a0 10.5\u00a0\n\n3\u00a0 Aspergillus\u00a0niger\u00a0 8.3\u00a0\n\n4\u00a0 Penicillium\u00a0citrinum\u00a0 3.8\u00a0\n\nTable\u00a01:\u00a0Frequency\u00a0of\u00a0fungi\u00a0isolated\u00a0from\u00a0sub\n\n\u2010epidermal\u00a0region\u00a0of\u00a0stored\u00a0wheat\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a09\u201013\u00a0\n\n12\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nFigure\u00a01:\u00a0\u00a0The\u00a0colonies\u00a0developed\u00a0on\u00a0the\u00a0Czapek\u2019s\u00a0solu\u2010\ntion\u00a0agar\u00a0of\u00a0the\u00a0stored\u00a0grain\u00a0fungi\u00a0\n\nFigure\u00a02:\u00a0Schematic\u00a0showing\u00a0isolated\u00a0fungal\u00a0colonies\u00a0under\u00a0micro\u2010\nscopic\u00a0view\u00a0i)\u00a0Aspergillus\u00a0fumigatus\u00a0ii)\u00a0Aspergillus\u00a0niger\u00a0iii)\u00a0Alternaria\u00a0\n\nalternate\u00a0iv)\u00a0Penicillium\u00a0citrinum\u00a0\n\nReferences\u00a0\u00a0\nBasu:\u00a0 Studies\u00a0 of\u00a0 microorganisms\u00a0 associated\u00a0 with\u00a0\n\nstored\u00a0wheat\u00a0and\u00a0 its\u00a0flour\u00a0 in\u00a0 India.\u00a0Research\u00a0paper\u00a0\n\npresentation\u00a0at\u00a0University\u00a0of\u00a0Lucknow,\u00a0India;\u00a01974.\u00a0\n\nBrook\u00a0PJ,\u00a0White,\u00a0EP:\u00a0Fungus\u00a0toxins\u00a0affecting\u00a0mammals.\u00a0\n\nAnn.\u00a0Rev.\u00a0Phytopathol.\u00a01966,\u00a04:\u00a0171\u2010194.\u00a0\n\nChristensen,CM:\u00a0 Deterioration\u00a0 of\u00a0 stored\u00a0 grains\u00a0 by\u00a0\n\nmoulds.\u00a0Wallerstein\u00a0Lab.\u00a0Commun.\u00a01956,\u00a019\u00a0:\u00a031\u201048.\u00a0\n\nDuggeli\u00a0 M:\u00a0 The\u00a0 mycological\u00a0 florainfection.\u00a0 Zentr.\u00a0\n\nMicrobial\u00a0Infection\u00a01904,\u00a012:\u00a0602\u2010614.\u00a0\n\nField\u00a0 RW\u00a0and\u00a0King\u00a0 TH:\u00a0 Influence\u00a0 of\u00a0 storage\u00a0 fungi\u00a0 \u00a0 on\u00a0\n\nthe\u00a0deterioration\u00a0of\u00a0corn\u00a0 in\u00a0storage\u00a0a\u00a0review.\u00a0Lowa\u00a0\n\nAcad.\u00a0Sci.\u00a01962,\u00a051:\u00a0265\u2010280.\u00a0\n\nFlannigan\u00a0B:\u00a0Distribution\u00a0of\u00a0seed\u00a0borne\u00a0microorganism\u00a0\n\nin\u00a0naked\u00a0barley\u00a0and\u00a0wheat\u00a0before\u00a0harvest.\u00a0Trans.\u00a0Br.\u00a0\u00a0\n\nMycol.\u00a0Soc.\u00a01974,\u00a062:\u00a051\u201058.\u00a0\n\nFrisvad\u00a0 JC:\u00a0 Mycotoxins\u00a0 and\u00a0 mycotoxigenic\u00a0 fungi\u00a0 in\u00a0\n\nstorage.\u00a0 In:\u00a0 Stored\u00a0 grain\u00a0 ecosystems.\u00a0 (D.S.\u00a0 Jayas,\u00a0\n\nN.D.G.\u00a0 White,\u00a0 and\u00a0 W.E\u00a0 .Muir,\u00a0 eds.).\u00a0 New\u00a0 York,\u00a0\n\nMarcel\u00a0Dekker.1995,\u00a0251\u2010288.", "start_char_idx": 13659, "end_char_idx": 15712, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "48cca47d-31a4-4b23-ac2a-9d028547f6d0": {"__data__": {"id_": "48cca47d-31a4-4b23-ac2a-9d028547f6d0", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "70c3e191-4f4b-42b1-9246-8f1e25ff084b", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "014c4eca964c08f89095805ab2b1e4d75b4ff9385a7c6f6088eeb8ca92f5835b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91f6282e-d082-41be-9cd8-0fda54ec37e3", "node_type": "1", "metadata": {}, "hash": "71373974ded13a5a5e6c845af30143def73670fcd2e333657e1ab152fe598ba3", "class_name": "RelatedNodeInfo"}}, "text": "Graves\u00a0 RR\u00a0 Rogers\u00a0 RF\u00a0 Lyons\u00a0 AJ\u00a0 Jr.,\u00a0 Hesseltine\u00a0 CW:\u00a0\n\nBacterial\u00a0 and\u00a0 actinomycetes\u00a0 flora\u00a0 of\u00a0 Kansas\u00a0 \u2013\u00a0\n\nNebraska\u00a0 and\u00a0 Pacific\u00a0 Northwest\u00a0 wheat\u00a0 and\u00a0 wheat\u00a0\n\nflour.\u00a0Cereal\u00a0Chem.\u00a01967\u00a044:\u00a0288\u2010299.\u00a0\n\nHesseltine\u00a0 CW:\u00a0 Flour\u00a0 and\u00a0 wheat:\u00a0 Research\u00a0 on\u00a0 their\u00a0\n\nmicrobiological\u00a0 flora,\u00a0 Bakers\u00a0 Dig.\u00a0 1968,\u00a0 42:\u00a0 40\u201042,\u00a0\n\n66.\u00a0\n\nHocking\u00a0 AD:\u00a0 Stored\u00a0 grain\u00a0 in\u00a0 Australia\u00a0 2003.\u00a0\u00a0\n\nProceedings\u00a0of\u00a0the\u00a0Australian\u00a0Postharvest\u00a0Technical\u00a0\n\nConference,\u00a0Canberra.\u00a0CSIRO\u00a0Stored\u00a0Grain\u00a0Research\u00a0\n\nLaboratory,\u00a0Canberra.\u00a02003;\u00a0pp.\u00a055\u201058.\u00a0\n\nHyde\u00a0 M,\u00a0 Olly\u00a0 B,\u00a0 Galleymore\u00a0 HB:\u00a0 The\u00a0 subepidermal\u00a0\n\nfungi\u00a0 of\u00a0 cereal\u00a0 grains\u00a0 II.\u00a0 The\u00a0 nature\u00a0 identify\u00a0 and\u00a0\n\norigin\u00a0 of\u00a0 the\u00a0 mycelium\u00a0 in\u00a0 wheat.\u00a0 Ann.\u00a0 Application\u00a0\n\nBiol.\u00a01951,\u00a08:\u00a0248\u2010356.\u00a0\n\nInagaski\u00a0N,\u00a0Ikeda\u00a0M:\u00a0Studies\u00a0on\u00a0the\u00a0fungi\u00a0isolated\u00a0from\u00a0\n\nfoods\u00a0 II.\u00a0 Identification\u00a0 of\u00a0 Penicillia\u00a0 and\u00a0 Aspergilli\u00a0\n\nfrom\u00a0flour.\u00a0Bull.\u00a0Nat.\u00a0Hygenic\u00a0Lab.\u00a0Tokyo.\u00a01959,\u00a0\u00a077:\u00a0\n\n347\u2010366.\u00a0\n\nJames\u00a0 N,\u00a0 Wilson,\u00a0 J,\u00a0 Stark\u00a0 E:\u00a0 The\u00a0 microflora\u00a0 of\u00a0 stored\u00a0\n\nwheat.\u00a0Can.\u00a0J.\u00a0Res.\u00a01946,\u00a024:\u00a0224\u2010233.\u00a0\n\nJayas\u00a0DS:\u00a0Stored\u2010grain\u00a0ecosystems.\u00a0New\u00a0York:\u00a0Dekker.\u00a0\n\n1995,\u00a0pp.\u00a0757.\u00a0\n\nKent\u2010Jones\u00a0 DW,\u00a0 Amos\u00a0 AJ:\u00a0 Preliminary\u00a0 studies\u00a0 in\u00a0\n\nbacteriology\u00a0 of\u00a0 wheat\u00a0 and\u00a0 flour.\u00a0 Analyst.\u00a0 1930,\u00a0 55:\u00a0\n\n248\u2010268.\u00a0\n\nMachacek\u00a0 JE,\u00a0 Greaney\u00a0 FJ:\u00a0 The\u00a0 block\u00a0 point\u00a0 of\u00a0 kernel\u00a0\n\nsmudge\u00a0disease\u00a0of\u00a0cereals.\u00a0Can.\u00a0J.\u00a0Res.\u00a01938,\u00a016:\u00a084\u2010\n\n113.\u00a0\n\nMagan\u00a0 N,\u00a0 Hope\u00a0 R,\u00a0 Cairns\u00a0 V,\u00a0 Aldred\u00a0 D:\u00a0 Post\u2010harvest\u00a0\n\nfungal\u00a0 ecology:\u00a0 impact\u00a0 of\u00a0 fungal\u00a0 growth\u00a0 and\u00a0\n\nmycotoxin\u00a0accumulation\u00a0in\u00a0stored\u00a0grain.\u00a0European\u00a0J.\u00a0\n\nPlant\u00a0Pathol.\u00a02003,\u00a0109:\u00a0723\u2010730.\u00a0\n\nMehrothra\u00a0 DK:\u00a0 Studies\u00a0 on\u00a0 some\u00a0 microrganisms\u00a0\n\nassociated\u00a0 with\u00a0 rice\u00a0 in\u00a0 storage.\u00a0 Paper\u00a0 presentation\u00a0\n\nAbstract\u00a0at\u00a0University\u00a0of\u00a0Lucknow.\u00a01974.\u00a0\n\nNeethirajan\u00a0 S,\u00a0 Karunakaran\u00a0 S,\u00a0 Jayas\u00a0 DS,White\u00a0 NDG:\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01\u00a0:\u00a09\u201013\u00a0\n\n13\u00a0\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nDetection\u00a0 techniques\u00a0 for\u00a0 stored\u2010product\u00a0 insects\u00a0 in\u00a0\n\ngrain.\u00a0Int.\u00a0Fd.\u00a0Control.\u00a02007,\u00a018:\u00a0157\u2010162.\u00a0\n\nPalni\u00a0 IK:\u00a0 Studies\u00a0 on\u00a0 the\u00a0 microflora\u00a0 associated\u00a0 with\u00a0\n\nwheat\u00a0 grains\u00a0 and\u00a0 various\u00a0 milling\u00a0 fraction.\u00a0 Paper\u00a0\n\npresentation.\u00a0Abstract,\u00a0University\u00a0of\u00a0Lucknow.\u00a01975.", "start_char_idx": 15715, "end_char_idx": 17889, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91f6282e-d082-41be-9cd8-0fda54ec37e3": {"__data__": {"id_": "91f6282e-d082-41be-9cd8-0fda54ec37e3", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "48cca47d-31a4-4b23-ac2a-9d028547f6d0", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "7857c410a48258c769aa54e0c8c699eafeed4054f775a54538fc3cf6f76ae4af", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "add9152a-ea13-4642-9270-153b80aeed4f", "node_type": "1", "metadata": {}, "hash": "8092fd4adc46bcae93646e8fd5564f710f3a41508e4d90925f0e173bd611ea9c", "class_name": "RelatedNodeInfo"}}, "text": "Pelhate\u00a0 J:\u00a0 Inventaire\u00a0 de\u00a0 la\u00a0 mycoflora\u00a0 des\u00a0 bles\u00a0 de\u00a0\n\nconservation\u00a0 \u00a0 (Inventory\u00a0 of\u00a0 mycoflora\u00a0 of\u00a0 stored\u00a0\n\nwheat)\u00a0Bull.\u00a0Soc.\u00a0Mycol.\u00a0France\u00a0\u00a01968,\u00a084:\u00a0127\u2010143.\u00a0\n\nPoisson\u00a0 J,\u00a0 Guilbot\u00a0 MA:\u00a0 Mycoflora\u00a0 des\u00a0 Farines\u00a0\n\nFranscaises.\u00a0Menuerie,\u00a0France\u00a01956,\u00a0pp\u00a042\u201052.\u00a0\n\nSankaran\u00a0 R:\u00a0 Mould\u00a0 Problems\u00a0 in\u00a0 the\u00a0 Storage\u00a0 of\u00a0 Food\u00a0\n\nGrains.\u00a0Symposium\u00a0\u2018Stratergies\u00a0on\u00a0Storage,\u00a0Handling\u00a0\n\nand\u00a0 Processing\u00a0 of\u00a0 Food\u00a0 Grains\u00a0 in\u00a0 the\u00a0 Present\u00a0\n\nContext'.\u00a0 Bangalore\u00a0 Chapter\u00a0 of\u00a0 Association\u00a0 of\u00a0 Food\u00a0\n\nScientists\u00a0and\u00a0Technologists,\u00a0India\u00a01976.\u00a0\n\nSankaran\u00a0 R,\u00a0 \u00a0 Sehgal\u00a0 DD,\u00a0 Parihar\u00a0 DB,\u00a0 Nath\u00a0 H:\u00a0 Cereal\u00a0\n\ngrain\u00a0 fungi\u00a0 and\u00a0 grain\u00a0 quality.\u00a0 \u00a0 Def.\u00a0 Sci.\u00a0 1976,\u00a0 \u00a0 26:\u00a0\u00a0\n\n161.\u00a0\n\nSankaran\u00a0R,\u00a0Mattada\u00a0RR,\u00a0Thangamani:\u00a0Mycoflora\u00a0and\u00a0\n\nchanges\u00a0of\u00a0wheat\u00a0seed\u00a0quality\u00a0during\u00a0storage.\u00a0 Itzd.\u00a0\n\nFd.\u00a0Packer.\u00a01975,\u00a029:\u00a01.\u00a0\n\nTsunoda\u00a0H:\u00a0Microorganisms\u00a0which\u00a0deteriorates\u00a0stored\u00a0\n\ncereals\u00a0 and\u00a0 grains.\u00a0 In:\u00a0 Toxic\u00a0 microorganisms\u00a0 (M.\u00a0\n\nHerzberg,\u00a0 ed.).\u00a0 Published\u00a0 by\u00a0 UJNR\u00a0 Joint\u00a0 Panels\u00a0 on\u00a0\n\ntoxic\u00a0 microorganisms\u00a0 and\u00a0 the\u00a0 U.S.\u00a0 Department\u00a0 of\u00a0\n\nInterior\u00a0Washington\u00a0D.C.\u00a01970.\u00a0\n\nTsuruta\u00a0 O:.\u00a0 Microorganisms\u00a0 in\u00a0 stored\u00a0 grains.\u00a0 Training\u00a0\n\nmanual\u00a0on\u00a0storage\u00a0and\u00a0preservation\u00a0of\u00a0wheat\u00a0grain\u00a0\n\n1970,\u00a0pp.\u00a0307.\u00a0\n\nTuite\u00a0 JF,\u00a0 Christensen\u00a0 CM:\u00a0 Grain\u00a0 storage\u00a0 studies\u00a0 24:\u00a0\n\nMoisture\u00a0 content\u00a0 of\u00a0 wheat\u00a0 seed\u00a0 in\u00a0 relation\u00a0 to\u00a0\n\ninvasion\u00a0of\u00a0seed\u00a0by\u00a0species\u00a0of\u00a0the\u00a0Aspergillus\u00a0glaucus\u00a0\n\ngroup\u00a0and\u00a0the\u00a0effect\u00a0of\u00a0invasion\u00a0upon\u00a0germination\u00a0of\u00a0\n\nthe\u00a0seed.\u00a0J.\u00a0Phytopathol.\u00a0\u00a01957,\u00a047:\u00a0323\u2010327.\u00a0\n\nU.S.\u00a0 Department\u00a0 of\u00a0 Agriculture:\u00a0 Annual\u00a0 World\u00a0\n\nProduction\u00a0Summary,\u00a0Grains,\u00a0http://www.usda.gov/\n\nAgriculture\u00a0crop\u00a0production.\u00a02003.\u00a0\n\nWallace\u00a0 HAH:\u00a0 Fungi\u00a0 and\u00a0 other\u00a0 organisms\u00a0 associated\u00a0\n\nwith\u00a0stored\u00a0grains\u00a0 in\u00a0grain\u00a0storage\u00a0part\u00a0of\u00a0a\u00a0system\u00a0\n\n(R.N.\u00a0Sinha\u00a0and\u00a0W.E.\u00a0Muir,\u00a0eds.).\u00a0AVI\u00a0publishing\u00a0Co.,\u00a0\n\nConnecticut\u00a0Sec.\u00a0II\u00a0Capt.\u00a01973,\u00a04\u00a0:\u00a071\u201078.\u00a0\n\nWallace\u00a0 HAH\u00a0 Sinha\u00a0 RN:\u00a0 Causal\u00a0 factors\u00a0 operative\u00a0 in\u00a0\n\ndistributional\u00a0 patterns\u00a0 and\u00a0 abundance\u00a0 of\u00a0 fungi:\u00a0 a\u00a0\n\nmultivariate\u00a0 study.\u00a0 In:\u00a0 The\u00a0 Fungal\u00a0 Community\u2010\u00a0 Its\u00a0\n\nOrganisation\u00a0 and\u00a0 Role\u00a0 in\u00a0 Ecosystems\u00a0 (D.T.\u00a0 Wicklow\u00a0\n\nand\u00a0 \u00a0 \u00a0 G.C.\u00a0 Carroll,\u00a0 eds.).\u00a0 \u00a0 Marcell\u00a0 Dekker\u00a0 Inc.,\u00a0 New\u00a0\n\nYork.\u00a01981,\u00a0pp.\u00a0233\u2010247.\u00a0\n\nWicklow\u00a0 DT:\u00a0 The\u00a0 mycology\u00a0 of\u00a0 stored\u00a0 grain:\u00a0 an\u00a0\necological\u00a0 perspective.", "start_char_idx": 17892, "end_char_idx": 20125, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "add9152a-ea13-4642-9270-153b80aeed4f": {"__data__": {"id_": "add9152a-ea13-4642-9270-153b80aeed4f", "embedding": null, "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-57", "node_type": "4", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "6dad564ec62bc5191aa4c4088db3a534e9d42a43a2634d84c482da938f0f3132", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91f6282e-d082-41be-9cd8-0fda54ec37e3", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "82e79a6e71088291b966b5c3c6905ee5c242df52995f890165ca8f97e8dfafa4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "0b6c57eb-d134-432d-9c2e-4442c1c40a81", "node_type": "1", "metadata": {}, "hash": "13ed189b756947bac91576b1dfb346812f878365a23fd6f888226266ae0a0499", "class_name": "RelatedNodeInfo"}}, "text": "In:\u00a0 Stored\u00a0 grain\u00a0 ecosystems\u00a0\n(D.\u00a0 S.\u00a0 Jayas,\u00a0 N.\u00a0 D.\u00a0 G.\u00a0 White\u00a0 and\u00a0 W.\u00a0 E.\u00a0 Muir,\u00a0 eds.).\u00a0\nNew\u00a0York,\u00a0Marcel\u00a0Dekker.\u00a01995,\u00a0pp.\u00a0197\u2010249.", "start_char_idx": 20127, "end_char_idx": 20266, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "0b6c57eb-d134-432d-9c2e-4442c1c40a81": {"__data__": {"id_": "0b6c57eb-d134-432d-9c2e-4442c1c40a81", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "add9152a-ea13-4642-9270-153b80aeed4f", "node_type": "1", "metadata": {"identifier": "njb-57", "author": "Mathew, Shiju; Thomas, George; Ahmad, Tufail", "title": "An Evaluation of the Fungi Isolated from Sub-epidermal Region of Post-harvested Stored Wheat Grains", "date": "2022-07-11", "file": "njb-57.pdf"}, "hash": "3c6796daf00835566cbb29e7d3863a0210245ead93839073ac9e7ba049d39716", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "2b134834-8a9a-4016-bade-798ced74e751", "node_type": "1", "metadata": {}, "hash": "6d8d0a99b2ab843b86072d5c9b698f12954e707a5b3a4e83c16faf79438bf7fb", "class_name": "RelatedNodeInfo"}}, "text": "1\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n1\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nORIGINAL\u00a0RESEARCH\u00a0ARTICLE\u00a0\n\nInformation\u00a0Theory\u00a0and\u00a0Multivariate\u00a0Techniques\u00a0for\u00a0\nAnalyzing\u00a0DNA\u00a0Sequence\u00a0Data:\u00a0An\u00a0Example\u00a0from\u00a0\n\nTomato\u00a0Genes\u00a0\n\nBal\u00a0K.\u00a0Joshi\u00a0and\u00a0Dilip\u00a0R.\u00a0Panthee\u00a0\nNorth\u00a0Carolina\u00a0State\u00a0University,\u00a0Raleigh,\u00a0North\u00a0Carolina\u2010\u00a027695,\u00a0USA\u00a0\n\nAbstract\u00a0\nDNA\u00a0and\u00a0amino\u00a0acid\u00a0sequences\u00a0are\u00a0alphabetic\u00a0symbols\u00a0having\u00a0no\u00a0underlying\u00a0metric.\u00a0Use\u00a0of\u00a0information\u00a0theory\u00a0is\u00a0\n\none\u00a0of\u00a0the\u00a0solutions\u00a0for\u00a0sequence\u00a0metric\u00a0problems.\u00a0The\u00a0reflection\u00a0of\u00a0DNA\u00a0sequence\u00a0complexity\u00a0in\u00a0phenotype\u00a0\n\nstability\u00a0 might\u00a0 be\u00a0 useful\u00a0 for\u00a0 crop\u00a0 improvement.\u00a0 Shannon\u2010Weaver\u00a0 index\u00a0 (Shannon\u00a0 Entropy,\u00a0 H\u2019)\u00a0 and\u00a0 mutual\u00a0\n\ninformation\u00a0 (MI)\u00a0 index\u00a0 were\u00a0 estimated\u00a0 from\u00a0 DNA\u00a0 sequences\u00a0 of\u00a0 22\u00a0 genes,\u00a0 consisted\u00a0 of\u00a0 two\u00a0 gene\u00a0 families\u00a0 of\u00a0\n\ntomato,\u00a0 namely\u00a0 disease\u00a0 resistance\u00a0 and\u00a0 fruit\u00a0 quality.\u00a0 Main\u00a0 objective\u00a0 was\u00a0 use\u00a0 of\u00a0 information\u00a0 theory\u00a0 and\u00a0\n\nmultivariate\u00a0 techniques\u00a0 to\u00a0 understand\u00a0 diversity\u00a0 among\u00a0 genes\u00a0 and\u00a0 relate\u00a0 the\u00a0 sequence\u00a0 complexity\u00a0 with\u00a0\n\nphenotypes.\u00a0The\u00a0normalized\u00a0H\u2019\u00a0value\u00a0ranged\u00a0from\u00a00.429\u00a0to\u00a00.461.\u00a0The\u00a0highest\u00a0diversity\u00a0was\u00a0observed\u00a0 in\u00a0the\u00a0\n\ngene\u00a0 Crtr\u2010B\u00a0 (beta\u00a0 carotene\u00a0 hydroxylase).\u00a0 Two\u00a0 principal\u00a0 components\u00a0 which\u00a0 accounted\u00a0 for\u00a0 36.65%\u00a0 variation\u00a0\n\nplaced\u00a0these\u00a0genes\u00a0into\u00a0four\u00a0groups.\u00a0Groupings\u00a0of\u00a0these\u00a0genes\u00a0by\u00a0both\u00a0principal\u00a0component\u00a0and\u00a0cluster\u00a0analyses\u00a0\n\nshowed\u00a0 clearly\u00a0 the\u00a0 similarity\u00a0 at\u00a0 phenotypes\u00a0 levels\u00a0 within\u00a0 cluster.\u00a0 Sequences\u00a0 similarity\u00a0 among\u00a0 genes\u00a0 was\u00a0\n\nobserved\u00a0within\u00a0a\u00a0family.\u00a0Diversity\u00a0assessment\u00a0of\u00a0genes\u00a0applying\u00a0information\u00a0theory\u00a0should\u00a0link\u00a0to\u00a0understand\u00a0\n\nthe\u00a0sequences\u00a0complexity\u00a0with\u00a0respect\u00a0to\u00a0gene\u00a0stability\u00a0for\u00a0example\u00a0stability\u00a0of\u00a0resistance\u00a0gene.\u00a0\u00a0\n\n\u00a0\nKey\u00a0words:\u00a0Diversity\u00a0analysis,\u00a0DNA\u00a0sequences,\u00a0principal\u00a0component\u00a0analysis,\u00a0tomato\u00a0genes\u00a0\n\n\u00a0\nCorrespondence\u00a0Author:\u00a0\nE\u2010mail:\u00a0joshibalak@yahoo.com\u00a0\n\nIntroduction\u00a0\nSequencing\u00a0of\u00a0genomic\u00a0DNA\u00a0has\u00a0been\u00a0started\u00a0in\u00a0many\u00a0\n\norganisms\u00a0in\u00a0the\u00a0world\u00a0and\u00a0most\u00a0of\u00a0the\u00a0sequences\u00a0are\u00a0\n\npublically\u00a0available.\u00a0 International\u00a0Solanaceae\u00a0Genome\u00a0\n\nProject\u00a0 (SOL)\u00a0 has\u00a0 started\u00a0 sequencing\u00a0 the\u00a0 genome\u00a0 of\u00a0\n\ntomato.\u00a0 Ten\u00a0 countries\u00a0 namely\u00a0 Korea,\u00a0 China,\u00a0 United\u00a0\n\nKingdom,\u00a0India,\u00a0The\u00a0Netherlands,\u00a0France,\u00a0Japan,\u00a0Spain,\u00a0\n\nItaly\u00a0and\u00a0the\u00a0United\u00a0States\u00a0are\u00a0involved\u00a0in\u00a0the\u00a0genome\u00a0\n\nsequencing\u00a0 project\u00a0 of\u00a0 tomato\u00a0 (Mueller\u00a0 et\u00a0 al.,\u00a0 2005).\u00a0\n\nTomato\u00a0is\u00a0the\u00a0model\u00a0plant\u00a0for\u00a0the\u00a0study\u00a0of\u00a0a\u00a0number\u00a0of\u00a0\n\neconomically\u00a0 important\u00a0 traits\u00a0 including\u00a0 fruit\u00a0\n\ndevelopment\u00a0 and\u00a0 plant\u00a0 defense\u00a0 (Li\u00a0 et\u00a0 al.,\u00a0 2001;\u00a0\n\nTanksley,\u00a0 2004).\u00a0 Major\u00a0 problem\u00a0 with\u00a0 these\u00a0 sequence\u00a0\n\ndata\u00a0 is\u00a0 metric\u00a0 problem\u00a0 i.e.", "start_char_idx": 45, "end_char_idx": 2677, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "2b134834-8a9a-4016-bade-798ced74e751": {"__data__": {"id_": "2b134834-8a9a-4016-bade-798ced74e751", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "0b6c57eb-d134-432d-9c2e-4442c1c40a81", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "32c874b4b804b6606d76f370ffd9335e4e666412ee8f6f945f1c27823812bd39", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "453d29aa-c9b9-4101-917b-bdc81ca27a11", "node_type": "1", "metadata": {}, "hash": "87e6df149babec79b83699e631ab2653e2085ff583e208e73a719acd6c1e3673", "class_name": "RelatedNodeInfo"}}, "text": "difficult\u00a0 to\u00a0 analyze\u00a0\n\nstatistically\u00a0 to\u00a0 extract\u00a0 the\u00a0 biologically\u00a0 meaningful\u00a0\n\ninformation.\u00a0 Use\u00a0 of\u00a0 information\u00a0 theory\u00a0 is\u00a0 one\u00a0 of\u00a0 the\u00a0\n\nways\u00a0for\u00a0handling\u00a0with\u00a0sequence\u00a0metric\u00a0problem\u00a0data.\u00a0\n\nInformation\u00a0theory\u00a0which\u00a0 is\u00a0based\u00a0on\u00a0probability\u00a0and\u00a0\n\nstatistics\u00a0quantifies\u00a0information\u00a0in\u00a0the\u00a0categorical\u00a0form\u00a0\n\nof\u00a0 data\u00a0 e.g.\u00a0 alphabetic\u00a0 sequences\u00a0 of\u00a0 DNA.\u00a0 Entropy\u00a0 is\u00a0\n\nthe\u00a0 key\u00a0 measure\u00a0 of\u00a0 information\u00a0 which\u00a0 quantifies\u00a0 the\u00a0\n\nuncertainty\u00a0 involved\u00a0 when\u00a0 encountering\u00a0 a\u00a0 random\u00a0\n\nvariable.\u00a0 Another\u00a0 element\u00a0 of\u00a0 information\u00a0 theory\u00a0 is\u00a0\n\nmutual\u00a0 information\u00a0 which\u00a0 is\u00a0 the\u00a0 amount\u00a0 of\u00a0\n\ninformation\u00a0in\u00a0common\u00a0between\u00a0two\u00a0random\u00a0variables\u00a0\n\n(Schneider,\u00a02003;\u00a0Atchley\u00a0et\u00a0al.,\u00a02000).\u00a0\n\nMany\u00a0 resistant\u00a0 varieties\u00a0 of\u00a0 tomato\u00a0 have\u00a0 been\u00a0\n\ndeveloped\u00a0through\u00a0the\u00a0introgression\u00a0of\u00a0resistant\u00a0genes\u00a0\n\neither\u00a0from\u00a0cultivated\u00a0or\u00a0from\u00a0wild\u00a0species\u00a0of\u00a0tomato.\u00a0\n\nMore\u00a0 than\u00a0 8\u00a0 years\u00a0 is\u00a0 necessary\u00a0 to\u00a0 develop\u00a0 resistant\u00a0\n\nvariety\u00a0 through\u00a0 conventional\u00a0 breeding\u00a0 system.\u00a0\n\nHowever,\u00a0due\u00a0to\u00a0the\u00a0high\u00a0mutation\u00a0rate\u00a0 in\u00a0pathogen,\u00a0\n\nresistant\u00a0gene\u00a0may\u00a0not\u00a0be\u00a0effective\u00a0for\u00a0a\u00a0 longer\u00a0time\u00a0\n\nbecause\u00a0 of\u00a0 the\u00a0 evolution\u00a0 of\u00a0 mutant\u00a0 pathogen.\u00a0 More\u00a0\n\nstable\u00a0 resistant\u00a0 gene,\u00a0 if\u00a0 possible\u00a0 to\u00a0 identify\u00a0 at\u00a0 early\u00a0\n\nstage\u00a0 may\u00a0 contribute\u00a0 greatly\u00a0 in\u00a0 crop\u00a0 improvement.\u00a0\n\nSimilarly,\u00a0there\u00a0is\u00a0a\u00a0high\u00a0variation\u00a0for\u00a0fruit\u00a0quality\u00a0traits\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n2\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nof\u00a0tomato.\u00a0Many\u00a0of\u00a0these\u00a0genes\u00a0have\u00a0been\u00a0sequenced\u00a0\n\nand\u00a0 data\u00a0 are\u00a0 available\u00a0 in\u00a0 the\u00a0 Solanaceae\u00a0 Genomics\u00a0\n\nNetwork\u00a0(SGN)\u00a0(http://sgn.cornell.edu)\u00a0(Mueller\u00a0et\u00a0al.,\u00a0\n\n2005a;\u00a0Mueller\u00a0et\u00a0al.,\u00a02005b).\u00a0DNA\u00a0sequences\u00a0data\u00a0are\u00a0\n\nadded\u00a0rapidly\u00a0to\u00a0the\u00a0SGN.\u00a0However,\u00a0utilization\u00a0of\u00a0these\u00a0\n\ndata\u00a0 is\u00a0 restricted\u00a0 due\u00a0 to\u00a0 lack\u00a0 of\u00a0 data\u00a0 analysis\u00a0 tools.\u00a0\n\nUsing\u00a0 the\u00a0 information\u00a0 theory,\u00a0 various\u00a0 indices\u00a0 and\u00a0\n\nvariability\u00a0 had\u00a0 been\u00a0 estimated\u00a0 from\u00a0 the\u00a0 amino\u00a0 acid\u00a0\n\nsequences\u00a0 data\u00a0 and\u00a0 subsequently\u00a0 analyzed\u00a0 using\u00a0\n\nmultivariate\u00a0techniques\u00a0(Atchley\u00a0et\u00a0al.,\u00a01999;\u00a0Atchley\u00a0et\u00a0\n\nal.,\u00a02000;\u00a0Atchley\u00a0and\u00a0Zhao,\u00a02007;\u00a0Atchley\u00a0et\u00a0al.,\u00a02005).\u00a0\n\nDNA\u00a0sequences\u00a0are\u00a0also\u00a0alphabetic\u00a0symbols\u00a0that\u00a0need\u00a0\n\nto\u00a0transform\u00a0these\u00a0symbols\u00a0to\u00a0biologically\u00a0meaningful\u00a0\n\nvariables.\u00a0 Approaches\u00a0 used\u00a0 by\u00a0 Atchley\u00a0 et\u00a0 al.\u00a0 (1999,\u00a0\n\n2000,\u00a0 2005)\u00a0 can\u00a0 be\u00a0 used\u00a0 to\u00a0 summarize\u00a0 the\u00a0 DNA\u00a0\n\nsequences\u00a0 data.\u00a0 Multivariate\u00a0 technique,\u00a0 for\u00a0 example\u00a0\n\nPrincipal\u00a0 Component\u00a0 Analysis\u00a0 (PCA)\u00a0 and\u00a0 cluster\u00a0\n\nanalysis\u00a0 would\u00a0 help\u00a0 greatly\u00a0 to\u00a0 draw\u00a0 inference\u00a0 from\u00a0\n\nthese\u00a0DNA\u00a0sequences\u00a0data.\u00a0The\u00a0objective\u00a0of\u00a0this\u00a0study\u00a0\n\nwas\u00a0 to\u00a0 measure\u00a0 the\u00a0 diversity\u00a0 and\u00a0 to\u00a0 study\u00a0 the\u00a0\n\nrelationship\u00a0 between\u00a0 sequence\u00a0 variation\u00a0 and\u00a0\n\nphenotype\u00a0 among\u00a0 two\u00a0 gene\u00a0 families\u00a0 namely\u00a0 disease\u00a0\n\nresistance\u00a0and\u00a0fruit\u00a0quality.", "start_char_idx": 2679, "end_char_idx": 5442, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "453d29aa-c9b9-4101-917b-bdc81ca27a11": {"__data__": {"id_": "453d29aa-c9b9-4101-917b-bdc81ca27a11", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "2b134834-8a9a-4016-bade-798ced74e751", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "5a8ba95afe2241c7294d7af775297d748cfbc8c4a9ae5d312ed57026bf381522", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4545b4a0-3334-4424-aced-bd48f6914985", "node_type": "1", "metadata": {}, "hash": "edf194e59611f61a1dddd33490a29da8f00467b19ba3ad7ea3c25a9ee94c0951", "class_name": "RelatedNodeInfo"}}, "text": "Multivariate\u00a0analysis\u00a0allows\u00a0using\u00a0all\u00a0available\u00a0variable\u00a0\n\ninformation\u00a0 simultaneously\u00a0 producing\u00a0 a\u00a0 single\u00a0\n\nparameter.\u00a0 It\u00a0 has\u00a0 been\u00a0 used\u00a0 for\u00a0 both\u00a0 qualitative\u00a0 and\u00a0\n\nquantitative\u00a0 characters\u00a0 to\u00a0 measure\u00a0 genetic\u00a0\n\nrelationships\u00a0 within\u00a0 cereal\u00a0 species\u00a0 e.g.\u00a0 barley\u00a0 (Cross,\u00a0\n\n1992;\u00a0 Hussaini\u00a0 et\u00a0 al.,\u00a0 1977)\u00a0 and\u00a0 rice\u00a0 (Kanwal\u00a0 et\u00a0 al.,\u00a0\n\n1983).\u00a0 The\u00a0 information\u00a0 generated\u00a0 can\u00a0 be\u00a0 useful\u00a0 for\u00a0\n\nidentifying\u00a0 groups\u00a0 of\u00a0 accessions\u00a0 that\u00a0 have\u00a0 desirable\u00a0\n\ncharacters\u00a0for\u00a0further\u00a0study\u00a0and\u00a0for\u00a0investigating\u00a0some\u00a0\n\naspects\u00a0 of\u00a0 crop\u00a0 evolution\u00a0 (Brown,\u00a0 1991;\u00a0 Cowen\u00a0 and\u00a0\n\nFrey,\u00a0 1987;\u00a0 Perry\u00a0 and\u00a0 McIntosh,\u00a0 1991).\u00a0 Among\u00a0 the\u00a0\n\ndifferent\u00a0 methods\u00a0 of\u00a0 multivariate\u00a0 analysis,\u00a0 PCA\u00a0 and\u00a0\n\ncluster\u00a0analysis\u00a0are\u00a0commonly\u00a0used.\u00a0PCA\u00a0is\u00a0a\u00a0technique\u00a0\n\nfor\u00a0analyzing\u00a0relationships\u00a0among\u00a0several\u00a0quantitative\u00a0\n\nvariables\u00a0 measured\u00a0 on\u00a0a\u00a0 number\u00a0 of\u00a0 objects\u00a0 (Ringn\u00e9r,\u00a0\n\n2008).\u00a0 It\u00a0 provides\u00a0 information\u00a0 about\u00a0 the\u00a0 relative\u00a0\n\nimportance\u00a0 of\u00a0 each\u00a0 variable\u00a0 in\u00a0 characterizing\u00a0 the\u00a0\n\nobjects.\u00a0 Cluster\u00a0 analysis\u00a0 can\u00a0 be\u00a0 used\u00a0 to\u00a0 group\u00a0 units\u00a0\n\naccording\u00a0to\u00a0the\u00a0similarity\u00a0for\u00a0certain\u00a0characteristics.\u00a0\u00a0\n\nAmong\u00a0 the\u00a0 different\u00a0 measures\u00a0 of\u00a0 diversity,\u00a0 Shannon\u2010\nWeaver\u00a0index\u00a0(Shannon\u00a0entropy,\u00a0H')\u00a0is\u00a0being\u00a0used\u00a0for\u00a0\nqualitative\u00a0 traits\u00a0 (Holcomb\u00a0 et\u00a0 al.,\u00a0 1977;\u00a0 Niwranski\u00a0 et\u00a0\nal.,\u00a02002;\u00a0Tolbert\u00a0et\u00a0al.,\u00a01979)\u00a0and\u00a0amino\u00a0acid\u00a0sequence\u00a0\ndata\u00a0(Atchley\u00a0et\u00a0al.,\u00a02000,\u00a02005).\u00a0Principally\u00a0H'\u00a0includes\u00a0\nboth\u00a0 species\u00a0 number\u00a0 and\u00a0 evenness,\u00a0 where\u00a0 a\u00a0 greater\u00a0\nnumber\u00a0of\u00a0species\u00a0 increase\u00a0diversity,\u00a0as\u00a0does\u00a0a\u00a0more\u00a0\n\nequitable\u00a0distribution\u00a0of\u00a0individuals\u00a0among\u00a0species.\u00a0As\u00a0\nspecies\u00a0 richness\u00a0 and\u00a0 evenness\u00a0 increase,\u00a0 so\u00a0 does\u00a0\ndiversity.\u00a0 Diversity\u00a0 index\u00a0 provides\u00a0 important\u00a0\ninformation\u00a0about\u00a0rarity\u00a0and\u00a0commonness\u00a0of\u00a0species\u00a0in\u00a0\na\u00a0 community.\u00a0 The\u00a0 ability\u00a0 to\u00a0 quantify\u00a0 diversity\u00a0 in\u00a0 this\u00a0\nway\u00a0 is\u00a0 an\u00a0 important\u00a0 tool\u00a0 for\u00a0 biologists.\u00a0 Alphabetic\u00a0\nsequence\u00a0data\u00a0can\u00a0be\u00a0summarized\u00a0more\u00a0precisely\u00a0with\u00a0\nthis\u00a0 idea\u00a0 and\u00a0 multivariate\u00a0 techniques.\u00a0 This\u00a0 paper\u00a0\ndescribes\u00a0 the\u00a0 gene\u00a0 diversity\u00a0 in\u00a0 tomato\u00a0 using\u00a0\ninformation\u00a0theory\u00a0and\u00a0multivariate\u00a0techniques.\u00a0\n\nMaterials\u00a0and\u00a0Methods\u00a0\nTomato\u00a0 Genetic\u00a0 Resource\u00a0 Center\u00a0 (TGRC)\u00a0 (http://\n\ntgrc.ucdavis.edu)\u00a0has\u00a0 listed\u00a0a\u00a0total\u00a0of\u00a01239\u00a0genes\u00a0and\u00a0\n\ntheir\u00a0 symbols\u00a0 along\u00a0 with\u00a0 their\u00a0 phenotypes.\u00a0 Among\u00a0\n\nthem,\u00a0there\u00a0are\u00a068\u00a0resistance\u00a0genes\u00a0related\u00a0to\u00a0diseases\u00a0\n\nand\u00a041\u00a0genes\u00a0related\u00a0to\u00a0fruit\u00a0quality\u00a0of\u00a0tomato.\u00a0A\u00a0total\u00a0\n\nof\u00a022\u00a0genes\u00a0(Table\u00a01)\u00a0consisting\u00a0of\u00a08\u00a0disease\u00a0resistance\u00a0\n\ngenes,\u00a012\u00a0fruit\u00a0quality\u00a0related\u00a0genes\u00a0and\u00a02\u00a0genes\u00a0from\u00a0\n\npotato\u00a0 genome\u00a0 were\u00a0 used\u00a0 in\u00a0 this\u00a0 study.\u00a0 DNA\u00a0\n\nsequences\u00a0along\u00a0with\u00a0other\u00a0traits\u00a0of\u00a0these\u00a0genes\u00a0were\u00a0\n\ndownloaded\u00a0 from\u00a0 Solanaceae\u00a0 Genomics\u00a0 Network\u00a0\n\n(http://sgn.cornell.edu).", "start_char_idx": 5446, "end_char_idx": 8136, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4545b4a0-3334-4424-aced-bd48f6914985": {"__data__": {"id_": "4545b4a0-3334-4424-aced-bd48f6914985", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "453d29aa-c9b9-4101-917b-bdc81ca27a11", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "40207ba7619e234e6387bc413cb3cc428c5b4a4bba5beeda8c053505f065c116", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "b5b64a9c-4160-4e67-a23d-c2e6762179f6", "node_type": "1", "metadata": {}, "hash": "c975aa493bafc74a3bf26e648d76ead361a833968bc77d7e51128b5b1b81a8d6", "class_name": "RelatedNodeInfo"}}, "text": "Multiple\u00a0 alignments\u00a0 of\u00a0 DNA\u00a0 sequences\u00a0 were\u00a0 done\u00a0 in\u00a0\nClustelX2\u00a0 (http://www.clustal.org/).\u00a0 ClustalX2\u00a0\ncalculates\u00a0 the\u00a0 best\u00a0 match\u00a0 for\u00a0 the\u00a0 selected\u00a0 sequences\u00a0\nbased\u00a0on\u00a0the\u00a0homology\u00a0concept,\u00a0and\u00a0lines\u00a0them\u00a0up\u00a0so\u00a0\nthat\u00a0the\u00a0 identities,\u00a0similarities\u00a0and\u00a0differences\u00a0can\u00a0be\u00a0\nseen.\u00a0 Shannon\u00a0 Entropy\u00a0 (H\u2019)\u00a0 was\u00a0 estimated\u00a0 for\u00a0 each\u00a0\ngene,\u00a0 not\u00a0 the\u00a0 site\u00a0 of\u00a0 nucleotides\u00a0 using\u00a0 the\u00a0\nFastaEntropy\u00a0 software.\u00a0 \u00a0 FastaEntropy\u00a0 was\u00a0 originally\u00a0\ndeveloped\u00a0for\u00a0estimating\u00a0H\u2019\u00a0and\u00a0MI\u00a0of\u00a0amino\u00a0acid\u00a0sites\u00a0\n(column\u00a0wise)\u00a0based\u00a0on\u00a0the\u00a020\u00a0amino\u00a0acids\u00a0(\u00a0Atchley\u00a0et\u00a0\nal.,\u00a01999,2000;\u00a0Butte\u00a0and\u00a0Kohane,\u00a02000).\u00a0We\u00a0used\u00a0this\u00a0\nsoftware\u00a0 in\u00a0DNA\u00a0sequences\u00a0data.\u00a0To\u00a0verify\u00a0the\u00a0use\u00a0of\u00a0\nFastaEntropy\u00a0in\u00a0DNA\u00a0sequences\u00a0data,\u00a0we\u00a0estimated\u00a0H\u2019\u00a0\nof\u00a0 translated\u00a0 amino\u00a0 acids\u00a0 and\u00a0 found\u00a0 the\u00a0 strong\u00a0\nassociation\u00a0 with\u00a0 DNA\u00a0 sequence\u2010based\u00a0 estimates.\u00a0\nNormalized\u00a0 entropy\u00a0 value\u00a0 was\u00a0 used\u00a0 to\u00a0 develop\u00a0 the\u00a0\nentropy\u00a0 profile\u00a0 of\u00a0 each\u00a0 gene.\u00a0 Normalized\u00a0 mutual\u00a0\ninformation\u00a0(MI)\u00a0matrix\u00a0among\u00a0the\u00a0genes\u00a0was\u00a0used\u00a0to\u00a0\ngenerate\u00a0 the\u00a0 scatter\u00a0 biplot\u00a0 considering\u00a0 Principal\u00a0\nComponents\u00a0 I\u00a0 and\u00a0 II\u00a0 using\u00a0 NTSYSpc\u00a0 (http://\nwww.exetersoftware.com/index.html).\u00a0Cluster\u00a0analysis\u00a0\nwas\u00a0also\u00a0carried\u00a0out\u00a0based\u00a0on\u00a0this\u00a0MI\u00a0in\u00a0NTSYSpc.\u00a0PCA\u00a0\npermits\u00a0 reduction\u00a0 of\u00a0 the\u00a0 complexity\u00a0 or\u00a0 dimension\u00a0 of\u00a0\nthe\u00a0 problem\u00a0 (Johnson\u00a0 and\u00a0 Wichern,\u00a0 1988;\u00a0 Ringn\u00e9r,\u00a0\n2008).\u00a0The\u00a0technique\u00a0consists\u00a0of\u00a0reducing\u00a0the\u00a0structure\u00a0\nof\u00a0 the\u00a0 data\u00a0 matrix\u00a0 starting\u00a0 from\u00a0 a\u00a0 linear\u00a0 model\u00a0 of\u00a0\ngetting\u00a0 new\u00a0 variables,\u00a0 referred\u00a0 to\u00a0 as\u00a0 principal\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n3\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\ncomponents.\u00a0 Those\u00a0 principal\u00a0 components\u00a0 with\u00a0 eigen\u00a0\nvalues\u00a0\u00b3\u00a01.0\u00a0were\u00a0selected.\u00a0Cluster\u00a0analysis\u00a0allows\u00a0one\u00a0\nto\u00a0 identify\u00a0 groups\u00a0 of\u00a0 objects\u00a0 or\u00a0 variables\u00a0 that\u00a0 are\u00a0\nsimilar\u00a0among\u00a0themselves\u00a0(Sneath\u00a0and\u00a0Sokal,\u00a01973).\u00a0 \n\nRESULTS\u00a0AND\u00a0DISCUSSION\u00a0\nGenerally\u00a0mapping\u00a0populations\u00a0in\u00a0tomato\u00a0is\u00a0generated\u00a0\n\nby\u00a0crossing\u00a0cultivated\u00a0species\u00a0with\u00a0wild\u00a0relatives\u00a0which\u00a0\n\nresults\u00a0in\u00a0maximizing\u00a0the\u00a0genetic\u00a0variation\u00a0particularly\u00a0\n\nfor\u00a0disease\u00a0resistance\u00a0and\u00a0fruit\u00a0quality\u00a0traits\u00a0\u00a0(Frary\u00a0et\u00a0\n\nal.,\u00a0 2000;\u00a0 Fridman\u00a0 et\u00a0 al.,\u00a0 2000).\u00a0 Therefore,\u00a0 we\u00a0 used\u00a0\n\nthese\u00a0 two\u00a0 gene\u00a0 families\u00a0 (disease\u00a0 resistance\u00a0 and\u00a0 fruit\u00a0\n\nquality)\u00a0 to\u00a0 assess\u00a0 and\u00a0 characterize\u00a0 the\u00a0 sequence\u00a0\n\nvariation.\u00a0All\u00a0available\u00a0genes\u00a0with\u00a0DNA\u00a0sequences\u00a0were\u00a0\n\nincluded\u00a0in\u00a0our\u00a0study.\u00a0\n\nTomato\u00a0is\u00a0a\u00a0diploid\u00a0with\u00a02n=\u00a024\u00a0chromosomes\u00a0and\u00a0self\u00a0\npollinated\u00a0species.\u00a0The\u00a0eight\u00a0disease\u00a0resistance\u00a0genes\u00a0\nare\u00a0located\u00a0in\u00a0chromosomes\u00a01,\u00a03,\u00a04,\u00a05\u00a0and\u00a06,\u00a0mostly\u00a0in\u00a0\nlong\u00a0arm\u00a0(Table\u00a01).\u00a0One\u00a0locus,\u00a0Pseudomonas\u00a0syringe\u00a0pv\u00a0\ntomato\u00a0 resistance\u00a0 has\u00a0 4\u00a0 alleles.", "start_char_idx": 8140, "end_char_idx": 10779, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "b5b64a9c-4160-4e67-a23d-c2e6762179f6": {"__data__": {"id_": "b5b64a9c-4160-4e67-a23d-c2e6762179f6", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4545b4a0-3334-4424-aced-bd48f6914985", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "ce854794af304f059fd0577b24fdeacbcb4d9893594a5434beec7ac0c3c477f4", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "68179942-6ac1-46b2-ba89-f7ddfe3ab813", "node_type": "1", "metadata": {}, "hash": "f2274d9f4e5c853427f32cfe87bc0516d5f4b6279a7d84ff70976cc12b968aa8", "class_name": "RelatedNodeInfo"}}, "text": "DNA\u00a0 sequences\u00a0 of\u00a0\nthese\u00a0genes\u00a0ranged\u00a0from\u00a01496\u00a0to\u00a03987\u00a0base\u00a0pairs.\u00a0The\u00a0\nnumber\u00a0of\u00a0sequences\u00a0of\u00a0alleles\u00a0of\u00a0Pto\u00a0were\u00a0the\u00a0same.\u00a0\nWith\u00a0regards\u00a0to\u00a012\u00a0fruit\u00a0quality\u00a0related\u00a0genes,\u00a0mostly\u00a0\nthey\u00a0are\u00a0located\u00a0in\u00a0long\u00a0arm\u00a0of\u00a0chromosomes\u00a03\u00a0and\u00a06.\u00a0\nBeta\u00a0carotene\u00a0gene\u00a0has\u00a04\u00a0alleles\u00a0with\u00a0the\u00a0same\u00a0number\u00a0\nof\u00a0 DNA\u00a0 sequences\u00a0 and\u00a0 Py\u20101\u00a0 gene\u00a0 has\u00a0 5\u00a0 alleles\u00a0 with\u00a0\ndifferent\u00a0number\u00a0of\u00a0sequences.\u00a0The\u00a0allele\u00a0prov5\u00a0of\u00a0Psy\u2010\n1\u00a0has\u00a0the\u00a0shortest\u00a0sequences\u00a0and\u00a0never\u00a0ripe,\u00a0nr\u00a0has\u00a0the\u00a0\nlongest\u00a0DNA\u00a0sequences.\u00a0Two\u00a0disease\u00a0resistance\u00a0genes\u00a0\nfrom\u00a0potato\u00a0genome\u00a0were\u00a0of\u00a01508\u00a0and\u00a0333\u00a0number\u00a0of\u00a0\nbase\u00a0pairs.\u00a0\u00a0\n\nShannon\u00a0entropy\u00a0profiles\u00a0along\u00a0with\u00a0their\u00a0index\u00a0(H\u2019)\u00a0of\u00a0\n\neach\u00a0 gene\u00a0 are\u00a0 given\u00a0 in\u00a0 Figure\u00a0 1.\u00a0 The\u00a0 normalized\u00a0 H\u2019\u00a0\n\nvalue\u00a0 ranged\u00a0 from\u00a0 0.4291\u00a0 to\u00a0 0.461.\u00a0 All\u00a0 the\u00a0 alleles\u00a0 of\u00a0\n\nbeta\u00a0carotene\u00a0and\u00a0Pto\u00a0have\u00a0the\u00a0same\u00a0H\u2019\u00a0values.\u00a0Both\u00a0\n\ngenes\u00a0 of\u00a0 potato\u00a0 indicated\u00a0 more\u00a0 gene\u00a0 diversity\u00a0 and\u00a0\n\ncomplexity\u00a0 as\u00a0 compared\u00a0 to\u00a0 tomato\u00a0 genes.\u00a0 Within\u00a0\n\ntomato\u00a0 genes,\u00a0 the\u00a0 highest\u00a0 diversity\u00a0 was\u00a0 observed\u00a0 in\u00a0\n\nthe\u00a0gene,\u00a0CrtR\u2010b\u00a0(Beta\u00a0carotene\u00a0hydroxylase)\u00a0and\u00a0the\u00a0\n\nlowest\u00a0diversity\u00a0 in\u00a0Phytoene\u00a0synthase\u20101\u00a0(prov5).\u00a0Gene\u00a0\n\nof\u00a0 white\u00a0 flower\u00a0 was\u00a0 the\u00a0 most\u00a0 variable\u00a0 and\u00a0 of\u00a0 yellow\u00a0\n\nflower\u00a0 was\u00a0 the\u00a0 least\u00a0 variable.\u00a0 The\u00a0 second\u00a0 highest\u00a0\n\ncomplexity\u00a0 in\u00a0 DNA\u00a0 sequences\u00a0 was\u00a0 observed\u00a0 in\u00a0 the\u00a0\n\ngene\u00a0 resistance\u00a0 to\u00a0 potato\u00a0 cyst\u00a0 nematode.\u00a0 Entropy\u00a0\n\nvalues\u00a0 of\u00a0 all\u00a0 the\u00a0 proteins,\u00a0 generated\u00a0 after\u00a0 translating\u00a0\n\nthese\u00a0 DNA\u00a0 sequences\u00a0 were\u00a0 higher\u00a0 than\u00a0 their\u00a0 genes,\u00a0\n\nbut\u00a0 strong\u00a0 correlation\u00a0 between\u00a0 directly\u00a0 estimated\u00a0 H\u2019\u00a0\n\nvalues\u00a0 of\u00a0 genes\u00a0 and\u00a0 H\u2019\u00a0 of\u00a0 their\u00a0 translated\u00a0 protein\u00a0\n\nindicates\u00a0that\u00a0FastaEntropy\u00a0can\u00a0be\u00a0used\u00a0to\u00a0estimate\u00a0H\u2019\u00a0\n\nfrom\u00a0DNA\u00a0sequences\u00a0(data\u00a0not\u00a0shown).\u00a0On\u00a0an\u00a0average,\u00a0\n\nH\u2019\u00a0value\u00a0of\u00a0disease\u00a0resistance\u00a0gene\u00a0family\u00a0was\u00a0higher\u00a0\n\nthan\u00a0fruit\u00a0quality\u00a0gene\u00a0family.\u00a0\n\nThe\u00a0higher\u00a0the\u00a0H\u2019\u00a0value,\u00a0the\u00a0more\u00a0sequence\u00a0complexity\u00a0\n\nin\u00a0 the\u00a0 gene.\u00a0 Based\u00a0 on\u00a0 this\u00a0 hypothesis\u00a0 the\u00a0 gene\u00a0 with\u00a0\n\nhigh\u00a0H\u2019\u00a0value\u00a0would\u00a0be\u00a0more\u00a0stable\u00a0or\u00a0such\u00a0gene\u00a0need\u00a0\n\nlong\u00a0time\u00a0to\u00a0take\u00a0place\u00a0the\u00a0evolutionary\u00a0changes.\u00a0This\u00a0\n\nis\u00a0very\u00a0useful\u00a0particularly\u00a0to\u00a0develop\u00a0disease\u00a0resistance\u00a0\n\nvariety.\u00a0Breeder\u00a0can\u00a0identify\u00a0the\u00a0more\u00a0stable\u00a0resistance\u00a0\n\ngenes\u00a0looking\u00a0on\u00a0the\u00a0Shannon\u00a0entropy\u00a0profile.\u00a0\u00a0\n\nMutual\u00a0information\u00a0values\u00a0for\u00a0genes\u00a0are\u00a0given\u00a0in\u00a0Table\u00a0\n2.\u00a0The\u00a0value\u00a0with\u00a01,\u00a0for\u00a0example,\u00a0among\u00a0the\u00a0alleles\u00a0of\u00a0\nPto\u00a0(Pseudomonas\u00a0syringae\u00a0pv\u00a0tomato\u00a0resistance)\u00a0and\u00a0\nB\u00a0 (beta\u00a0 carotene)\u00a0 indicates\u00a0 that\u00a0 the\u00a0 alleles\u00a0 of\u00a0 these\u00a0\ngenes\u00a0have\u00a0the\u00a0same\u00a0sequences.", "start_char_idx": 10781, "end_char_idx": 13232, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "68179942-6ac1-46b2-ba89-f7ddfe3ab813": {"__data__": {"id_": "68179942-6ac1-46b2-ba89-f7ddfe3ab813", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "b5b64a9c-4160-4e67-a23d-c2e6762179f6", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "53598bd2f97a6f2355f376756e1564dcfaa23f46668f37eb8a61d34c858c13f3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "d2e40bee-6504-44c5-87f1-2c5b7e693834", "node_type": "1", "metadata": {}, "hash": "9d2daa4baee29275c0029058a75364bb2cd452f9d4fa71aa7b55dcbcdc0136cb", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0associations\u00a0 \u00a0of\u00a0\nPsy\u20101\u2010prov5\u00a0(yellow\u00a0color\u00a0of\u00a0ripe\u00a0fruit\u00a0flesh)\u00a0with\u00a0Psy\u20101\u2010\n2s\u00a0 and\u00a0 Psy\u20101\u2010prov4\u00a0 were\u00a0 the\u00a0 highest.\u00a0 Association\u00a0 of\u00a0\nRanGAP\u20102\u00a0was\u00a0 lower\u00a0with\u00a0most\u00a0of\u00a0the\u00a0genes.\u00a0Mutual\u00a0\ninformation\u00a0 values\u00a0 describe\u00a0 the\u00a0 extent\u00a0 of\u00a0 association\u00a0\u00a0\n\nFigure\u00a01.\u00a0\nNormalized\u00a0\nShannon\u00a0entropy\u00a0\nprofile\u00a0of\u00a0tomato\u00a0\ngenes\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n4\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nSN\u00a0 Gene\u00a0 Allele\u00a0 Locus\u00a0Name\u00a0 Chromosome\u2010arm\u00a0 Phenotype\u00a0 Seq.\u00a0Length\u00a0\nDisease\u00a0resistance\u00a0genes\u00a0 \u00a0\u00a0 \u00a0\u00a0\n\n\u00a01\u00a0 Asc\u00a0 \u2010\u2010\u00a0\nAlternaria\u00a0stem\u00a0\ncanker\u00a0resistance\u00a0\n\n\u00a03\u2010long\u00a0 Resistance\u00a0to\u00a0Alternaria\u00a0stem\u00a0canker\u00a0 1496\u00a0\n\n\u00a02\u00a0 Cf\u20109\u00a0 \u2010\u2010\u00a0\nCladosporium\u00a0\nfulvum\u00a0resistance\u00a0\n\n\u00a01\u2010short\u00a0\nResistance\u00a0to\u00a0specific\u00a0races\u00a0of\u00a0\nCladosporium\u00a0fulvum\u00a0\n\n2906\u00a0\n\n\u00a03\u00a0 Hero\u00a0 \u2010\u2010\u00a0\nHeterodera\u00a0\nrostochiensis\u00a0\n\n\u00a04\u2010short\u00a0\nResistance\u00a0to\u00a0potato\u00a0cyst\u00a0nematode\u00a0\n(Globodera\u00a0rostochiensis)\u00a0\n\n4280\u00a0\n\n\u00a04\u00a0 Pto\u00a0 1\u00a0\nPseudomonas\u00a0\nsyringae\u00a0pv\u00a0tomato\u00a0\nresistance\u00a0\n\n\u00a05\u2010long\u00a0\nResistance\u00a0to\u00a0Pseudomonas\u00a0syringae\u00a0\npv\u00a0tomato,\u00a0race\u00a0zero\u00a0sensitive\u00a0to\u00a0\ninsecticide\u00a0Fenthion\u00a0\n\n2466\u00a0\n\n\u00a05\u00a0 Pto\u00a0 2\u00a0\nPseudomonas\u00a0\nsyringae\u00a0pv\u00a0tomato\u00a0\nresistance\u00a0\n\n\u00a05\u2010long\u00a0\nResistance\u00a0to\u00a0Pseudomonas\u00a0syringae\u00a0\npv\u00a0tomato\u00a0\n\n2466\u00a0\n\n\u00a06\u00a0 Pto\u00a0 h\u00a0\nPseudomonas\u00a0\nsyringae\u00a0pv\u00a0tomato\u00a0\nresistance\u00a0\n\n5\u2010long\u00a0\nResistance\u00a0to\u00a0Pseudomonas\u00a0syringae\u00a0\nwithout\u00a0Fenthion\u00a0sensitivity\u00a0\n\n2466\u00a0\n\n\u00a07\u00a0 Pto\u00a0 Pto\u20102\u00a0\nPseudomonas\u00a0\nsyringae\u00a0pv\u00a0tomato\u00a0\nresistance\u00a0\n\n\u00a05\u2010long\u00a0\nResistance\u00a0to\u00a0Pseudomonas\u00a0syringae\u00a0\npv\u00a0tomato\u00a0\n\n2466\u00a0\n\n\u00a08\u00a0 Mi\u20101.2\u00a0 \u00a0\u00a0\nLeucine\u00a0zipper,\u00a0\nnucleotide\u00a0binding,\u00a0\nleucine\u2010rich\u00a0repeat\u00a0\n\n6\u2010short\u00a0\n\nR\u00a0gene\u00a0that\u00a0confers\u00a0resistance\u00a0\nagainst\u00a0some\u00a0species\u00a0of\u00a0root\u00a0knot\u00a0\nnematode\u00a0and\u00a0specific\u00a0isolates\u00a0of\u00a0\npotato\u00a0aphid\u00a0and\u00a0white\u00a0fly\u00a0\n\n3987\u00a0\n\nFruit\u00a0quality\u00a0genes\u00a0 \u00a0\u00a0 \u00a0\u00a0 \u00a0\u00a0\n\n\u00a09\u00a0 B\u00a0 1\u00a0 Beta\u2010carotene\u00a0 \u00a06\u2010long\u00a0\nFlesh\u00a0of\u00a0ripe\u00a0fruit\u00a0orange,\u00a0due\u00a0to\u00a0high\u00a0\nB\u2010carotene,\u00a0low\u00a0lycopene\u00a0\nconcentrations\u00a0\n\n1772\u00a0\n\n\u00a010\u00a0 B\u00a0 c\u00a0 Beta\u2010carotene\u00a0 6\u2010long\u00a0\nIncreased\u00a0fruit\u00a0lycopene\u00a0content,\u00a0\nphenotype\u00a0similar\u00a0to\u00a0B\u2010og\u00a0\n\n1772\u00a0\n\n\u00a011\u00a0 B\u00a0 m\u00a0 Beta\u2010carotene\u00a0 6\u2010long\u00a0\nHigh\u00a0B\u2010carotene,\u00a0low\u00a0lycopene\u00a0in\u00a0ripe\u00a0\nfruit\u00a0\n\n1772\u00a0\n\n\u00a012\u00a0 B\u00a0 og\u00a0 Beta\u2010carotene\u00a0 6\u2010long\u00a0\nCorolla\u00a0tawny\u00a0orange,", "start_char_idx": 13233, "end_char_idx": 15321, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "d2e40bee-6504-44c5-87f1-2c5b7e693834": {"__data__": {"id_": "d2e40bee-6504-44c5-87f1-2c5b7e693834", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "68179942-6ac1-46b2-ba89-f7ddfe3ab813", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "87575c4f347b10b14989ca07e9754251be2b24b27f160440b28be6258371399e", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "4da73f3a-049b-4031-8765-c68045131744", "node_type": "1", "metadata": {}, "hash": "48cc5b8519cc3ae2772dd38b6adc6dcd54ddb8ed4b55e6e29cafff5fae6840fa", "class_name": "RelatedNodeInfo"}}, "text": "increased\u00a0fruit\u00a0\nlycopene\u00a0\n\n1772\u00a0\n\n\u00a013\u00a0 CrtR\u2010b\u00a0 wf\u00a0\nBeta\u00a0carotene\u00a0\nhydroxylase\u00a0\n\n3\u2010short\u00a0 White\u00a0flower\u00a0 1340\u00a0\n\n\u00a014\u00a0 nr\u00a0 \u2010\u2010\u00a0 Never\u00a0ripe\u00a0 \u00a09\u2010long\u00a0\n\nFruits\u00a0turn\u00a0color\u00a0at\u00a0normal\u00a0time,\u00a0but\u00a0\ndevelop\u00a0full\u00a0pigmentation\u00a0slowly\u00a0and\u00a0\nnever\u00a0assume\u00a0as\u00a0deep\u00a0a\u00a0color\u00a0as\u00a0\nnormal\u00a0\n\n3240\u00a0\n\n\u00a015\u00a0 Psy\u00a01\u00a0 (1s)\u00a0 Phytoene\u00a0synthase\u00a01\u00a0 \u00a03\u2010short\u00a0 Yellow\u00a0color\u00a0of\u00a0ripe\u00a0fruit\u00a0flesh\u00a0 942\u00a0\n\n\u00a016\u00a0 Psy\u00a01\u00a0 (2s)\u00a0 Phytoene\u00a0synthase\u00a01\u00a0 \u00a03\u2010short\u00a0\nYellow\u00a0fruit\u00a0flesh,\u00a0lighter\u00a0yellow\u00a0\nflowers\u00a0\n\n1730\u00a0\n\n\u00a017\u00a0 Psy\u00a0\u00a01\u00a0 prov4\u00a0 Phytoene\u00a0synthase\u00a01\u00a0 3\u2010short\u00a0 Yellow\u00a0color\u00a0of\u00a0ripe\u00a0fruit\u00a0flesh\u00a0 1939\u00a0\n\u00a018\u00a0 Psy\u00a01\u00a0 prov5\u00a0 Phytoene\u00a0synthase\u00a01\u00a0 3\u2010short\u00a0 Yellow\u00a0color\u00a0of\u00a0ripe\u00a0fruit\u00a0flesh\u00a0 213\u00a0\n\n\u00a019\u00a0 Psy\u00a01\u00a0 y\u00a0 Phytoene\u00a0synthase\u00a01\u00a0 3\u2010short\u00a0\nLikely\u00a0allele\u00a0of\u00a0r\u00a0with\u00a0reddish\u00a0flesh\u00a0\ntones\u00a0in\u00a0ripe\u00a0fruit\u00a0\n\n303\u00a0\n\n\u00a020\u00a0 rin\u00a0 1\u00a0 Ripening\u00a0inhibitor\u00a0 \u00a05\u2010long\u00a0\nFruits\u00a0green\u00a0at\u00a0maturity,\u00a0later\u00a0turning\u00a0\nbright\u00a0yellow,\u00a0retarded\u00a0ripening\u00a0\n\n1192\u00a0\n\nPotato\u00a0genes\u00a0 \u00a0\u00a0 \u00a0\u00a0 \u00a0\u00a0\n\n\u00a021\u00a0 Star\u00a0 \u00a0\u00a0\nSolanum\u00a0tubersum\u00a0\nankyrin\u00a0repeat\u00a0\n\n\u00a0\u00a0\nPutatively\u00a0involved\u00a0in\u00a0quantitative\u00a0\nresistance\u00a0to\u00a0Phytophthora\u00a0infestans\u00a0\n\n1508\u00a0\n\n\u00a022\u00a0\nRanGA\nP2\u00a0\n\n\u00a0\u00a0\nGTPase\u2010activating\u00a0\nprotein\u00a0\n\n\u00a0\u00a0\nA\u00a0GTPase\u2010activating\u00a0protein\u00a0that\u00a0\ninteracts\u00a0with\u00a0Rx,\u00a0the\u00a0potato\u00a0virus\u00a0X\u00a0\nresistance\u00a0gene\u00a0\n\n333\u00a0\n\nTable\u00a01.\u00a0List\u00a0of\u00a0genes\u00a0and\u00a0phenotypes\u00a0along\u00a0with\u00a0their\u00a0location\u00a0related\u00a0to\u00a0diseases\u00a0resistance\u00a0and\u00a0fruit\u00a0quality\u00a0of\u00a0\n\ntomato\u00a0(http://tgrc.ucdavis.edu/Data/Acc/Genes.aspx)\u00a0used\u00a0in\u00a0this\u00a0study\u00a0\n\nbetween\u00a0 gene\u00a0 pair\u00a0 and\u00a0 zero\u00a0 value\u00a0 between\u00a0 them\u00a0\n\nmeans\u00a0 they\u00a0 are\u00a0 independent\u00a0 (Atchley\u00a0 et\u00a0 al,\u00a0 1999,\u00a0\n\n2000).\u00a0 We\u00a0 found\u00a0 all\u00a0 genes\u00a0 having\u00a0 some\u00a0 degree\u00a0 of\u00a0\n\nassociation\u00a0with\u00a0each\u00a0others.\u00a0\u00a0\n\nResult\u00a0of\u00a0principal\u00a0component\u00a0analysis\u00a0of\u00a0sequences\u00a0of\u00a0\n\nDNA\u00a0 is\u00a0 given\u00a0 in\u00a0 Figure\u00a0 2.\u00a0 The\u00a0 first\u00a0 seven\u00a0 principal", "start_char_idx": 15322, "end_char_idx": 17021, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "4da73f3a-049b-4031-8765-c68045131744": {"__data__": {"id_": "4da73f3a-049b-4031-8765-c68045131744", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "d2e40bee-6504-44c5-87f1-2c5b7e693834", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "9fc1e1efbcedb0a8cd4e45343a2389300f49257550f453b86451211cf47f4077", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7896c77b-efa2-418a-95c7-fe4ef6c1ec22", "node_type": "1", "metadata": {}, "hash": "df63def84cdf555e6241fa32acfebb39b53e8985a9b98345660db5c3a524ae71", "class_name": "RelatedNodeInfo"}}, "text": "The\u00a0 first\u00a0 seven\u00a0 principal\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n5\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\ncomponents\u00a0with\u00a0\u2265\u00a01\u00a0eigen\u00a0value\u00a0accounted\u00a0for\u00a070.03%\u00a0\n\nof\u00a0 the\u00a0 total\u00a0 variance\u00a0 among\u00a0 the\u00a0 genes\u00a0 based\u00a0 on\u00a0 the\u00a0\n\nDNA\u00a0 sequences.\u00a0 The\u00a0 first\u00a0 principal\u00a0 component\u00a0\n\nexplained\u00a0 18.61%\u00a0 and\u00a0 second\u00a0 component\u00a0 accounted\u00a0\n\nfor\u00a018.03%\u00a0variance.\u00a0Plotting\u00a0of\u00a0these\u00a0accessions\u00a0along\u00a0\n\nfirst\u00a0 and\u00a0 second\u00a0 principal\u00a0 components\u00a0 indicated\u00a0 that\u00a0\n\nthese\u00a0 genes\u00a0 were\u00a0 placed\u00a0 in\u00a0four\u00a0 groups.\u00a0Most\u00a0 of\u00a0the\u00a0\n\nindividual\u00a0 genes\u00a0 make\u00a0 a\u00a0 separate\u00a0 cluster\u00a0 (Figure\u00a0 3).\u00a0\n\nClustering\u00a0 these\u00a0 genes\u00a0 can\u00a0 be\u00a0 helpful\u00a0 to\u00a0 identify\u00a0 the\u00a0\n\nsimilarity\u00a0among\u00a0the\u00a0genes.\u00a0\u00a0\n\nBoth\u00a0 PCA\u00a0 and\u00a0 cluster\u00a0 analysis\u00a0 separated\u00a0 these\u00a0 genes\u00a0\n\ninto\u00a0 four\u00a0 groups.\u00a0 These\u00a0 grouping\u00a0 were\u00a0 also\u00a0 similar\u00a0\n\nphenotypically.\u00a0 For\u00a0 example,\u00a0 genes\u00a0 related\u00a0 to\u00a0 beta\u00a0\n\ncarotene\u00a0 make\u00a0 a\u00a0 single\u00a0 cluster\u00a0 and\u00a0 genes\u00a0 related\u00a0 to\u00a0\n\nfruit\u00a0 characters\u00a0 tended\u00a0 to\u00a0 appear\u00a0 together.\u00a0 This\u00a0\n\nindicates\u00a0that\u00a0the\u00a0nature\u00a0of\u00a0variation\u00a0in\u00a0DNA\u00a0sequences\u00a0\n\nreflect\u00a0the\u00a0similar\u00a0variation\u00a0at\u00a0phenotypic\u00a0levels.\u00a0\u00a0\n\nGenes\u00a0 within\u00a0 cluster\u00a0 were\u00a0 phenotypically\u00a0 similar.\u00a0\n\nHowever,\u00a0 one\u00a0 group\u00a0 consisted\u00a0 of\u00a0 both\u00a0 disease\u00a0\n\nresistance\u00a0and\u00a0fruit\u00a0quality\u00a0genes.\u00a0This\u00a0might\u00a0be\u00a0due\u00a0to\u00a0\n\nrole\u00a0 of\u00a0 secondary\u00a0 metabolites\u00a0 in\u00a0 fruit\u00a0 quality\u00a0 and\u00a0\n\ndisease\u00a0resistance.\u00a0For\u00a0example\u00a0tomato\u00a0fruit\u00a0contains\u00a0\n\nantioxidant,\u00a0mainly\u00a0pigment\u00a0which\u00a0is\u00a0also\u00a0necessary\u00a0in\u00a0\n\ndefense\u00a0 mechanism.\u00a0 Potato\u00a0 gene,\u00a0 RanGAP2\u00a0 made\u00a0 an\u00a0\n\nFigure\u00a02.\u00a0Plotting\u00a0of\u00a0tomato\u00a0genes\u00a0considering\u00a0Principal\u00a0Component\u00a0I\u00a0(18.61%)\u00a0and\u00a0Principal\u00a0Component\u00a0II\u00a0\n(18.03%)\u00a0based\u00a0on\u00a0mutual\u00a0information\u00a0index\u00a0calculated\u00a0from\u00a0the\u00a0DNA\u00a0sequences\u00a0\u00a0\n\nFigure\u00a03.\u00a0UPGMA\u00a0cluster\u00a0analysis\u00a0of\u00a0tomato\u00a0genes\u00a0generated\u00a0from\u00a0the\u00a0mutual\u00a0information\u00a0matrix\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.\u00a01:\u00a01\u20108\u00a0\n\n6\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nG\nen\n\ne\u00a0\nP\nto\n\u00a0\n\n2\u00a0\n3\u00a0\n\n4\u00a0\n5\u00a0\n\n6\u00a0\n7\u00a0\n\n8\u00a0\n9\u00a0\n\n10\n\u00a0\n\n11\n\u00a0\n\n12\n\u00a0\n\n13\n\u00a0\n\n14\n\u00a0\n\n15\n\u00a0\n\n16\n\u00a0\n\n17\n\u00a0\n\n18\n\u00a0\n\n19\n\u00a0\n\n20\n\u00a0\n\n21\n\u00a0\n\nP\nto\n2\u00a0\n\n1\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nP\nto\n\u20102\n\u00a0\n\n1\u00a0\n1\u00a0\n\n1\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nP\nto\n\u2010h\n\u00a0\n\n1\u00a0\n1\u00a0\n\n1\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nP\nsy\n\u20101\n\u20102\ns\u00a0\n\n0.\n02\n\n7\u00a0\n0.\n02\n\n7\u00a0\n0.\n02\n\n7\u00a0\n0.", "start_char_idx": 16993, "end_char_idx": 19301, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7896c77b-efa2-418a-95c7-fe4ef6c1ec22": {"__data__": {"id_": "7896c77b-efa2-418a-95c7-fe4ef6c1ec22", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "4da73f3a-049b-4031-8765-c68045131744", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "becedabeffe8c959b0872ecea72ddcd9165d017c902cbe5bba17f6d36d3a161f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "ec37c0ab-17ed-418c-ab29-ac7643dc98c2", "node_type": "1", "metadata": {}, "hash": "fa5e123513bd393a7c6451a08b0b65ec77b9096b128d52516169aa6af762312b", "class_name": "RelatedNodeInfo"}}, "text": "02\n\n7\u00a0\n0.\n02\n\n7\u00a0\n0.\n02\n\n7\u00a0\n0.\n02\n\n7\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nP\nsy\n\u20101\n\u2010\n\np\nro\nv5\n\n\u00a0\n0.\n08\n\n1\u00a0\n0.\n08\n\n1\u00a0\n0.\n08\n\n1\u00a0\n0.\n08\n\n1\u00a0\n0.\n90\n\n8\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nP\nsy\n\u20101\n\u2010\n\np\nro\nv4\n\n\u00a0\n0.\n02\n\n5\u00a0\n0.\n02\n\n5\u00a0\n0.\n02\n\n5\u00a0\n0.\n02\n\n5\u00a0\n0.\n26\n\n7\u00a0\n0.\n90\n\n8\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nP\nsy\n\u20101\n\u2010y\n\u00a0\n\n0.\n05\n\n6\u00a0\n0.\n05\n\n6\u00a0\n0.\n05\n\n6\u00a0\n0.\n05\n\n6\u00a0\n0.\n63\n\n7\u00a0\n0.\n75\n\n6\u00a0\n0.\n65\n\n\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nH\ner\no\n\u00a0\n\n0.\n00\n\n9\u00a0\n0.\n00\n\n9\u00a0\n0.\n00\n\n9\u00a0\n0.\n00\n\n9\u00a0\n0.\n01\n\n3\u00a0\n0.\n03\n\n1\u00a0\n0.\n01\n\n5\u00a0\n0.\n01\n\n6\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nM\ni\u2010\n1.\n2\u00a0\n\n0.\n01\n\n\u00a0\n0.\n01\n\n\u00a0\n0.\n01\n\n\u00a0\n0.\n01\n\n\u00a0\n0.\n01\n\n\u00a0\n0.\n01\n\n7\u00a0\n0.\n01\n\n4\u00a0\n0.\n01\n\n3\u00a0\n0.\n23\n\n\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nB\n\u20101\n\u00a0\n\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n03\n\n4\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n07\n\n7\u00a0\n0.\n06\n\n4\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nB\n\u2010c\n\u00a0\n\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n03\n\n4\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n07\n\n7\u00a0\n0.\n06\n\n4\u00a0\n1\u00a0\n\n1\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nB\n\u2010m\n\n\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n03\n\n4\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n07\n\n7\u00a0\n0.\n06\n\n4\u00a0\n1\u00a0\n\n1\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nB\n\u2010o\ng\u00a0\n\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n03\n\n4\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n2\u00a0\n0.\n07\n\n7\u00a0\n0.\n06\n\n4\u00a0\n1\u00a0\n\n1\u00a0\n1\u00a0\n\n1\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nP\nsy\n\u20101\n\u20101\ns\u00a0\n\n0.\n00\n\n7\u00a0\n0.\n00\n\n7\u00a0\n0.\n00\n\n7\u00a0\n0.\n00\n\n7\u00a0\n0.\n02\n\n2\u00a0\n0.\n02\n\n1\u00a0\n0.\n02\n\n3\u00a0\n0.\n02\n\n4\u00a0\n0.\n00\n\n9\u00a0\n0.\n01\n\n\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nn\nr\u2010\n1\u00a0\n\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n01\n\n\u00a0\n0.\n03\n\n6\u00a0\n0.\n01\n\n2\u00a0\n0.\n00\n\n9\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n5\u00a0\n0.", "start_char_idx": 19272, "end_char_idx": 20808, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "ec37c0ab-17ed-418c-ab29-ac7643dc98c2": {"__data__": {"id_": "ec37c0ab-17ed-418c-ab29-ac7643dc98c2", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7896c77b-efa2-418a-95c7-fe4ef6c1ec22", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "79699c055eb28c046188fa28a73a68335368b32e533aa80e3b5dc4b4b3a84124", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "f7591895-f2bd-41ac-b9f7-c1d7864ba09c", "node_type": "1", "metadata": {}, "hash": "1f7e8bfb2970622612b66a3e94cbd9df70e63dd5215a0c074dea93a930c757f7", "class_name": "RelatedNodeInfo"}}, "text": "00\n\n5\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n01\n\n1\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nri\nn\n\u20101\n\u00a0\n\n0.\n01\n\n2\u00a0\n0.\n01\n\n2\u00a0\n0.\n01\n\n2\u00a0\n0.\n01\n\n2\u00a0\n0.\n01\n\n2\u00a0\n0.\n02\n\n9\u00a0\n0.\n01\n\n4\u00a0\n0.\n01\n\n9\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n7\u00a0\n0.\n01\n\n\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\u00a0\n\nSt\nar\n\u00a0\n\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n01\n\n8\u00a0\n0.\n02\n\n1\u00a0\n0.\n01\n\n8\u00a0\n0.\n05\n\n4\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n01\n\n4\u00a0\n0.\n01\n\n4\u00a0\n0.\n01\n\n4\u00a0\n0.\n01\n\n4\u00a0\n0.\n02\n\n7\u00a0\n0.\n01\n\n\u00a0\n0.\n01\n\n\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\n\u00a0\n\nA\nsc\n\u00a0\n\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n3\u00a0\n0.\n02\n\n2\u00a0\n0.\n00\n\n5\u00a0\n0.\n02\n\n\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n2\u00a0\n0.\n01\n\n1\u00a0\n0.\n01\n\n8\u00a0\n1\u00a0\n\n\u00a0\n\u00a0\n\nC\nrt\nR\n\u2010w\n\nf\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n8\u00a0\n0.\n02\n\n1\u00a0\n0.\n01\n\n3\u00a0\n0.\n01\n\n2\u00a0\n0.\n00\n\n7\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n9\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n5\u00a0\n0.\n02\n\n\u00a0\n0.\n00\n\n4\u00a0\n1\u00a0\n\n\u00a0\n\nR\nan\n\nG\nA\nP\n\u20102\n\u00a0\n\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n7\u00a0\n0.\n02\n\n8\u00a0\n0.\n00\n\n7\u00a0\n0.\n02\n\n2\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n3\u00a0\n0.\n01\n\n4\u00a0\n0.\n00\n\n3\u00a0\n0.\n00\n\n7\u00a0\n1\u00a0\n\nC\nf\u2010\n9\u00a0\n\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n7\u00a0\n0.\n02\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n02\n\n3\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n5\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n8\u00a0\n0.\n00\n\n7\u00a0\n0.\n00\n\n4\u00a0\n0.\n00\n\n4\u00a0\n0.\n03\n\n2\u00a0\n0.\n00\n\n6\u00a0\n0.\n00\n\n7\u00a0\n0.\n00\n\n3\u00a0\n\nTa\nb\nle\n\u00a02\n.\u00a0M\n\nu\ntu\nal\n\u00a0in\nfo\nrm\n\nat\nio\nn\n\u00a0m\n\nat\nri\nx\u00a0\nam\n\no\nn\ng\u00a0\n22\n\n\u00a0g\nen\n\nes\n\u00a0o\nf\u00a0\nto\nm\nat\no\n\u00a0a\nn\nd\n\u00a0p\no\nta\nto\n\u00a0e\nst\nim\n\nat\ned\n\n\u00a0f\nro\nm\n\u00a0D\nN\nA\n\u00a0s\neq\n\nu\nen\n\nce\ns\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 20779, "end_char_idx": 22307, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "f7591895-f2bd-41ac-b9f7-c1d7864ba09c": {"__data__": {"id_": "f7591895-f2bd-41ac-b9f7-c1d7864ba09c", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "ec37c0ab-17ed-418c-ab29-ac7643dc98c2", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "5a5c10e49fce8597b691b2ad97723fe6845c042bbf9bc3faa1a014179c8996eb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "1b7dec42-e808-4503-83f0-a1dcc6d328f5", "node_type": "1", "metadata": {}, "hash": "5051834f691a9efdb25c27441e6badfff9c22ed9d91f76a97dadaac2be7f7fa7", "class_name": "RelatedNodeInfo"}}, "text": "1:\u00a01\u20108\u00a0\n\n7\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nindividual\u00a0 cluster.\u00a0 Phytophthora\u00a0 infestans\u00a0 resistance\u00a0\n\ngene\u00a0from\u00a0potato\u00a0clustered\u00a0with\u00a0Cladosporium\u00a0fulvum\u00a0\n\nresistance\u00a0gene\u00a0of\u00a0tomato.\u00a0\u00a0\n\nSequences\u00a0of\u00a0DNA\u00a0are\u00a0being\u00a0deposited\u00a0in\u00a0the\u00a0website\u00a0\n\nby\u00a0large\u00a0amount\u00a0and\u00a0such\u00a0data\u00a0has\u00a0a\u00a0sequence\u00a0metric\u00a0\n\nproblem.\u00a0This\u00a0problem\u00a0could\u00a0now\u00a0be\u00a0handled\u00a0following\u00a0\n\nthe\u00a0method\u00a0of\u00a0estimating\u00a0variability\u00a0and\u00a0covariability,\u00a0\n\nmutual\u00a0 information\u00a0 index\u00a0 and\u00a0 applying\u00a0 dimension\u00a0\n\nreduction\u00a0 approaches\u00a0 as\u00a0 used\u00a0 in\u00a0 amino\u00a0 acids\u00a0\n\nsequences\u00a0 (Atchley\u00a0 et\u00a0 al.,\u00a0 2000,\u00a0 2005;\u00a0 Atchley\u00a0 and\u00a0\n\nZhao,\u00a0 2006).\u00a0 Atchley\u00a0 et\u00a0 al.\u00a0 (2000)\u00a0 summarized\u00a0 the\u00a0\n\namino\u00a0 acids\u00a0 sites\u00a0 of\u00a0 bHLH\u00a0 protein\u00a0 by\u00a0 estimating\u00a0\n\nentropy\u00a0 and\u00a0 mutual\u00a0 information\u00a0 values.\u00a0 Information\u00a0\n\ntheory\u00a0is\u00a0useful\u00a0to\u00a0look\u00a0for\u00a0pattern\u00a0in\u00a0DNA\u00a0and\u00a0protein\u00a0\n\nsequences\u00a0 (Schneider,\u00a0 1999).\u00a0 Information\u00a0 theory\u00a0 has\u00a0\n\nbeen\u00a0 applied\u00a0 to\u00a0 the\u00a0 analysis\u00a0 of\u00a0 DNA\u00a0 and\u00a0 protein\u00a0\n\nsequences\u00a0for\u00a0analyzing\u00a0sequence\u00a0complexity\u00a0from\u00a0the\u00a0\n\nShannon\u2010Weaver\u00a0 indices\u00a0 and\u00a0 comparing\u00a0 homologous\u00a0\n\nsites\u00a0 in\u00a0 a\u00a0 set\u00a0of\u00a0 aligned\u00a0 sequences\u00a0 by\u00a0 means\u00a0 of\u00a0 their\u00a0\n\ninformation\u00a0content.\u00a0\u00a0\n\nStrong\u00a0relationship\u00a0was\u00a0detected\u00a0in\u00a0the\u00a0present\u00a0study\u00a0\nbetween\u00a0 DNA\u00a0 variation\u00a0 and\u00a0 phenotype.\u00a0 A\u00a0 large\u00a0\namount\u00a0of\u00a0genetic\u00a0and\u00a0molecular\u00a0information\u00a0has\u00a0been\u00a0\ndeposited\u00a0 at\u00a0 http://sgn.cornell.edu\u00a0 (Jim\u00e9nez\u2010G\u00f3mez\u00a0\nand\u00a0 Maloof,\u00a0 2009)\u00a0 that\u00a0 might\u00a0 be\u00a0 useful\u00a0 to\u00a0 explore\u00a0\npossible\u00a0 relationships\u00a0 using\u00a0 this\u00a0 technique.\u00a0\nRelationship\u00a0 among\u00a0 the\u00a0 diversity\u00a0 index,\u00a0 sequence\u00a0\ncomplexity\u00a0and\u00a0phenotype\u00a0stability\u00a0of\u00a0the\u00a0gene\u00a0should\u00a0\nbe\u00a0considered\u00a0in\u00a0the\u00a0future\u00a0work\u00a0to\u00a0enhance\u00a0the\u00a0crop\u00a0\nimprovement\u00a0efforts.\u00a0\n\nREFERENCES\u00a0\nAtchley,\u00a0 W.R.,\u00a0 W.\u00a0 Terhalle\u00a0 and\u00a0 A.\u00a0 Dress.\u00a0 1999.\u00a0\n\nPositional\u00a0dependence,\u00a0cliques\u00a0and\u00a0predictive\u00a0motifs\u00a0\n\nin\u00a0 the\u00a0 bHLH\u00a0 protein\u00a0 domain.\u00a0 Journal\u00a0 of\u00a0 Molecular\u00a0\n\nEvolution\u00a048:\u00a0501\u2010516.\u00a0\n\nAtchley,\u00a0W.R.,\u00a0K.R.\u00a0Wollenberg,\u00a0W.M.\u00a0Fitch,\u00a0W.\u00a0Terhalle\u00a0\n\nand\u00a0 A.W.\u00a0 Dress.\u00a0 2000.\u00a0 Correlations\u00a0 among\u00a0 amino\u00a0\n\nacid\u00a0sites\u00a0 in\u00a0bHLH\u00a0protein\u00a0domains:\u00a0An\u00a0 information\u00a0\n\ntheoretic\u00a0 analysis.\u00a0 Molecular\u00a0 Biology\u00a0 and\u00a0 Evolution\u00a0\n\n17:\u00a0164\u2010178.\u00a0\n\nAtchley,\u00a0W.R.\u00a0and\u00a0J.\u00a0Zhao.\u00a02007.\u00a0Molecular\u00a0architecture\u00a0\n\nof\u00a0 the\u00a0 DNA\u2010binding\u00a0 region\u00a0 and\u00a0 its\u00a0 relationship\u00a0 to\u00a0\n\nclassification\u00a0 of\u00a0 basic\u00a0 helix\u2010loop\u2010helix\u00a0 proteins.\u00a0\n\nMolecular\u00a0Biology\u00a0and\u00a0Evolution\u00a024:\u00a0192\u2010202.\u00a0\n\nAtchley,\u00a0 W.R.,\u00a0 J.\u00a0 Zhao,\u00a0 A.D.\u00a0 Fernandes\u00a0 and\u00a0 T.\u00a0 Dr\u00fcke.\u00a0\n\n2005.\u00a0Solving\u00a0the\u00a0protein\u00a0sequence\u00a0metric\u00a0problem.\u00a0\n\nProceedings\u00a0 of\u00a0 the\u00a0 National\u00a0 Academy\u00a0 of\u00a0 Sciences\u00a0\n\n102:\u00a06395\u20106400.\u00a0\n\nBrown,\u00a0 J.S.\u00a0 1991.", "start_char_idx": 22308, "end_char_idx": 24851, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "1b7dec42-e808-4503-83f0-a1dcc6d328f5": {"__data__": {"id_": "1b7dec42-e808-4503-83f0-a1dcc6d328f5", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "f7591895-f2bd-41ac-b9f7-c1d7864ba09c", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "ab6f18c38f35d0770ea549a603b121f3f78418fb828b365af30b0d9b5c50f074", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c556b206-98ac-4d4d-98fa-c4074b981652", "node_type": "1", "metadata": {}, "hash": "9d7b9044fc0f5c5ead2ae9980639e0624738475e63c1c24212b9d121a35a45bf", "class_name": "RelatedNodeInfo"}}, "text": "Brown,\u00a0 J.S.\u00a0 1991.\u00a0 Principal\u00a0 component\u00a0 and\u00a0 cluster\u00a0\n\nanalysis\u00a0 of\u00a0 cotton\u00a0 cultivar\u00a0 variability\u00a0 across\u00a0 the\u00a0 US\u00a0\n\ncotton\u00a0belt.\u00a0Crop\u00a0Science\u00a031:\u00a0915\u2010922.\u00a0\n\nButte,\u00a0A.J.\u00a0and\u00a0 I.S.\u00a0Kohane.\u00a02000.\u00a0Mutual\u00a0 information\u00a0\n\nrelevance\u00a0 networks:\u00a0 Functional\u00a0 genomic\u00a0 clustering\u00a0\n\nusing\u00a0 pairwise\u00a0 entropy\u00a0 measurements.\u00a0 Pacific\u00a0\n\nSymposium\u00a0on\u00a0Bio\u2010computing\u00a0p.\u00a0418\u2010429.\u00a0\n\nCowen,\u00a0 N.M.\u00a0 and\u00a0 K.J.\u00a0 Frey.\u00a0 1987.\u00a0 Relationships\u00a0\n\nbetween\u00a0 three\u00a0 measures\u00a0 of\u00a0 genetic\u00a0 distance\u00a0 and\u00a0\n\nbreeding\u00a0behaviour\u00a0in\u00a0oats\u00a0(Avena\u00a0sativa\u00a0L).\u00a0Genome\u00a0\n\n29:\u00a097\u2010106.\u00a0\n\nCross,\u00a0 R.J.\u00a0 1992.\u00a0 A\u00a0 proposed\u00a0 revision\u00a0 of\u00a0 the\u00a0 IBPGR\u00a0\n\nbarley\u00a0 descriptor\u00a0 list.\u00a0 Theoretical\u00a0 and\u00a0 Applied\u00a0\n\nGenetics\u00a084:\u00a0501\u2010507.\u00a0\n\nFrary,\u00a0A.,\u00a0T.C.\u00a0Nesbitt,\u00a0S.\u00a0Grandillo,\u00a0E.\u00a0Knaap,\u00a0B.\u00a0Cong,\u00a0J.\u00a0\n\nLiu,\u00a0J.\u00a0Meller,\u00a0R.\u00a0Elber\u00a0and\u00a0K.B.\u00a0Alpert.\u00a02000.\u00a0fw2.\u00a02:\u00a0a\u00a0\n\nquantitative\u00a0 trait\u00a0 locus\u00a0 key\u00a0 to\u00a0 the\u00a0 evolution\u00a0 of\u00a0\n\ntomato\u00a0fruit\u00a0size.\u00a0Science\u00a0289:\u00a085\u201088.\u00a0\n\nFridman,\u00a0 E.,\u00a0 T.\u00a0 Pleban\u00a0 and\u00a0 D.\u00a0 Zamir.\u00a0 2000.\u00a0 A\u00a0\n\nrecombination\u00a0 hotspot\u00a0 delimits\u00a0 a\u00a0 wild\u2010species\u00a0\n\nquantitative\u00a0trait\u00a0 locus\u00a0for\u00a0tomato\u00a0sugar\u00a0content\u00a0to\u00a0\n\n484\u00a0bp\u00a0within\u00a0an\u00a0invertase\u00a0gene.\u00a0Proceedings\u00a0of\u00a0the\u00a0\n\nNational\u00a0Academy\u00a0of\u00a0Sciences\u00a097:\u00a04718\u20104723.\u00a0\n\nHolcomb,\u00a0 J.,\u00a0 D.M.\u00a0 Tolbert\u00a0 and\u00a0 S.K.\u00a0 Jain.\u00a0 1977.\u00a0 A\u00a0\n\ndiversity\u00a0 analysis\u00a0 of\u00a0 genetic\u00a0 resources\u00a0 in\u00a0 rice.\u00a0\n\nEuphytica\u00a026:\u00a0441\u2010450.\u00a0\n\nHussaini,\u00a0S.H.,\u00a0M.M.\u00a0Goodman\u00a0and\u00a0D.H.\u00a0Timothy.\u00a01977.\u00a0\n\nMultivariate\u00a0 analysis\u00a0 and\u00a0 the\u00a0 geographical\u00a0\n\ndistribution\u00a0 of\u00a0 the\u00a0 world\u00a0 collection\u00a0 of\u00a0 finger\u00a0 millet.\u00a0\n\nCrop\u00a0Science\u00a017:\u00a0257\u2010263.\u00a0\n\nJim\u00e9nez\u2010G\u00f3mez,\u00a0J.M.\u00a0and\u00a0J.N.\u00a0Maloof.\u00a02009.\u00a0Sequence\u00a0\n\ndiversity\u00a0in\u00a0three\u00a0tomato\u00a0species:\u00a0SNPs,\u00a0markers,\u00a0and\u00a0\n\nmolecular\u00a0evolution.\u00a0BMC\u00a0Plant\u00a0Biology\u00a09:\u00a085.\u00a0\n\nJohnson,\u00a0 R.A.\u00a0 and\u00a0 D.W.\u00a0 Wichern.\u00a0 1988.\u00a0 Applied\u00a0\n\nmultivariate\u00a0statistical\u00a0analysis.\u00a02nd\u00a0ed.\u00a0Prentice\u00a0Hall,\u00a0\n\nEnglewood\u00a0Cliffs,\u00a0NJ.\u00a0\n\nKanwal,\u00a0K.S.,\u00a0R.M.\u00a0Singh\u00a0and\u00a0J.\u00a0Singh.\u00a01983.\u00a0Divergent\u00a0\n\ngene\u00a0 pools\u00a0 in\u00a0 rice\u00a0 improvement.\u00a0 Theoretical\u00a0 and\u00a0\n\nApplied\u00a0Genetics\u00a065:\u00a0263\u2010267.\u00a0\n\nAtchley,\u00a0 W.R.,\u00a0 W.\u00a0 Terhalle\u00a0 and\u00a0 A.\u00a0 Dress.\u00a0 1999.\u00a0\n\nPositional\u00a0dependence,\u00a0cliques\u00a0and\u00a0predictive\u00a0motifs\u00a0\n\nin\u00a0 the\u00a0 bHLH\u00a0 protein\u00a0 domain.\u00a0 Journal\u00a0 of\u00a0 Molecular\u00a0\n\n\n\nNepal\u00a0Journal\u00a0of\u00a0Biotechnology.\u00a0Jan.\u00a02011,\u00a0Vol.\u00a01,\u00a0No.", "start_char_idx": 24832, "end_char_idx": 27006, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c556b206-98ac-4d4d-98fa-c4074b981652": {"__data__": {"id_": "c556b206-98ac-4d4d-98fa-c4074b981652", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "1b7dec42-e808-4503-83f0-a1dcc6d328f5", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "3595de3dee3912bd46314cf6ca1cfec587378f29f93c9f0ca6324893285eaf80", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "41e6172d-5d2a-41dc-8c2e-98ee4e6cd5c8", "node_type": "1", "metadata": {}, "hash": "35074d369a3a35d9086b4e564827b004db27d357f6a6586aa85c112f07c4f6e5", "class_name": "RelatedNodeInfo"}}, "text": "1:\u00a01\u20108\u00a0\n\n8\u00a0\n\nBiotechnology\u00a0Society\u00a0of\u00a0Nepal\u00a0(BSN),\u00a0All\u00a0rights\u00a0reserved\u00a0\u00a0\n\nEvolution\u00a048:\u00a0501\u2010516.\u00a0\n\nAtchley,\u00a0W.R.,\u00a0K.R.\u00a0Wollenberg,\u00a0W.M.\u00a0Fitch,\u00a0W.\u00a0Terhalle\u00a0\n\nand\u00a0 A.W.\u00a0 Dress.\u00a0 2000.\u00a0 Correlations\u00a0 among\u00a0 amino\u00a0\n\nacid\u00a0sites\u00a0 in\u00a0bHLH\u00a0protein\u00a0domains:\u00a0An\u00a0 information\u00a0\n\ntheoretic\u00a0 analysis.\u00a0 Molecular\u00a0 Biology\u00a0 and\u00a0 Evolution\u00a0\n\n17:\u00a0164\u2010178.\u00a0\n\nAtchley,\u00a0W.R.\u00a0and\u00a0J.\u00a0Zhao.\u00a02007.\u00a0Molecular\u00a0architecture\u00a0\n\nof\u00a0 the\u00a0 DNA\u2010binding\u00a0 region\u00a0 and\u00a0 its\u00a0 relationship\u00a0 to\u00a0\n\nclassification\u00a0 of\u00a0 basic\u00a0 helix\u2010loop\u2010helix\u00a0 proteins.\u00a0\n\nMolecular\u00a0Biology\u00a0and\u00a0Evolution\u00a024:\u00a0192\u2010202.\u00a0\n\nAtchley,\u00a0 W.R.,\u00a0 J.\u00a0 Zhao,\u00a0 A.D.\u00a0 Fernandes\u00a0 and\u00a0 T.\u00a0 Dr\u00fcke.\u00a0\n\n2005.\u00a0Solving\u00a0the\u00a0protein\u00a0sequence\u00a0metric\u00a0problem.\u00a0\n\nProceedings\u00a0 of\u00a0 the\u00a0 National\u00a0 Academy\u00a0 of\u00a0 Sciences\u00a0\n\n102:\u00a06395\u20106400.\u00a0\n\nBrown,\u00a0 J.S.\u00a0 1991.\u00a0 Principal\u00a0 component\u00a0 and\u00a0 cluster\u00a0\n\nanalysis\u00a0 of\u00a0 cotton\u00a0 cultivar\u00a0 variability\u00a0 across\u00a0 the\u00a0 US\u00a0\n\ncotton\u00a0belt.\u00a0Crop\u00a0Science\u00a031:\u00a0915\u2010922.\u00a0\n\nButte,\u00a0A.J.\u00a0and\u00a0 I.S.\u00a0Kohane.\u00a02000.\u00a0Mutual\u00a0 information\u00a0\n\nrelevance\u00a0 networks:\u00a0 Functional\u00a0 genomic\u00a0 clustering\u00a0\n\nusing\u00a0 pairwise\u00a0 entropy\u00a0 measurements.\u00a0 Pacific\u00a0\n\nSymposium\u00a0on\u00a0Bio\u2010computing\u00a0p.\u00a0418\u2010429.\u00a0\n\nCowen,\u00a0 N.M.\u00a0 and\u00a0 K.J.\u00a0 Frey.\u00a0 1987.\u00a0 Relationships\u00a0\n\nbetween\u00a0 three\u00a0 measures\u00a0 of\u00a0 genetic\u00a0 distance\u00a0 and\u00a0\n\nbreeding\u00a0behaviour\u00a0in\u00a0oats\u00a0(Avena\u00a0sativa\u00a0L).\u00a0Genome\u00a0\n\n29:\u00a097\u2010106.\u00a0\n\nCross,\u00a0 R.J.\u00a0 1992.\u00a0 A\u00a0 proposed\u00a0 revision\u00a0 of\u00a0 the\u00a0 IBPGR\u00a0\n\nbarley\u00a0 descriptor\u00a0 list.\u00a0 Theoretical\u00a0 and\u00a0 Applied\u00a0\n\nGenetics\u00a084:\u00a0501\u2010507.\u00a0\n\nFrary,\u00a0A.,\u00a0T.C.\u00a0Nesbitt,\u00a0S.\u00a0Grandillo,\u00a0E.\u00a0Knaap,\u00a0B.\u00a0Cong,\u00a0J.\u00a0\n\nLiu,\u00a0J.\u00a0Meller,\u00a0R.\u00a0Elber\u00a0and\u00a0K.B.\u00a0Alpert.\u00a02000.\u00a0fw2.\u00a02:\u00a0a\u00a0\n\nquantitative\u00a0 trait\u00a0 locus\u00a0 key\u00a0 to\u00a0 the\u00a0 evolution\u00a0 of\u00a0\n\ntomato\u00a0fruit\u00a0size.\u00a0Science\u00a0289:\u00a085\u201088.\u00a0\n\nFridman,\u00a0 E.,\u00a0 T.\u00a0 Pleban\u00a0 and\u00a0 D.\u00a0 Zamir.\u00a0 2000.\u00a0 A\u00a0\n\nrecombination\u00a0 hotspot\u00a0 delimits\u00a0 a\u00a0 wild\u2010species\u00a0\n\nquantitative\u00a0trait\u00a0 locus\u00a0for\u00a0tomato\u00a0sugar\u00a0content\u00a0to\u00a0\n\n484\u00a0bp\u00a0within\u00a0an\u00a0invertase\u00a0gene.\u00a0Proceedings\u00a0of\u00a0the\u00a0\n\nNational\u00a0Academy\u00a0of\u00a0Sciences\u00a097:\u00a04718\u20104723.\u00a0\n\nHolcomb,\u00a0 J.,\u00a0 D.M.\u00a0 Tolbert\u00a0 and\u00a0 S.K.\u00a0 Jain.\u00a0 1977.\u00a0 A\u00a0\n\ndiversity\u00a0 analysis\u00a0 of\u00a0 genetic\u00a0 resources\u00a0 in\u00a0 rice.\u00a0\n\nEuphytica\u00a026:\u00a0441\u2010450.\u00a0\n\nHussaini,\u00a0S.H.,\u00a0M.M.\u00a0Goodman\u00a0and\u00a0D.H.\u00a0Timothy.\u00a01977.\u00a0\n\nMultivariate\u00a0 analysis\u00a0 and\u00a0 the\u00a0 geographical\u00a0\n\ndistribution\u00a0 of\u00a0 the\u00a0 world\u00a0 collection\u00a0 of\u00a0 finger\u00a0 millet.\u00a0\n\nCrop\u00a0Science\u00a017:\u00a0257\u2010263.", "start_char_idx": 27007, "end_char_idx": 29264, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "41e6172d-5d2a-41dc-8c2e-98ee4e6cd5c8": {"__data__": {"id_": "41e6172d-5d2a-41dc-8c2e-98ee4e6cd5c8", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "c556b206-98ac-4d4d-98fa-c4074b981652", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "aef28fd438932f4145319e90ed634f326ffae2b53d81d9ca5c9f6c78b6eaa59f", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "57a8c025-0a09-442a-a1f0-54afd38090f7", "node_type": "1", "metadata": {}, "hash": "367bf88afb66d462c6154b2ff905403cbceed842a20766ac67e735f0b9d3da3e", "class_name": "RelatedNodeInfo"}}, "text": "Crop\u00a0Science\u00a017:\u00a0257\u2010263.\u00a0\n\nJim\u00e9nez\u2010G\u00f3mez,\u00a0J.M.\u00a0and\u00a0J.N.\u00a0Maloof.\u00a02009.\u00a0Sequence\u00a0\n\ndiversity\u00a0in\u00a0three\u00a0tomato\u00a0species:\u00a0SNPs,\u00a0markers,\u00a0and\u00a0\n\nmolecular\u00a0evolution.\u00a0BMC\u00a0Plant\u00a0Biology\u00a09:\u00a085.\u00a0\n\nJohnson,\u00a0 R.A.\u00a0 and\u00a0 D.W.\u00a0 Wichern.\u00a0 1988.\u00a0 Applied\u00a0\n\nmultivariate\u00a0statistical\u00a0analysis.\u00a02nd\u00a0ed.\u00a0Prentice\u00a0Hall,\u00a0\n\nEnglewood\u00a0Cliffs,\u00a0NJ.\u00a0\n\nKanwal,\u00a0K.S.,\u00a0R.M.\u00a0Singh\u00a0and\u00a0J.\u00a0Singh.\u00a01983.\u00a0Divergent\u00a0\n\ngene\u00a0 pools\u00a0 in\u00a0 rice\u00a0 improvement.\u00a0 Theoretical\u00a0 and\u00a0\n\nApplied\u00a0Genetics\u00a065:\u00a0263\u2010267.\u00a0\n\nLi,\u00a0 L.,\u00a0 C.\u00a0 Li\u00a0 and\u00a0 G.A.\u00a0 Howe.\u00a0 2001.\u00a0 Genetic\u00a0 analysis\u00a0 of\u00a0\n\nwound\u00a0signaling\u00a0in\u00a0tomato.\u00a0Evidence\u00a0for\u00a0a\u00a0dual\u00a0role\u00a0\n\nof\u00a0jasmonic\u00a0acid\u00a0in\u00a0defense\u00a0and\u00a0female\u00a0fertility.\u00a0Plant\u00a0\n\nPhysiology\u00a0127:\u00a01414\u20101417.\u00a0\n\nMueller,\u00a0 L.A.,\u00a0 T.H.\u00a0 Solow,\u00a0 N.\u00a0 Taylor,\u00a0 B.\u00a0 Skwarecki,\u00a0 R.\u00a0\n\nBuels,\u00a0J.\u00a0Binns,\u00a0C.\u00a0Lin,\u00a0M.H.\u00a0Wright,\u00a0R.\u00a0Ahrens\u00a0and\u00a0Y.\u00a0\n\nWang.\u00a0 2005a.\u00a0 The\u00a0 SOL\u00a0 genomics\u00a0 network.\u00a0 A\u00a0\n\ncomparative\u00a0 resource\u00a0 for\u00a0 Solanaceae\u00a0 biology\u00a0 and\u00a0\n\nbeyond.\u00a0Plant\u00a0Physiology\u00a0138:\u00a01310\u20101317.\u00a0\n\nMueller,\u00a0L.A.,\u00a0S.D.\u00a0Tanksley,\u00a0J.J.\u00a0Giovannoni,\u00a0J.\u00a0Van\u00a0Eck,\u00a0\n\nS.\u00a0Stack,\u00a0D.\u00a0Choi,\u00a0B.D.\u00a0Kim,\u00a0M.\u00a0Chen,\u00a0Z.\u00a0Cheng\u00a0and\u00a0C.\u00a0\n\nLi.\u00a0 2005b.\u00a0 The\u00a0 tomato\u00a0 sequencing\u00a0 project,\u00a0 the\u00a0 first\u00a0\n\ncornerstone\u00a0of\u00a0the\u00a0International\u00a0Solanaceae\u00a0Project\u00a0\n\n(SOL).\u00a0Comparative\u00a0and\u00a0Functional\u00a0Genomics\u00a06.\u00a0\n\nNiwranski,\u00a0K.,\u00a0P.G.\u00a0Kevan\u00a0and\u00a0A.\u00a0Fjellberg.\u00a02002.\u00a0Effects\u00a0\n\nof\u00a0vehicle\u00a0disturbance\u00a0and\u00a0soil\u00a0compaction\u00a0on\u00a0Arctic\u00a0\n\ncollembolan\u00a0 abundance\u00a0 and\u00a0 diversity\u00a0 on\u00a0 Igloolik\u00a0\n\nIsland,\u00a0 Nunavut,\u00a0 Canada.\u00a0 European\u00a0 Journal\u00a0 of\u00a0 Soil\u00a0\n\nBiology\u00a038:\u00a0193\u2010196.\u00a0\n\nPerry,\u00a0 M.C.\u00a0 and\u00a0 M.S.\u00a0 McIntosh.\u00a0 1991.\u00a0 Geographical\u00a0\n\npatterns\u00a0 of\u00a0 variation\u00a0 in\u00a0 the\u00a0 USDA\u00a0 soybean\u00a0\n\ngermplasm\u00a0 collection:\u00a0 I.\u00a0 Morphological\u00a0 traits.\u00a0 Crop\u00a0\n\nScience\u00a031:\u00a01350\u20101355.\u00a0\n\nRingn\u00e9r,\u00a0 M.\u00a0 2008.\u00a0 What\u00a0 is\u00a0 principal\u00a0 component\u00a0\n\nanalysis?\u00a0Nature\u00a0Biotechnology\u00a026:\u00a0303.\u00a0\n\nSchneider,\u00a0T.D.\u00a01999.\u00a0Information\u00a0Theory\u00a0Primer.\u00a0Web\u00a0\n\nDocument:\u00a01\u20109.\u00a0\n\nSneath,\u00a0 P.H.A.\u00a0 and\u00a0 R.R.\u00a0 Sokal.\u00a0 1973.\u00a0 Numerical\u00a0\n\ntaxonomy.\u00a0Springer.\u00a0\n\nTanksley,\u00a0 S.D.\u00a0 2004.\u00a0 The\u00a0 genetic,\u00a0 developmental,\u00a0 and\u00a0\n\nmolecular\u00a0bases\u00a0of\u00a0fruit\u00a0size\u00a0and\u00a0shape\u00a0variation\u00a0 in\u00a0\n\ntomato.\u00a0The\u00a0Plant\u00a0Cell\u00a0Online\u00a016:\u00a0S181\u2010189.", "start_char_idx": 29239, "end_char_idx": 31313, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "57a8c025-0a09-442a-a1f0-54afd38090f7": {"__data__": {"id_": "57a8c025-0a09-442a-a1f0-54afd38090f7", "embedding": null, "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-58", "node_type": "4", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "54a0a7dde4b521f0fac4c2e5def3d20d7a32e2f231d42de44a8f77d2841355b2", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "41e6172d-5d2a-41dc-8c2e-98ee4e6cd5c8", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "405409feafa6c73afb6fb6e8ee5438066098bd14b969703c9cd3ac6a24483efb", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "5da867eb-4a7e-455d-aa09-51ac015398f0", "node_type": "1", "metadata": {}, "hash": "3753bc53864eff55c8efaee05d1d4dad0adc0af4808018814643922504582b47", "class_name": "RelatedNodeInfo"}}, "text": "Tolbert,\u00a0D.M.,\u00a0C.O.\u00a0Qualset,\u00a0S.K.\u00a0Jain\u00a0and\u00a0J.C.\u00a0Craddock.\u00a0\n1979.\u00a0 A\u00a0 diversity\u00a0 analysis\u00a0 of\u00a0 a\u00a0 world\u00a0 collection\u00a0 of\u00a0\nbarley.\u00a0Crop\u00a0Science\u00a019:\u00a0789\u2010794.", "start_char_idx": 31316, "end_char_idx": 31469, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "5da867eb-4a7e-455d-aa09-51ac015398f0": {"__data__": {"id_": "5da867eb-4a7e-455d-aa09-51ac015398f0", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "57a8c025-0a09-442a-a1f0-54afd38090f7", "node_type": "1", "metadata": {"identifier": "njb-58", "author": "Joshi, Bal K; Panthee, Dilip R", "title": "Information Theory and Multivariate Techniques for Analyzing DNA Sequence Data: An Example from Tomato Genes", "date": "2022-07-11", "file": "njb-58.pdf"}, "hash": "ab12268a355d4ae50478a3371e6869ff0f5b27ec9506966b8b859eebe7c72789", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "664d397b-fe6a-4228-a03a-17b8fb24fe6e", "node_type": "1", "metadata": {}, "hash": "9f795677689d1917b68da09bf941ecd19f870ecf34711f38b31d1e2c93836dff", "class_name": "RelatedNodeInfo"}}, "text": "Nepal J Biotechnol. 2 0 2 0  J u l y ; 8(1):17-28    DOI: https://doi.org/10.3126/njb.v8i1.30206     \n\n\u00a9NJB, BSN 17 \n\nResearch article \n\nPhytochemical Evaluation, Antioxidant and Antimicrobial \nActivities of Various Extracts from Leaves and Stems of \nBryophyllum pinnatum \nImaobong E. Daniel  , Ekemini I. Akpan, Edidiong C. Utam \n\nDepartment of Chemistry, University of Uyo, PMB 1017, Uyo, Akwa Ibom State, Nigeria \nArticle history:- Received: 5 Mar 2020; Revised: 7 Jul 2020; Accepted: 20 Jul 2020; Published online: 31 Jul 2020 \n\nAbstract \nAntioxidant and antimicrobial activities of different extracts (methanol and ethyl acetate) of leaf and stem of \nBryophyllum pinnatum were studied. The screening for the secondary metabolites was carried out using the \nstandard methods. The antioxidant capacities of the different extracts were assessed using DPPH (2,2-diphenyl-\n1-picrylhydrazyl) radicals and Ferric reducing antioxidant power (FRAP) while the antimicrobial activity of \nthe extracts obtained were screened against Gram-positive, Gram-negative bacteria and fungi (Staphylococcus \naureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio cholerae, Candida albicans and Aspergillus \nniger) using Agar well diffusion method. Both extracts obtained from leaf and stem of B. pinnatum contained \nmost of the phytochemical compounds tested for. However, anthocyanins and anthraquinone were not \ndetected in leaf extracts while coumarin was absent in stem extracts. Quantification of bioactive compounds \nshowed that both extracts contained the highest concentration of polyphenols (34.49 \u00b10.47 mg GAE/g and 32.32 \n\u00b11.2 mg GAE/g for methanol leaf and stem extracts respectively) while the least concentration was recorded \nfor alkaloids (0.03\u00b10.02 mg/g for methanol stem extract). Results revealed that the extracts showed dose-\ndependent scavenging of DPPH as well as the ability of the extracts to reduce FeCl3 solution, with methanol \nextracts exhibiting the highest scavenging and reducing capacity. However the leaves of B. pinnatum had \n\ngreater antioxidant activity than the stem by DPPH and ferric reducing assays, with IC50 values ranging from \n3.147\u00b5g/ml to 3.80\u00b5g/ml for DPPH and 331.9 - 451 \u00b5g/ml for FRAP assays. The antimicrobial activity of \nvarious solvent extracts of leaf and stem reveal that microorganisms exhibited different sensitivities towards \nthese extracts in a dose-dependent manner. Methanol leaf extract showed no activity against E. coli while P. \naeruginosa was insensitive to ethyl acetate leaf extract. For stem extracts, A. niger, V. cholerae and P. aeruginosa \nwere resistant to methanol extract while A. niger, Salmonella spp. and P. aeruginosa was resistant towards ethyl \nacetate stem extract. The results obtained in this study showed that B. pinnatum is a reservoir of bioactive \n\ncompounds and both extracts exhibited significant antimicrobial and antioxidant activity. \n\nKeywords: Bryophyllum pinnatum, Antioxidant activity, Agar well diffusion, Aspergillus niger, Polyphenols, \nFerric reducing power assay. \n\n Corresponding author, email: imaudoekwere@gmail.com \n\nIntroduction \nA medicinal plant is any plant in which one or more \n\nof its organs contain substances that can be used for \n\nthe synthesis of useful drugs, and also serve as a \n\nmajor lead for modern drug design [1, 2].  Numerous \n\nresearchers on medicinal plants and herbal drug \n\nproduction reported that bioactive components of \n\nmedicinal plant occur in the leaves, flowers, roots, \n\nstem, bark or wood.", "start_char_idx": 48, "end_char_idx": 3582, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "664d397b-fe6a-4228-a03a-17b8fb24fe6e": {"__data__": {"id_": "664d397b-fe6a-4228-a03a-17b8fb24fe6e", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "5da867eb-4a7e-455d-aa09-51ac015398f0", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "00bb32085b52080137d3f2e49a6d295612e99f5aaa6ae4bdc49be7db3cdcf667", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "91aa64ff-9276-4590-9fc4-f32dabe6729d", "node_type": "1", "metadata": {}, "hash": "ddc18d338853a14c098675d40b2329a30010f49f53f728344e3410f4e602a1d8", "class_name": "RelatedNodeInfo"}}, "text": "These bioactive compounds \n\ncommonly known as phytochemicals include \n\nterpenes, alkaloids, flavonoid, bioflavonoid, \n\nbenzophonones, xanthenes as well as some \n\nmetabolites such as tannins, saponins, cyanates, \n\noxalate and anthraquinones [3, 4, 5, 6]. Several plants \n\ncontaining secondary metabolites possess \n\nantioxidant, antimicrobial and other biological \n\npotentials [7]. Natural antioxidants, such as phenolic \n\ncompounds are gaining importance due to their \n\nbenefits for human health, decreasing the risk of \n\ndegenerative diseases by reduction of oxidative \n\nstress and inhibition of macromolecular oxidation [8, \n\n9]. Several studies indicate that medicinal plants \n\nwhich are rich reservoir of bioactive compounds \n\ncontain compounds that are significant in \n\ntherapeutic application against human and animal \n\npathogen, including bacteria, fungi and viruses [10, \n\n11]. \n\nNepal Journal of Biotechnology \nPublisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  \n\nJournal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 \n\nmailto:imaudoekwere@gmail.com\nhttps://orcid.org/0000-0001-6097-901X\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):17-28 Daniel et al. \n\n\u00a9NJB, BSN 18 \n\nStudies show that nearly 80% of the world\u2019s \n\npopulation still relies on traditional medicines for \n\nprimary health care, most of which is the use of plant \n\nextracts [12]. In the past few years, uncertainties \n\nconcerning the safety of synthetic antimicrobial \n\ndrugs have led to an increase in the demand for \n\nnatural compounds such as plants rich in \n\nantimicrobials [13]. This is because herbal medicines \n\nhave been reported to be safe, affordable, acceptable, \n\navailable and without any adverse side effects \n\nespecially when compared with synthetic drugs [14, \n\n15]. In developing countries, there is a gradual \n\nrevival of interest in the use of medicinal plants \n\nespecially herbal preparations in the local healthcare \n\nsystems because of the increasing problems of Multi-\n\nDrug Resistance (MDR) to human pathogenic \n\nbacteria [16, 17]. One of such plants used in the \n\ntreatment of a wide range of ailments is Bryophyllum \n\npinnatum (B. pinnatum). \n\nB. pinnatum belongs to the family Crassulaceae and \n\nthe common names include life plant, love plant, \n\nmiracle leaf, and Canterbury bells. It is widely \n\ndistributed in tropical Africa, America, Hawaii, \n\nIndia, China, Australia, and Madagascar [18]. B. \n\npinnatum is a succulent plant, 50 \u2013 200 cm tall and \n\nabout 3.2 cm wide, and reproduces via seeds and \n\nalso vegetatively from leaf bulbils [19, 20]. They are \n\nmedium green above blotched with purple \n\nunderneath. It has flashy, dark green leaves. Its \n\nflower is in paniculate cymes 20 \u2013 80 cm long. It has \n\nfruit whose follicles are 10 -14 mm long enclosed in \n\nthe persistent papery calyx. The seeds are numerous \n\nin each fruit. The leaves and leaf juice have been \n\nused traditionally as anti-inflammatory, antipyretic, \n\nantimicrobial, antioxidant, antitumor, antidiabetic, \n\nanti-ulcer, antiseptic, hypocholesterolemic, and \n\ncough suppressant [21]. In Nigeria, the plant is \n\nparticularly known for its effective wound healing \n\nproperties and detachment of the umbilicus of \n\ninfants, for the treatment of earache, burns, \n\nabscesses, ulcer, insect\u2019s bites, whitlow, diarrhea and \n\nlithiasis [19, 20]. The lightly roasted leaves are used \n\nexternally for skin fungus and inflammations and \n\nthe leaf infusion is an internal remedy for fevers [22]. \n\nB. pinnatum leaves are used to expel worms, cure \n\nacute and chronic bronchitis, pneumonia and other \n\nforms of respiratory tract infections [23].", "start_char_idx": 3583, "end_char_idx": 7224, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "91aa64ff-9276-4590-9fc4-f32dabe6729d": {"__data__": {"id_": "91aa64ff-9276-4590-9fc4-f32dabe6729d", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "664d397b-fe6a-4228-a03a-17b8fb24fe6e", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "69225955e7049be0c5fbe62185ee5c695c0ef122320d4468c981a67c006da680", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "98b89dc9-3227-4cb8-ba7e-ad11462ed644", "node_type": "1", "metadata": {}, "hash": "b47befbb2e3d832edcf7a1827a384744cf40a43000caa1acfc49a2ea89668a72", "class_name": "RelatedNodeInfo"}}, "text": "It is used \n\nfor all sorts of respiratory conditions such as asthma, \n\ncough and bronchitis. It is employed for the \n\ntreatment of kidney stones, gastric ulcers and \n\noedema of the leg [24]. \n\nMaterials and Methods  \nCollection of Plant Materials \n\nFresh leaves and stem of B. pinnatum were obtained \n\nfrom Etinan and Ikot Ekpene local government areas \n\nin Akwa Ibom State, Nigeria, and authenticated by \n\nthe Department of Botany, University of Uyo, \n\nNigeria, and a voucher specimen [Voucher No: \n\nUUPH27(a)] was kept in the herbarium for future \n\nreference.  \n\nExtraction Procedure \n\nThe leaves and stem were thoroughly washed with \n\ndistilled water and air-dried for 2 weeks. The dried \n\nparts were pulverized and the powdered plant parts \n\nwere separately divided into portions. 750g of the \n\ndifferent plant parts were macerated with 1.7 L each \n\nof methanol and ethyl acetate at room temperature \n\nfor 72 hrs. After 72 hrs, the different extracts were \n\nfiltered separately off through a cotton plug and \n\nfinally with a Whatman No. 1 filter paper. The liquid \n\nfiltrates were concentrated and evaporated to \n\ndryness using a rotary evaporator (WG- tv311-V, \n\nWilmad-LabGlass, USA) at 40 \u00b0C, and each extract \n\nwas transferred into well-labeled sterile glass vials \n\nand stored at 4 \u00b0C before use [25]. \n\nPhytochemical Screening of Plant Extracts \nThe leaf and stem extracts (methanol and ethyl \n\nacetate) were screened for the presence of various \n\nbioactive components (phytochemicals) using \n\nstandard procedures [6, 26, 27]. \n\nTest for Anthraquinones \n\nTo 6g of the different plant parts in this study, 10\u2009ml \n\nof benzene was added. After 10 minutes, the solution \n\nwas filtered and 10 ml of 10% ammonia was added \n\nto the filtrates and shaken.  The presence of pink, \n\nviolet, or red color signified the presence of \n\nanthraquinones in the ammonia phase [26]. \n\nDetermination of Tannins \n10\u2009ml each of bromine water was added to the 0.5\u2009g \n\nof leaf and stem extracts of B. pinnatum. The \n\ndiscoloration of bromine water indicated the \n\npresence of tannins. \n\nTest for Saponins \n5.0\u2009ml of distilled water was added to the different \n\nplant extracts in a test tube. The froth formed was \n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):17-28 Daniel et al. \n\n\u00a9NJB, BSN 19 \n\nmixed with few drops of olive oil. The formation of \n\nfoams showed the presence of saponins. \n\nTests for Flavonoids \nShinodaTest. Magnesium strip and HCl were mixed \n\nwith plant extracts. The development of pink colour \n\nconfirmed the presence of flavonoid. \n\nTests for Glycosides \nLiebermann\u2019s Test. 2.0\u2009ml of acetic acid and 2\u2009ml of \n\nchloroform were added to the different extracts. The \n\nmixtures were allowed to cool after which \n\nconcentrated H2SO4 was added. The appearance of \n\ngreen color signified the presence of aglycone, \n\nsteroidal part of glycosides. \n\nKeller-Kiliani Test. A solution of glacial acetic acid \n\n(4.0\u2009ml) with 1 drop of 2.0% FeCl3 mixture as well as \n\n1\u2009ml H2SO4 was added to 10\u2009ml of the different plant \n\nextracts. A brown ring formed between the layers \n\nindicated the presence of cardiac steroidal \n\nglycosides. \n\nSalkowski\u2019s Test. 2\u2009ml of Conc. H2SO4 was added to \n\nthe plant crude extract. A reddish-brown color \n\nformed indicated the presence of the steroidal \n\naglycone part of the glycoside.", "start_char_idx": 7225, "end_char_idx": 10537, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "98b89dc9-3227-4cb8-ba7e-ad11462ed644": {"__data__": {"id_": "98b89dc9-3227-4cb8-ba7e-ad11462ed644", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "91aa64ff-9276-4590-9fc4-f32dabe6729d", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "b5eaf610de39b992131189850b1dba934ec3b9b48dc180f30b572b5f0546eb32", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "331eeadb-20db-43f3-9cd2-0ca07c8c6693", "node_type": "1", "metadata": {}, "hash": "6086c798dc03f9cfdfb658f2168538594975e62f887c7f91e06a505a2dced18d", "class_name": "RelatedNodeInfo"}}, "text": "Test for Terpenoid \n2.0\u2009ml of chloroform was added to 5\u2009ml plant extracts \n\nand evaporated on the water path. The mixture was \n\nboiled with 3\u2009ml of H2SO4. A grey color formed \n\nconfirmed the presence of terpenoids. \n\nTest for Steroids \n2\u2009ml of chloroform and concentrated H2SO4 were \n\nadded with the 5\u2009ml plant crude extract. In the lower \n\nchloroform layer, red color appeared that indicated \n\nthe presence of steroids. \n\nTest for Coumarins \n1ml of 10% sodium hydroxide solution was added to \n\n1ml of the different plant extracts. Formation of \n\nyellow colour indicated the presence of coumarins \n\nTest for Reducing Sugar (De- Oxy sugars) \n0.5g of each extract was macerated with 20 ml of \n\ndistilled water and filtered. To 1 ml of the filtrates, 1 \n\nml of alkaline copper reagent was added. The \n\nmixture was boiled for 5 min and allowed to cool. \n\nThen 1 ml of phosphomolybdic acid reagent and 2 \n\nml of distilled water was added and the absorbance \n\nread at 420 nm \n\nTest for phenols \nThe extract (500 mg) was dissolved in 5ml of distilled \n\nwater. To this, few drops of neutral 5% ferric \n\nchloride solution were added. A dark green color \n\nindicated the presence of phenolic compounds. \n\nTest for Quinone \n1ml of each of the various extracts was treated \n\nseparately with alcoholic potassium hydroxide \n\nsolution. Quinones give coloration ranging from red \n\nto blue. \n\nTest for amino acids and proteins  \nBiuret test: To 0.5 mg of extract equal volume of 40% \n\nNaOH solution and two drops of 1% copper \n\nsulphate solution was added. The appearance of \n\nviolet colour indicated the presence of protein. \n\nNinhydrin test: About 0.5 mg of extract was taken \n\nand 2 drops of freshly prepared 0.2% ninhydrin \n\nreagent was added and heated. The appearance of \n\npink or purple colour indicated the presence of \n\nproteins, peptides or amino acids. \n\nAnthocyanins \n2 ml of 2 N HCl and ammonia was added to 2 ml of \n\nthe different extracts. The appearance of pink-red \n\nwhich later turned blue-violet indicated the presence \n\nof anthocyanins. \n\nTest for Alkaloids  \nExtracts were dissolved individually in dilute \n\nhydrochloric acid and filtered. The filtrates were \n\nused to test the presence of alkaloids.  \n\nDragendorff\u2019s Test: 1ml of the filtrate was treated \n\nwith few drops Dragendorff\u2019s reagent. Formation of \n\norange-brown precipitate indicated the presence of \n\nalkaloids.  \n\nMayer\u2019s test: Filtrates were treated with Mayer\u2019s \n\nreagent. The formation of a yellow cream precipitate \n\nindicated the presence of alkaloids. \n\nQuantitative Estimation of \nPhytoconstituents \nThe phytochemicals which are present in the \n\nmethanol and ethyl acetate stem and leaf extracts of \n\nB. pinnatum was quantitatively determined using \n\nstandard Protocols. \n\nDetermination of Polyphenols \nThe total phenolic content in the extracts were \n\ndetermined by the modified Folin-Ciocalteu method \n\nas described by Singleton and Rossi [28] and \n\nmodified by Ayoola et al. [29]. Sample extract was \n\ndissolved in methanol (1 mg/ml). An aliquot of 0.5 \n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):17-28 Daniel et al. \n\n\u00a9NJB, BSN 20 \n\nml of each plant extract (1 mg/ml) was mixed with 5 \n\nml of Folin- Ciocalteu reagent which was previously \n\ndiluted with distilled water (1:10 v/v). The mixture \n\nwas shaken slightly and allowed to stand at 22 \u00b0C for \n\n5mins.", "start_char_idx": 10540, "end_char_idx": 13880, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "331eeadb-20db-43f3-9cd2-0ca07c8c6693": {"__data__": {"id_": "331eeadb-20db-43f3-9cd2-0ca07c8c6693", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "98b89dc9-3227-4cb8-ba7e-ad11462ed644", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "14d51edf5b6235aff9f9f13199fb6e2c5cf903b84c6858b3f2f8d35a1254c5bc", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "958b6631-c6c4-420e-a1b7-b10fcf77baa0", "node_type": "1", "metadata": {}, "hash": "c110a3c17680bb7c82a9594dc07cdf8157c710e1c441a0a8727fcb13e71a3023", "class_name": "RelatedNodeInfo"}}, "text": "After, 4 ml (75 g/l) of sodium carbonate \n\n(Na2CO3) was added, and the tubes containing the \n\nmixtures were allowed to stand for 30 min at 40 \u00b0C \n\nto develop colour. Absorbance was then read at 765 \n\nnm using the UV spectrophotometer (Shimadzu, \n\nJapan). Results were expressed as Gallic acid \n\nequivalent in (mg/g) of extracts (10\u2013100 mg/ml). All \n\nsamples were analyzed in triplicate. \n\nDetermination of total flavonoids \nTotal flavonoid contents were determined using \n\nAluminium chloride colorimetric method [30]. A \n\nvolume of 0.5 ml of 2% AlCl3 ethanol solution was \n\nadded to 0.5 ml of sample solution. After one hour at \n\nroom temperature, the absorbance was measured at \n\n420 nm. Using UV spectrophotometer (Shimadzu, \n\nJapan). Yellow color indicated the presence of \n\nflavonoids. Total flavonoid content was calculated \n\nas quercetin equivalent (mg/g). The calibration \n\ncurve ranged from 10 \u2013 100 mg/ml. \n\nDetermination of total alkaloids \nTotal alkaloids were determined according to the \n\nstandard method as described by Harbone [27]. 200 \n\nml of 10% acetic acid in ethanol was added to 5 g of \n\ndifferent plant extracts. The mixtures were covered \n\nand allowed to stand for 4 h. The solutions were \n\ndecanted and filtered and the extracts were further \n\nconcentrated in a water bath until one-quarter of the \n\noriginal volume was obtained. Concentrated \n\nammonium hydroxide was added dropwise to the \n\nconcentrated extract until the precipitation was \n\ncomplete. The precipitate was collected and washed \n\nwith dilute ammonium hydroxide and then filtered. \n\nThe residue was dried and weighed.  \n\nDetermination of total tannins \n\n50 ml of distilled water was added to 500 mg of the \n\nplant extracts and was shaken for 1 h in a mechanical \n\nshaker. The solution obtained was filtered into a 50 \n\nml volumetric flask and made up to the mark. 5 ml \n\nof the filtrate was pipetted out into a test tube and \n\nmixed with 2 ml of 0.1 M FeCl3 in 0.1 N HCl and \n\n0.008 M potassium ferrocyanide. The absorbance \n\nwas measured at 120 nm within 10 min [31].  \n\nDetermination of total saponins \nTo 20 g of each plant extracts, 100 cm3 of 20% \n\naqueous ethanol were added. The samples were \n\nheated at 55\u00b0C for 4 h with continuous stirring. The \n\nmixture was filtered and the residue re-extracted \n\nwith 200 ml 20% ethanol. The combined extracts \n\nwere reduced to 40 ml over water bath at about 90\u00b0C. \n\n20 ml of diethyl ether was added to the concentrates \n\nand shaken vigorously. The aqueous layer recovered \n\nwas further purified by adding 60 ml of n-butanol. \n\nThe combined n-butanol extracts were washed twice \n\nwith 10 ml of 5% aqueous sodium chloride and \n\nheated in a water bath. After evaporation, the \n\nsamples were dried in the oven to a constant weight \n\nand the saponin content was calculated [32]. \n\nIn vitro Antioxidant Assay Antioxidant \nactivity by DPPH Assay  \nThe free radical scavenging activity of the different \n\nextracts was measured in vitro by 2,2\u2019-diphenyl-1-\n\npicrylhydrazyl (DPPH) method as described by \n\nBrand-Williams et al. [33]. 0.5 Mm solution of DPPH \n\nwas added to sample solutions at different \n\nconcentrations (20 \u2013 100 \u03bcg/ml). A control (Abs \n\nControl) containing methanol and DPPH solution \n\nwas also prepared. All solutions obtained were then \n\nincubated for 1 hour at room temperature. \n\nAbsorbance was measured at 517 nm. Vitamin C was \n\nused as standard and the same concentrations of it \n\nwere prepared as the test solutions.", "start_char_idx": 13881, "end_char_idx": 17348, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "958b6631-c6c4-420e-a1b7-b10fcf77baa0": {"__data__": {"id_": "958b6631-c6c4-420e-a1b7-b10fcf77baa0", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "331eeadb-20db-43f3-9cd2-0ca07c8c6693", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "29fc4076afb23f48e28caf789a11c1e34620393f52afee09557ca7ecf61f02c3", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "7954a0e4-0f7b-466c-836e-39d5264c0694", "node_type": "1", "metadata": {}, "hash": "7172fc9387d400068970476728193ac9aa6029fdece0c91c27174aee7faccd94", "class_name": "RelatedNodeInfo"}}, "text": "The percentage \n\nof inhibition of samples was calculated from \n\nobtained absorbance by the equation: \n\n\ud835\udc43\ud835\udc52\ud835\udc5f\ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc4e\ud835\udc54\ud835\udc52 \ud835\udc5c\ud835\udc53 \ud835\udc3c\ud835\udc5b\u210e\ud835\udc56\ud835\udc4f\ud835\udc56\ud835\udc61\ud835\udc56\ud835\udc5c\ud835\udc5b = \n\ud835\udc34\ud835\udc4f\ud835\udc60 \ud835\udc50\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59 \u2212 \ud835\udc34\ud835\udc4f\ud835\udc60 \ud835\udc61\ud835\udc52\ud835\udc60\ud835\udc61/\ud835\udc60\ud835\udc61\ud835\udc4e\ud835\udc5b\ud835\udc51\ud835\udc4e\ud835\udc5f\ud835\udc51\n\n\ud835\udc34\ud835\udc4f\ud835\udc60 \ud835\udc50\ud835\udc5c\ud835\udc5b\ud835\udc61\ud835\udc5f\ud835\udc5c\ud835\udc59\n\u00d7 100 \n\nA percent inhibition versus concentration curve was \n\nplotted and the concentration of sample required for \n\n50% inhibition was determined and represented as \n\nIC50 value for each of the test solutions.  \n\nFerric reducing/antioxidant power \n(FRAP) Assay \nThe reducing property of the extract was determined \n\nby assessing the ability of the extracts to reduce FeCl3 \n\nsolution [34]. Briefly appropriate concentrations of \n\nthe extracts were mixed with 2.5 ml of 200 mM of \n\nsodium phosphate buffer (pH 6.6) and 2.5 ml of 1% \n\npotassium ferrocyanide. The mixture was incubated \n\nat 50\u00b0C for 20 min after which 2.5 ml of 10% \n\ntrichloroacetic acid was added. The mixture was \n\nthen centrifuged at 650 rpm for 10 min. Supernatant \n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):17-28 Daniel et al. \n\n\u00a9NJB, BSN 21 \n\n(The upper layer) (5 ml) was mixed with equal \n\nvolume of deionized water and 1 ml of 0.1% ferric \n\nchloride, and the absorbance was measured at 700 \n\nnm. The ferric-reducing-power capacities of the \n\nplant extracts and standard antioxidants were \n\nexpressed graphically by plotting absorbance \n\nagainst concentration [34]. Ascorbic acid was used as \n\na positive reference. The experiment was done in \n\ntriplicate. \n\nAntimicrobial Activity \nCollection of Test Organisms \n\nMicroorganisms used were obtained from the \n\nmicrobial stock collection unit of the Department of \n\nMicrobiology, University of Uyo, Akwa Ibom State. \n\nThe test organisms used were 1 Gram-positive \n\nbacterium (Staphylococcus aureus), 4 Gram-negative \n\nbacteria (Escherichia coli, Pseudomonas aeruginosa, \n\nSalmonella spp., Vibrio cholerae) and two fungi \n\n(Candida albicans, Aspergillus niger). These organisms \n\nwere sub cultured to obtain pure and fresh isolates. \n\nThe pure bacterial cultures were maintained on \n\nnutrient agar medium and fungal culture on potato \n\ndextrose agar (PDA) medium. Isolates were \n\nidentified using standard microbiological \n\nprocedures by carrying out Gram\u2019s reaction and \n\nbiochemical tests to confirm the species. \n\nPreparation of test organisms before inoculation \nMcFarland standard was used as a reference to \n\nadjust the turbidity of bacterial suspensions. The \n\nbacterial suspensions were standardized following \n\nthe CLSI guidelines for aerobic bacteria. All of the \n\ntest microorganisms were grown in Mueller Hinton \n\nbroth for 18\u201324 h, followed by the matching of \n\nbacterial suspension to the turbidity equivalent to \n\n0.5 McFarland solutions (1-2\u00d7108 cfu/ml). Different \n\nconcentrations (10, 20, 40, 60 and 80 mg/ml) of the \n\nextracts were prepared and kept in corked test tubes. \n\nSeeding of Muller \u2013 Hinton Agar plates \n0.1 ml of each diluted isolates was aseptically \n\ntransferred into Mueller\u2013Hinton agar (Oxoid, UK) \n\nplates and aseptically spread evenly using sterile \n\nHockey stick.", "start_char_idx": 17349, "end_char_idx": 20350, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "7954a0e4-0f7b-466c-836e-39d5264c0694": {"__data__": {"id_": "7954a0e4-0f7b-466c-836e-39d5264c0694", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "958b6631-c6c4-420e-a1b7-b10fcf77baa0", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "efd0f9d33135a00a2d398d662eed49702ef38c31ad699bb22f43ef419336910b", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "c2596582-a1e7-4c0e-a271-dc290b887bd6", "node_type": "1", "metadata": {}, "hash": "bde47736e302ea4968f767aef1ca25acd00498406b4031c8c56cae02e2867323", "class_name": "RelatedNodeInfo"}}, "text": "The seeded plates were left for 30 mins \n\nfor the isolates to diffuse into the medium. Sterile \n\ncork borer of 5 mm was used to bore holes on the \n\nagar plates. 0.1 ml of each of the extracts was then \n\ndropped in the holes and labeled accordingly. The \n\ndiameters of inhibition zones were measured [35]. \n\nResults \nPhytochemical screening \nThe results of the qualitative phytochemical \n\nscreening of methanol and ethyl acetate of leaf and \n\nstem extracts of  B. pinnatum as presented in Table 1 \n\nrevealed the presence of amino acids, quinone, \n\nphenol, reducing sugar, coumarin, steroids, \n\nterpenoids, glycosides, flavonoids, saponins, tannins \n\nand alkaloids in both plant parts. However, \n\nanthocyanins and anthraquinones were not detected \n\nin the methanol and ethyl acetate leaf extracts. \n\nThe results of the quantitative phytochemical \n\nscreening of methanol and ethyl acetate of leaf and \n\nstem extracts of  B. pinnatum in Table 2 shows that \n\nthe concentration of alkaloids ranged from 0.03\u00b10.02 \n\nto 0.90\u00b10.13 mg/g. alkaloids were not detected in the \n\nethyl acetate stem extract. The concentration of \n\nsaponins varied from 0.45\u00b10.43 to 1.12\u00b10.21 mg/g, \n\nwith methanol leaf extracts having the highest \n\nconcentration (1.12\u00b10.21 mg/g). \n\nThe results of flavonoids (y=0.142x-0.0.177: \n\nR2=0.983) in both plant part extracts showed that the \n\nstem extracts contained the highest concentration of \n\nflavonoids (0.53\u00b10.13 and 0.31\u00b10.01 mg for methanol \n\nand ethyl acetate extracts respectively) while the  \n\nTable 2: Quantification of the Phytochemicals in the extracts of \nBryophyllum pinnatum \n\nPhytochemica\nls (mg/g) \n\nLeaf extracts Stem extracts \n\nMethanol \nEthyl \n\nacetate \nMethanol \n\nEthyl \nacetate \n\nAlkaloids 0.16\u00b10.01 0.90\u00b10.13 0.03\u00b10.02 ND \nSaponins 1.12\u00b10.21 1.02\u00b10.22 1.05\u00b10.11 0.45\u00b10.43 \nFlavonoids 0.21\u00b10.14 0.11\u00b10.14 0.53\u00b10.13 0.31\u00b10.01 \nTannins 4.98\u00b11.31 2.8\u00b1\u00b10.21 0.64\u00b10.24 0.25\u00b10.12 \nPoly phenols 34.49\u00b10.47 21.2\u00b12.2 32.32\u00b11.2 17.9\u00b10.62 \n\nND = not detected \n\nTable 1: Qualitative Analysis of Different Parts of Bryophyllum \npinnatum \n\nPlant Parts Leaf Extract Stem Extract \nSolvents Methanol Ethyl \n\nAcetate \nMethanol Ethyl \n\nAcetate \nAnthocyanins Absent Absent Present Present \nAmino acid Present Present Present Present \nQuinone Present Present Present Present \nPhenol Present Present Present Present \nReducing sugar Present Present Present Present \nCoumarin Present present Present Present \nSteroids  Present Present Present Present \nTerpenoids Present Present Present Present \n\nGlycosides Present Present Present Present \nFlavonoids Present Present Present Present \nSaponins Present Present Present Present \nTannins Present Present Present Present \nAnthraquinone Absent  Absent  Present Present \nAlkaloids Present Present Present Absent  \n\n\n\nNepal J Biotechnol. J u l y  2 0 2 0 ; 8(1):17-28 Daniel et al. \n\n\u00a9NJB, BSN 22 \n\nleast concentration was present in the leaf extracts \n\nwith (0.21\u00b10.14 and 0.11\u00b10.14 mg/g for methanol  \n\nand ethyl acetate extracts respectively).", "start_char_idx": 20351, "end_char_idx": 23352, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "c2596582-a1e7-4c0e-a271-dc290b887bd6": {"__data__": {"id_": "c2596582-a1e7-4c0e-a271-dc290b887bd6", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "d0d7f7a03b82874ad6eb950af75fa272b2adc42f6e68b3a2f4d0c0f534900409", "class_name": "RelatedNodeInfo"}, "2": {"node_id": "7954a0e4-0f7b-466c-836e-39d5264c0694", "node_type": "1", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "hash": "ffdf2a84f6e0798e0738b81338d31a3286bca2ba2df9387c62d6ca8031ddd98a", "class_name": "RelatedNodeInfo"}, "3": {"node_id": "8eda33e2-9a0f-4fe7-b845-17cc8c509def", "node_type": "1", "metadata": {}, "hash": "e2685bca89f75418e336b3a0f8b4886ff63b3ac42bd89d97ae768c05e5b57e0c", "class_name": "RelatedNodeInfo"}}, "text": "Tannin \n\ncontent (y=0.168x -0.094: R2=0.975) was higher in \n\nmethanol leaf extract (4.98\u00b11.31 mg/g) while the \n\nleast concentration was recorded for ethyl acetate \n\nstem extract (0.25\u00b10.12 mg/g). Methanol leaf extract \n\nrecorded the highest concentration of polyphenols \n\n(34.49\u00b10.47 mg/g) while ethyl acetate stem extract  \n\ncontained the least concentration (17.9\u00b10.62 mg/g). \n\n(y=0.149\u20130.121 x: R2 = 0.967). \n\nAntimicrobial activity \nTables 3 and Table 4 show the result of the \n\nantibacterial activity of leaf and stem extracts of B. \n\npinnatum respectively tested against five bacterial \n\nand two fungi strains at different concentrations (10, \n\n20, 40, 60 and 80 mg/ml). All the extracts showed \n\nstrong antimicrobial activity against test organisms \n\nin a dose-dependent manner. Results from Table 3 \n\nshowed that S. aureus, C. albican, V. cholerae, \n\nSalmonella spp. and P. aeruginosa were susceptible to \n\nthe methanol leaf extract of B. pinnatum at all \n\nconcentrations. However, antibacterial activity was \n\nnot observed against E. coli. For ethyl acetate leaf \n\nextracts, the tested microorganisms showed varying \n\ndegree of susceptibility at various concentrations \n\nexcept V. cholerae and P. aeruginosa, which were \n\nresistant to ethyl acetate extract at all concentrations. \n\nTable 4 showed that A. niger, V. cholera and P. \n\naeruginosa were resistant to methanol stem extract at \n\nall concentrations while Salmonella spp. was resistant \n\nat 10 mg/ml. the zone of inhibition of S. aureus \n\nranged from 7\u00b10.11 to 15\u00b10.01 mg/ml while that of \n\nTable 3: Antimicrobial Activity of methanol and ethyl acetate leaf extracts of Bryophyllum pinnatum against the human \npathogenic bacteria by disc diffusion method. \n\nCrude extracts Isolates Zone of inhibition (mm) \n\n10 mg/ml 20 mg/ml 40 mg/ml 60 mg/ml 80 mg/ml \nMethanol S. aureus 8.00\u00b10.01 10.00\u00b10.3 12.0\u00b11.2 14\u00b10.2 20\u00b10.5 \n\nA. niger NA NA NA 10\u00b10.05 14\u00b10.3 \nCandida albican 16\u00b10.34 20\u00b10.21 23\u00b11.2 30\u00b11.2 34\u00b11.41 \nVibrio cholera 8\u00b10.03 10\u00b10.25 11\u00b10.23 15\u00b10.11 22\u00b11.21 \nEscherichia coli NA NA NA NA NA \nSalmonella spp. 10\u00b10.12 13\u00b10.22 16\u00b11.3 20\u00b11.31 23\u00b11.10 \nP. aeruginosa 14\u00b10.01 20\u00b11.22 23\u00b11.2 27\u00b11.22 30\u00b10.9 \n\nEthylacetate S. aureus 8\u00b10.01 9\u00b10.01 10\u00b10.13 13\u00b10.03 14\u00b10.12 \nA. niger 8\u00b10.1 9\u00b10.11 10\u00b10.6 12\u00b10.11 13\u00b10.21 \nCandida albican 13\u00b10.3 17\u00b10.33 21\u00b10.02 26\u00b10.22 30\u00b10.12 \nVibrio cholera NA NA NA NA NA \nEscherichia coli 13\u00b10.01 15\u00b10.01 17\u00b10.03 20\u00b10.41 25\u00b10.61 \nSalmonella spp.", "start_char_idx": 23353, "end_char_idx": 25792, "text_template": "{metadata_str}\n\n{content}", "metadata_template": "{key}: {value}", "metadata_seperator": "\n", "class_name": "TextNode"}, "__type__": "1"}, "8eda33e2-9a0f-4fe7-b845-17cc8c509def": {"__data__": {"id_": "8eda33e2-9a0f-4fe7-b845-17cc8c509def", "embedding": null, "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum", "date": "2020-07-31", "file": "njb-62.pdf"}, "excluded_embed_metadata_keys": [], "excluded_llm_metadata_keys": [], "relationships": {"1": {"node_id": "njb-62", "node_type": "4", "metadata": {"identifier": "njb-62", "author": "Daniel, Imaobong E.; Akpan, Ekemini I.; Utam, Edidiong C.", "title": "Phytoch