6 Microbiome Composition and Circadian Rhythm Disruption Alters Epithelial Barrier Integrity Elisha Pinker1, Timur Tuganbaev2 1Columbia University, New York, NY, USA 2Lab of Dr. Eran Elinav; Department of Immunology, Weizmann Institute of Science, Israel ABSTRACT: The intestine is home to one of the most complex ecological communities, termed the human gut microbiome. The gut microbiome modulates a wide range of human diseases from diabetes to neurological disorders to cancer. Separating the host and the gut microbiome is the epithelial barrier. The intestinal epithelium serves as an adaptive interaction hub between the host and microbiome that plays an important role in deciding the outcome of host-microbi- ome interactions. Regulation of epithelial barrier permeability to ions, nutrients and microbiome metabolites is known to be a tightly controlled process on the host side. While previous stud- ies have identified the microbiome as a factor for epithelial permeability, the exact mechanisms through which it mediates remains less clear. Here, we show that alterations in microbiota com- position by treatment with antibiotics selectively targeting specific members of the microbiome community impacts the permeability of the intestine. Additionally, modulating the microbiome through other methods such as altering diet composition shows changes in permeability of the epithelial barrier. As daily feeding rhythm entrains diurnal fluctuations in microbiome, we have set out to measure how epithelial barrier permeability disrupts the clock. We have discovered that the permeability of the intestinal epithelial barrier exhibits circadian rhythms in mice. Dis- ruption of these rhythms, through jet lag or genetic deficiencies in circadian machinery, alters epithelial barrier integrity. Together, these findings provide evidence that disruptions in circadian rhythms as well as alterations in microbiome composition have direct consequences in intestinal permeability, and that microbiome might serve as a tool in regulating epithelium permeability. INTRODUCTION The human body is colonized by a complex community of microorganisms, termed the hu- man microbiome, that includes bacteria, ar- chaea, viruses and fungi [1]. This ecological community exists primarily on mucosal sites, like the gastrointestinal tract, skin, lungs and urogenital system [2]. This community is such where the microbiome bacteria outnumber eu- karyotic cells in the entire human body 1.3:1 [3]. The microbiota is responsible for the regu- lation of many physiological processes, includ- ing digestion, absorption [4], host metabolism [5], the maturation and function of the immune system [4], and even host behavior and cogni- tive function [6]. However, when the balanced microbial composition is disrupted, dysbiosis can cause intestinal diseases [7]. Modulating © 2021 Pinker, Tuganbaev. This is an open access article distributed under the terms of the Creative Commons At- tribution License, which permits the user to copy, distribute, and transmit the work provided that the original authors and source are credited. KEYWORDS: Circadian rhythms, intestinal permeability, microbiome 7 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. the microbiome through the diet is one way to induce dysbiosis. Treatment with high-fat diet (HFD), containing 60% of caloric energy derived from fat (a model of a “western diet”), is one such example where the balanced gut microbial community is altered to the point of dysbiosis resulting in metabolic dysfunction consequently inducing disease, such as obe- sity and type 2 diabetes as well as exacer- bating inflammatory bowel diseases (IBD) [7]. But, when treated with broad-spectrum antibi- otics, obesity and glucose intolerance in mice fed with HFD is abrogated [8]. Previous stud- ies show, that when microbiome from mice fed HFD were transplanted into germ-free mice (GF: free of microorganisms) significantly more weight is put on by these mice compared to GF mice transplanted with microbiome from lean donor mice fed NC (normal chow) [9]. Additionally, GF condition in mice causes sig- nificant immune system deficiency [7]. Tools like gnotobiotic animal models (GF mice) and broad-spectrum antibiotics, have enabled the discovery that microbiota contributions to health and disease span multiple organ systems. It has previously been shown that the mammalian intestinal microbiota composition exhibits diurnal oscillations both in terms of its composition and metabolic activity [8]. The circadian clock is the top of a hierarchical in- ternal clock system that is responsible for the circadian biological processes ranging from dictating the rhythmic gene expression in or- gans to behavior, metabolism and immunity [10][11]. When working with circadian rhythms, scientists devised Zeitgeber times (ZT) which measure time on a 24-hour light/dark cycle, so ZT12 and ZT0 denote “dusk” and dawn”, re- spectively. The molecular mechanisms behind the circadian clocks in mammals is orchestrat- ed under a transcriptional autoregulatory feed- back loop comprised of the ‘core’ clock genes CLOCK and BMAL1, responsible for encod- ing activators, and PER1, PER2, CRY1 and CRY2, responsible for encoding repressors [12]. A disruption of the circadian clock in hu- mans, environmentally or by gene deficiency, is associated with an array of diseases such as, obesity and diabetes [13] [14], cancer [15], cardiovascular disease [16], IBD (inflammatory bowel disease) exacerbation [17] and suscepti- bility to infection. One common feature among the diseases associated with circadian rhythm disruption, is that they appear to be triggered or promoted by inflammatory processes. Desyn- chronized interaction between mucosal organs and a complex community of microorganisms colonizing them have emerged as a potential source of inflammation underlining diseases promoted by circadian clock disruption. The source of this inflammation can be located at the gut epithelium [18]. The intestinal epithelium is comprised of a single-cell layer constituting the largest and most important barrier against the external environment [19]. Acting as a se- lectively permeable barrier, the intestinal epi- thelium can permit the absorption of nutrients, electrolytes and water, while simultaneously defending against toxins [19]. The epithelium maintains an effective barrier through the help of protein-protein networks forming complexes like tight junctions [19]. A compromised epithe- lial barrier allowing the passage of toxins, an- tigens and bacteria into the blood stream cre- ates what is commonly known as “leaky gut” [20]. There is therefore an urgent need to better identify the players behind the disruption and upkeep of intestinal barrier function, to devise methods to combat negative barrier alteration. In this project we studied different factors af- fecting permeability in the epithelial barrier, specifically in the small intestine, the part of the intestine responsible for the majority of nutri- ent absorption [21]. Recent studies on type 2 diabetes and obesity have elucidated the link between microbiome composition and small in- testine permeability; however, the mediators of these effects have yet to be determined [22]. Over the course of this project, we demonstrated the consequences that genetic and environmental circadian disruption have on gut barrier integrity in mice. Furthermore, we 8 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. found evidence to suggest that altering the mi- crobiome, either with diet intervention or antibiot- ic treatment, can disrupt the diurnal oscillations and alter permeability. Understanding the key players behind circadian disruption and intesti- nal barrier maintenance has potential in thera- peutic and preventative medicine interventions. METHODS Mice Under normal conditions, mice had strict light- dark cycles lasting 12 hours each. Lights were turned on either at 11:30 AM or 11:30 PM, ZT0 or ZT12 respectively. For the induction of jet lag, mice were shifted between both control light conditions every 3 days for 3 weeks at a consis- tent but arbitrary time. Jet lag mice were moved between the different light schedules every 3 days for 3 weeks. For consistency, jet lag mice were only experimented on at 1:30 PM, which was synchronized with the ZTs of the control group (i.e., ZT14 of jet lag mice corresponded to ZT14 of control mice, as all mice were exposed to the same light-dark conditions at the onset of sample collection). HFD mice were placed in ZT2 and ZT14 light-dark cycles to measure two time points upon sacrifice. These mice were fed a strict HFD for 3 weeks. Mice receiving antibi- otic treatment, either received exclusively van- comycin (1 g/l), ampicillin (1 g/l), neomycin (1 g/l), and metronidazole (1 g/l) or a combination of all four in drinking water for 3 weeks. Per 1/2- /- (KO) and Per1/2-/+ (WT) were housed un- der normal conditions and sacrificed at ZT 14. Fecal Microbiome Transplantation (FMT) Microbiome samples were collected and stored in Eppendorf tubes containing 20% glycerol. Fecal samples were homogenized and stored in dry ice before long-term storage at -80° C. For the microbiome transplantation, 0.1g of thawed fecal sample was mixed in 1mL sterile PBS and 200 μL of the diluted sample was gavaged per mouse. HFD microbiome samples were tak- en from mice fed ad libitum HFD for 4 weeks. Measuring Permeability with FITC-dextran Food was removed from mice 2 hours be- fore gavage (starting at 9 AM). The solution was made with 4kDA fluorescein isothiocy- anate (FITC)-dextran dissolved in a phos- phate buffered saline (PBS) with a concen- tration of 40 mg/ml. At 11 AM, mice were gavaged with 200 μl dextran. After 2.5 hours (1:30 PM, ZT14), blood was drawn and centri- fuged to recover the serum. The fluorescence of the serum was quantified at an excitation wavelength of 485 nm and emission wave- length of 533 nm using a microplate reader. Ussing Chamber The Ussing chamber is an instrument used to measure epithelial resistance which is a measure of epithelial barrier permeability. The procedure followed was in accordance with the manufacturer’s instructions (Warner Instruments, P2300). Small intestine tissue is excised and immediately mounted in the chamber. Current is then applied to the sys- tem and voltage clamp recordings were tak- en. Small intestine was carefully cleaned and handled minimally to prevent microfractur- ing the tissue and compromising the results. Additionally, samples were mounted within three hours of tissue excise to limit cell death. PRR stimulation measures degree of mi- crobial presence at systemic sites PRR reporter cell lines were obtained from Invi- vogen (HEK-Blue TLR (Toll-like Receptors) and NLR (Nod-like Receptors) reported cell lines): TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, NOD1, NOD2. Extracts from spleen, liver, and serum were homogenized and added to reporter cell lines incubated with KEL-Blue detection medium according to manufacturer’s instructions. Detec- tion is a measurement of specific binding indi- cating extent of microbial leakage out of the gut. Statistical Analysis Data is presented as the mean ± standard deviation. Mann-Whitney U-tests were per- 9 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. formed to compare groups. ANOVA with post hoc test was used for comparison between multiple groups with a Bonferroni correc- tion. p < 0.05 and q < 0.1 were the thresh- old for being considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. RESULTS State of Small Intestinal Microbiome Deter- mines Epithelial Barrier Function With a GF mouse model, we initially set out to establish the role a microbiome has on the per- meability of the small intestine epithelial barrier. Mice underwent fecal transplantation of either normal chow (NC) microbiome or high fat diet (HFD) fed donor specific pathogen free (SPF) microbiome, while the control group received phosphate buffered saline (PBS) (Figure 1A). Small intestine samples were taken and epithe- lial resistance was measured with the Ussing Chamber. Interestingly, mice with HFD microbi- ome showed increased permeability while mice with PBS (no microbiome) showed reduced permeability (Figure 1B). Together, this data suggests that microbiome shaped by diet is an active influencer over intestinal permeability. We next set out to further analyze which subsets of bacteria within the microbiome com- munity are most influential in barrier function modulation. With an antibiotic murine model, we could target specific subsets of bacteria to determine the impact the presence or lack thereof has on epithelial permeability in the in- testine. We treated 5 groups of WT mice, fed with normal chow, with selective antibiotics that target specific members of microbiome: metronidazole (anaerobic bacteria), vancomy- cin (Gram-positive bacteria), ampicillin (broad spectrum), neomycin (Gram-negative bacte- ria), or a combination of all four for a duration of 3 weeks delivered in their drinking water (Figure 1C). The group containing all four an- tibiotics is a crude simulation of the germ-free mouse model. After 3 weeks, a FITC-dextran assay was performed, which entails orally ad- ministering mice with fluorescent dye coated 4 kDa beads and measuring fluorescence in the peripheral blood, at ZT14, as a proxy for epithelial barrier permeability. Significant re- duction in optical density (OD) was observed in four of the antibiotic groups compared to the control (Figure 1D). Additionally, vanco- mycin treated mice had significantly higher OD than neomycin and metronidazole groups (Figure 1D). The lack of a significant reduction in permeability in the combination treatment, containing all four antibiotics, was likely due to outliers. Two days after blood was taken, and fluorescence was no longer detectable, mice were sacrificed, at ZT14, and small in- testine samples were removed for the Ussing Chamber measurements. Only the jejunum re- gion of the small intestine was used for Uss- ing Chamber. All five antibiotic groups showed significantly reduced permeability and vanco- mycin, again, showed higher permeability than two of the other antibiotic groups (Figure 1E). Together, these results uncover that the state of microbial composition directly alters the barrier integrity in the small intestine and the entire system. Additionally, vancomycin-treat- ed groups experienced the smallest reduction in permeability suggesting, that gram-positive bacteria play the smallest role in intestinal epithelial barrier function compared to oth- er bacterial subtypes in the gut microbiome. Genetic and Environmental Disruption of Circadian Clocks Impacts Epithelial Barrier Integrity It has been shown that a functional circadian clock of a host is required for normal diurnal rhythmicity in microbiota composition and func- tion ‎[8]. Building off our previous observations, the next step is to examine if circadian clock dis- ruption will likewise compromise the intestinal epithelial barrier. Testing of Per1/2-/- mice, ge- netically deficient in a functional host clock, has previously shown to disrupt diurnal rhythmicity in microbiota composition ‎[8]. With our own group of Per1/2-/- mice, we ran a FITC-dextran assay. 10 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. The OD for Per 1/2-/- was significantly higher (Figure 2A) demonstrating that circadian clock disruption results in increased intestinal per- meability, a feature of a “leaky gut” syndrome. Environmental clock disruptions such as chronic jet lag and shift work are behavioral patterns associated with an increased risk for obesity, diabetes and cardiovascular disease [23][18][14][16][24]. It has been previously es- tablished that a loss of microbiota oscillations and dysbiosis is associated with jet lag in mice [8]. So, we set out to learn if these losses of oscillations and dysbioses, in jet lag mice, also impact barrier function in the intestinal epithe- lial barrier. Prior to our jet lag experiment, we performed a 24-hour circadian experiment on WT mice measuring intestinal permeability with two independent methods – one measur- ing the electrical permeability of the epitheli- um to ions (the Ussing Chamber assay) and one measuring the concentration of microbial products in peripheral blood (Toll-like Recep- tor (TLR) assay) (Figure 2B, Figure 2C). Our findings demonstrate that just like microbial composition behaves in diurnal oscillations, so too does permeability. From this data we chose ZT2 and ZT14 as the lowest and high- est points of permeability respectively and prepared control groups at each of these time points. This makes sense due to the nocturnal behaviors of mice where ZT2 is during the day- light when mice are least active while ZT14 is during the night is when mice are most active. We then set out to mimic the situation of shift work and chronic jet lag by using a jet lag model in which mice were exposed to a 12-hour time shift every 3 days. Using a cir- cadian cabinet, which allows us to individually control the light cycles of each compartment, a group of mice were initially started in one of two A B C ED Figure 1. State of Small Intestinal Microbiome Determines Epithelial Barrier Function. (A) Schematic summarizing transplantation of microbiota from HFD, NC and PBS into germ-free recipient mice. (B) Ussing Chamber recording of small intestines from GF mice; measuring current (mA) as a measure- ment of permeability. (C) Schematic showing a murine antibiotic treatment setup. (D) FITC (fluorescein isothiocyanate)-dextran recovered from the serum of antibiotic treated WT mice and control. (E) Ussing Chamber recording of small intestines from antibiotic treated WT mice and control. Means ± SD are plotted. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney U test. 11 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. compartments (ZT2 or ZT14) with light cycle’s 12-hours apart. Every 3 days for 3 weeks the cages of mice were switched between the two compartments until they were “jet lagged”. The mice were then bled for FITC-dextran assay at the same time, ZT2 and ZT14, depending on the compartment. However, if the mice were jet lagged properly, and their circadian clock was disrupted, the ZT time at which blood was drawn would not matter. The results showed that the OD from FITC-dextran between jet lag groups ZT2 and ZT14 were non-signifi- cant (Figure 2D). Additionally, compared to the controls, the jet lag groups’ ODs was closer to Control ZT14 (highest measured permeability) than Control ZT2 (lowest measured permeabil- ity). When we excised small intestine samples, for Ussing chamber assay, we observed sim- ilar trends: non-significant permeability mea- surements between jet lag groups (sign of suc- cessful jet lag) and overall permeability levels aligned with ZT14 control levels (Figure 2E). Altogether, these data suggest that chronic environmental (jet lag model) or genet- ic disruption (Per1/2-/-) of host light-dark cycles exhibits significant alterations in intestinal epi- thelial barrier function and as a result increasing barrier permeability. This has relevance for fre- quent fliers traveling across different time zones and people who perform shift work. Additionally, our data suggests an explanation for clinical ob- servations, finding shift workers, with irregular sleep/wake schedules, to experience exacer- bated inflammatory bowel disease (IBD) due to the effect endogenous misalignment of circadi- an rhythms has on intestinal homeostasis ‎[26]. Metabolic Syndrome Induced by HFD Re- sults in Circadian Disruption and Intestinal Barrier Dysfunction Metabolic syndromes, like obesity and diabe- tes, have impacted hundreds of millions of peo- ple worldwide and consequently taken the lives of millions annually ‎[25]. Previous studies have uncovered the associations metabolic syn- dromes have with the functionality of the intes- Figure 2. Genetic and Environmental Dis- ruption of Circadian Clocks Impacts Epi- thelial Barrier Integrity. (A) FITC (fluorescein isothiocyanate)-dextran re- covered from the serum of Per1/2-/- and WT. (B) Ussing Chamber recording of small intestines from naïve WT over 24 hours. Arrows mark the low- est (ZT2) and highest (ZT14) points of permeability. (C) PRR stimulation by spleen, liver, serum ex- tracts from naïve WT mice. Arrows mark the lowest (ZT2) and highest (ZT14) points of permeability. (D) FITC (fluorescein isothiocyanate)-dextran recovered from the serum of jet lag mice and their respective controls. (E) Ussing Chamber recording of small intestines from jet lag mice and their respective controls. Means ± SD are plotted. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney U test. Figures 2D and 2E: 2-way ANOVA test. A B C D E 12 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. tinal barrier, specifically its state of permeability [27]. The study highlights the implications an influx of immune-stimulatory microbial ligands, leaking out of a permeable intestinal barrier can have, such as increased inflammation and risk of infection [27]. However, the tissue under scru- tiny was the colon. In our studies, we focused our attention on the small intestine, which one could argue is of more significance due to it be- ing the primary site of nutrient absorption [21]. In order to mimic human dietary habits, which would predispose one to a metabolic syndrome, we fed mice with an HFD for 3 weeks. In a pre- viously referenced chart (Figure 1B), where germ-free mice received fecal transplantation of an HFD microbiome we saw a significant in- crease in small intestine permeability than mice with a normal diet (NC) microbiome, suggesting that a microbial composition associated with an HFD diet causes intestinal barrier dysfunction. Previous studies linked circadian rhythm disruption to metabolic syndrome when they found enhanced weight gain and exacer- bated glucose intolerance in jet lag mice fed HFD [8]. In an attempt to determine if an HFD would alter permeability in a circadian context we set out to perform another permeability ex- periment on HFD mice but under circadian con- ditions. We fed two groups of mice with HFD for 3 weeks. Utilizing the circadian cabinet, each group was placed in different compartments and light-dark cycles were set 12-hours apart at the previously observed lowest and highest times of permeability, ZT2 and ZT14 respective- ly (Figure 3A). FITC-dextran assay was run and blood was drawn at ZT2 and ZT14 depending on which compartment the mice were in. From the FITC-dextran data we see no significant dif- ference in OD between HFD diet groups which suggests that the altered diet disrupted circa- dian rhythms (Figure 3B). Additionally, we see OD levels of HFD groups to be at levels clos- er to Control group ZT14 reaffirming previous data showing increased permeability in HFD mice (Figure 3B). When the Ussing Chamber is performed on small intestine samples, we like- wise see no significance between HFD groups and overall HFD permeability levels correlate Figure 3. Metabolic Syndrome Induced by HFD Results in Circadian Disruption and Intes- tinal Barrier Dysfunction. (A) Schematic showing the typical configuration of a circa-dian cabinet. (B) FITC (fluorescein isothiocyanate)-dextran recovered from the serum of HFD mice at different ZT times and their respective controls. (C) Ussing Chamber recording of small intestines of HFD mice at different ZT times and their respective controls. Means ± SD are plotted. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney U test. Fig- ure 3B and 3C: 2-way ANOVA test. A B C 13 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. more strongly with ZT14 than ZT2 (Figure 3C). Together, these observed results draw attention to a serious issue. Metabolic syn- dromes, which are usually onset by poor di- ets such as those high in fat, are only exac- erbated by the same stimuli that caused the development of the disease. Our data signi- fies metabolic syndromes’ role in both dis- rupting circadian rhythms and heightening intestinal barrier permeability that can lead to increased inflammation and infection. DISCUSSION & CONCLUSION In this study, we describe that the mammalian intestinal barrier function displays diurnal oscil- lations and is dependent on microbiome com- position. If microbiome composition is distorted, as in the case of antibiotic treatment or HFD, we see alterations in intestinal barrier integrity as well as disruptions in the circadian rhythms of epithelial permeability. While previous stud- ies sought to understand the consequences, di- urnal disruption has on microbial composition, here we found direct evidence of the role micro- bial composition plays in intestinal permeability. Next, we wanted to study the direct implications distorting circadian rhythms, either with genet- ic clock deficiency or time-shifted induced jet lag, have on intestinal barrier function. We not only found that these conditions significantly impaired the circadian rhythms of permeability, but also, increased overall permeability result- ing in greater pathobiont exposure and inflam- mation. Lastly, we found that inducing meta- bolic syndromes via HFD intervention disrupts circadian rhythms in the intestinal barrier as well as causes significant barrier dysfunction resulting in increased epithelial permeability. Altogether, these studies have uncov- ered numerous future paths. While this study has broadly implicated microbiome composi- tion as a mediator of gut permeability, in order to learn more about the key bacterial players in permeability mediation, we can perform se- quencing on the microbial compositions found in our antibiotic treated groups to reveal the unique bacteria having the biggest impact on reducing or increasing permeability. Under- standing which bacteria to promote and which to remove in the microbiota could be pivotal in suppressing symptoms in patients with IBD and metabolic syndromes, to name a few, caused by leakages in the intestinal epithelial. Exploit- ing antibiotic or probiotic treatments to target these bacteria could help combat diseases ex- acerbated by gut leakiness. Lastly, our observa- tions showing intestinal permeability function- ing under diurnal oscillations has applications from drug consumption to nutritional/dietary strategies. Further unravelling the roles, reg- ulators and mechanisms of intestinal permea- bility could serve to one day aid therapies for illnesses affected by the “leaky gut” syndrome. AUTHOR INFORMATION Corresponding Author *Elisha Pinker ep2858@columbia.edu Funding Sources Columbia College Class of 1939 Fellowship Competing Interests The authors declare no competing financial and non-financial interests. ACKNOWLEDGMENTS E.P. would like to thank Columbia College and the Weizmann Institute of Science for support- ing this research. REFERENCES [1] Marietta E, Rishi A, Taneja V. Immunoge- netic control of the intestinal microbiota. Im- munology. 2015;145(3):313-322. doi:10.1111/ imm.12474. [2] Neish AS. Mucosal Immunity and the Microbiome. Annals of the American Tho- racic Society. 2014;11(Suppl 1):S28-S32. doi:10.1513/AnnalsATS.201306-161MG. 14 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. [3] Sender R, Fuchs S, Milo R. Revised Estimates for the Number of Human and Bacteria Cells in the Body. PLoS Biology. 2016;14(8):e1002533. doi:10.1371/journal. pbio.1002533. [4] Clemente, J.C., Ursell, L.K., Parfrey, L.W., and Knight, R. (2012). The impact of the gut microbiota on human health: an integrative view. Cell [5] Sommer, F., and Backhed, F. (2013). The gut microbiota—masters of host development and physiology. Nat. Rev. Microbiol [6] Hsiao, E.Y., McBride, S.W., Hsien, S., Sha- ron, G., Hyde, E.R., McCue, T., Codelli, J.A., Chow, J., Reisman, S.E., Petrosino, J.F., et al. (2013). Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell [7] Shi N, Li N, Duan X, Niu H. Interaction between the gut microbiome and mucosal immune system. Military Medical Research. 2017;4:14. doi:10.1186/s40779-017-0122-9. [8] Thaiss C.A., Zeevi D., Levy M., Zilber- man-Schapira G., Suez J., Tengeler A.C. Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell. 2014;159(3):514–529 [9] de Groot PF, Frissen MN, de Clercq NC, Nieuwdorp M. Fecal microbiota transplantation in metabolic syndrome: history, present and future. Gut Microbes (2017) [10] Dibner, C., Schibler, U., and Albrecht, U. (2010). The mammalian circadian timing sys- tem: organization and coordination of central and peripheral clocks. Annu. Rev. Physiol. [11] Keller, M., Mazuch, J., Abraham, U., Eom, G.D., Herzog, E.D., Volk, H.D., Kramer, A., and Maier, B. (2009). A circadian clock in macrophages controls inflammatory immune responses. Proc. Natl. Acad. Sci. [12] Takahashi JS. Transcriptional architec- ture of the mammalian circadian clock. Na- ture reviews Genetics. 2017;18(3):164-179. doi:10.1038/nrg.2016.150. [13] Buxton, O.M., Cain, S.W., O’Connor, S.P., Porter, J.H., Duffy, J.F., Wang, W., Czeisler, C.A., and Shea, S.A. (2012). Adverse meta- bolic consequences in humans of prolonged sleep restriction combined with circadian dis- ruption. Sci. Transl. Med [14] Fonken, L.K., Workman, J.L., Walton, J.C., Weil, Z.M., Morris, J.S., Haim, A., and Nelson, R.J. (2010). Light at night increases body mass by shifting the time of food intake. Proc. Natl. Acad. Sc [15] Savvidis C, Koutsilieris M. Circadian Rhythm Disruption in Cancer Biology. Mo- lecular Medicine. 2012;18(1):1249-1260. doi:10.2119/molmed.2012.00077. [16] Scheer, F.A., Hilton, M.F., Mantzoros, C.S., and Shea, S.A. (2009). Adverse metabol- ic and cardiovascular consequences of circadi- an misalignment. Proc. Natl. Acad. Sci [17] Swanson, GR., Burgess, HJ. (2017). Sleep and circadian hygiene and inflammatory bowel disease. Gastroenterology Clinics of North America.46(4):881893. doi:10.1016/j.gtc.2017.08.014 [18] Koch S, Nusrat A. The life and death of epithelia during inflammation: lessons learned from the gut. Ann. Rev. Pathol. 2012;7:35–60. doi: 10.1146/annurev-pathol-011811-120905. [19] Groschwitz KR, Hogan SP. Intestinal Barrier Function: Molecular Regulation and Disease Pathogenesis. The Journal of allergy and clinical immunology. 2009;124(1):3-22. 15 Columbia Undergraduate Science Journal Vol. 15, 2021 Pinker et. al. doi:10.1016/j.jaci.2009.05.038. [20] Mu Q, Kirby J, Reilly CM, Luo XM. Leaky Gut As a Danger Signal for Autoimmune Dis- eases. Frontiers in Immunology. 2017;8:598. doi:10.3389/fimmu.2017.00598. [21] Kiela PR, Ghishan FK. Physiology of Intestinal Absorption and Secretion. Best practice & research Clinical gastroenter- ology. 2016;30(2):145-159. doi:10.1016/j. bpg.2016.02.007. [22] Chakaroun RM, Massier L, Kovacs P. Gut Microbiome, Intestinal Permeability, and Tissue Bacteria in Metabolic Disease: Perpe- trators or Bystanders? Nutrients. 2020;12(4). doi:10.3390/nu12041082 [23] Archer, S.N., Laing, E.E., Mo¨ ller-Levet, C.S., van der Veen, D.R., Bucca, G., Lazar, A.S., Santhi, N., Slak, A., Kabiljo, R., von Schantz, M., et al. (2014). Mistimed sleep disrupts circadian regulation of the human transcriptome. Proc. Natl. Acad. Sci [24] Suwazono, Y., Dochi, M., Sakata, K., Oku- bo, Y., Oishi, M., Tanaka, K., Kobayashi, E., Kido, T., and Nogawa, K. (2008). A longitudinal study on the effect of shift work on weight gain in male Japanese workers. Obesity [25] Obesity and Overweight. https://www. who.int/news-room/fact-sheets/detail/obesi- ty-and-overweight. Accessed 20 Dec. 2020. [26] Nojkov, Borko, et al. “The Impact of Ro- tating Shift Work on the Prevalence of Irritable Bowel Syndrome in Nurses.” The American Journal of Gastroenterology, vol. 105, no. 4, Apr. 2010, pp. 842–47. PubMed Central, doi:10.1038/ajg.2010.48. [27] Thaiss CA, Levy M, Grosheva I, Zheng D, Soffer E, Blacher E, et al. Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection. Science. 2018;359:1376–1