Volume 14 | 2020 | cusj.columbia.edu Atomic Materials Analysis: Correction of Self-Absorption Distortion in XANES PAGE 9 Administration Letter from the Editor-In-Chief…………………………………………..3 Letter from the President………………………………………………...5 Faculty Advisory Board and Editorial Board…………………………...7 Articles Characterization and Correction of Self Absorption Distortion in XANES Aidan P. Reddy, Apurva Mehta………………………………………….9 Protein-Polymer Complex Coacervates as Synthetic Membraneless Organelles Hansen Tjo, Nicholas Zervoudis, Allie C. Obermeyer……………....21 I’m Upset, Not Ashamed: An Investigation into Adolescent Shame Acknowledgement Olivia Putnam, Keely Lake……………………………………………..27 Contents 1 2 Dear Readers, It is with great excitement that I am able to present to you the CUSJ Volume 14 - 2020 Edition. Since our very first volume in 2006, it has been the mission of CUSJ to provide an accessible and professional, open access platform for undergraduates to communicate their research advances in various scientific fields. We CUSJ editors believe, as a community of young scientists and writers, that it is absolutely essential for researchers of all ages and levels of experience to have the opportunity to share their ideas and learn from their peers. Creating a space to facilitate that discussion is exactly what CUSJ aims to achieve not only with each volume, but also with our outreach to the public and numerous research oriented events. COVID-19 has posed an incredibly difficult and tragic challenge to communities all over the world, scientists and non-scientists alike. With the shutdowns of universities accompanied by sudden changes in employment status and housing, the perseverance and determination shown by both our authors and editing team has been a true testament to their character, work ethic, and resilience. The hard work evident in each article of this volume, from experimental design to data interpretation, is a display of the talent and strength of our authors, and their ability to push on through an unprecedented global emergency. These undergraduates have shown that even during the most trying of times, while physically isolated, we can all still come together and communicate through science. Volume 14 of CUSJ is diverse in scientific fields represented, ranging from biochemistry and the development of enzymatic nanoreactors for industrial synthesis, to the social science of shame in teens. Our cover features a graph from author Aidan Reddy’s cutting-edge investigation into X-ray absorption spectroscopy data processing, in which he develops a new method for spectra correction. His work should allow for more accurate analysis of various materials’ atomic properties, advancing the fields of both materials science and physics. It has been a pleasure to serve as the Editor-In-Chief of CUSJ for the 2019-2020 academic year. My sincere thank you to everyone involved in the creation of this volume for their passion and commitment. Julia Parsley Editor-in-Chief Letter 3 4 Dear Readers, Founded in 2006, the Columbia Undergraduate Science Journal was created by a team of graduate students who wanted to provide a collegiate platform for undergraduates to publish their scholarship and to educate students about the academic publication process. With guidance from advisors and faculty, the editorial board strove to produce a professional-level, open-access science journal that provides participants with a valuable experience in publication. Since then our board has shifted to being solely composed of undergraduates. While still maintaining our core editorial standards, the journal has evolved to more explicitly try and foster a supportive student research community on our Morningside campus. It is this notion of community that can help us cope and process the unfamiliar circumstances of a global pandemic. In quarantine, student researchers can reflect on the utility of science and its capacity to contain and eradicate this crisis. The collegiate scientists of today should recognize this moment for its affirmation in the power of collaboration and community. Recall this moment as you go onto make the next breakthroughs in the natural and applied sciences. This year, the Columbia Undergraduate Science Journal formalized our intentions for community building, made inroads in connecting previous authors and alumni of our publication, and developed our editorial process further with open journal infrastructure. I was impressed by the quality of our submissions this year and I want to thank each of the authors for their cooperation in the compilation of our journal. I am incredibly proud of what we have accomplished this year. Thank you all for your support in the growth of the student research community. The Columbia Undergraduate Science Journal editorial board is proud to present the 14th annual Columbia Undergraduate Science Journal. Congratulations to the authors, thank you to our readers, and enjoy! Sincerely, Jason Mohabir President CUSJ Letter 5 6 Faculty Advisory Board Hugh Ducklow…………………………….……….Professor of Earth and Environmental Sciences Ivana Hughes………..Director of Frontiers of Science, Senior Lecturer in Discipline - Chemistry Marko Jovanovic………………………………………………...….Professor of Biological Sciences Laura Kaufman………………………………………………………….……...Professor of Chemistry Patricia Lindemann...Lecturer in Discipline and Director of Undergraduate Studies - Psychology Kyle Mandli……………………………………………….………..Professor of Applied Mathematics Matthew Palmer…………………...Senior Lecturer and Director of Undergraduate Studies - E3B Gerard Parkin……………………………………………………………...…...Professor of Chemistry Ron Prywes………………………………………………………….Professor of Biological Sciences Angela Rasmussen…….…….Associate Research Scientist - Center for Infection and Immunity Editors Julia Parsley…………..….Editor-In-Chief, CUSJ Kate Johnson….……..…...Editor-In-Chief, CJSJ Jason Mohabir…………....…………….President Alex Ying………....……………Director of Events Arya Rao…………......Director of Internal Affairs Ethan Chen……...…………..Director of Finance Maria Trifas……..………….Director of Outreach Neeraj Sakhrani…..Director of Communications Ilaria Simeone………………....…………...Editor Vikas Chelur……...…………....…………...Editor Huck Jun Hong……………......…………...Editor Noah Krever………………....…...………...Editor Elin Hu………………................…………...Editor Harrison Zhang…………..…....…………...Editor Elena Gribelyuk………….…....…………...Editor Brendon Choy………………....…………...Editor Isabella Leite…………..……....…………...Editor Jacy Fang……………...……....…………...Editor Katie Long…………..………....…………...Editor People Ashley Rosenberg……...…....…………...Editor Sophia A Ladyzhets…....…....…………...Editor Deena Shefter……………......…………...Editor Kanav Kalucha…………….....…………...Editor Sahith Vadada……………......…………...Editor Lydia Wu………………….......…………...Editor Arjun Kudinoor……………….........……...Editor Mayeesa Rahman……………....………...Editor Aswath Suryanarayanan…....…………...Editor Chloe Gong………………......…………...Editor Ellen Ren…………...…….......…………...Editor Avi Gupta…………...…….......…………...Editor Shivali Verma…………...........…………...Editor Hibah Vora…………….….......…………...Editor Unal Yigit Ozulku………….....……….…...Editor Michael Wang……………….......………...Editor Caroline Haoud……………….…………...Editor Shamara Yearwood……….....…………...Editor 7 8 &KDUDFWHUL]DWLRQ &RUUHFWLRQ RI 6HOI�$EVRUSWLRQ 'LVWRUWLRQ LQ ;$1(6 $LGDQ 3� 5HGG\ DQG $SXUYD 0HKWD 6/$& 1DWLRQDO $FFHOHUDWRU /DERUDWRU\� ���� 6DQG +LOO 5G� 0HQOR 3DUN� &$ ����� 7KLV LV QRW WKH DXWKRU¶V SUHVHQW DGGUHVV ;$1(6� ;�5$< $%62537,21 63(&7526&23<� 1($5 ('*( 6758&785( $%675$&7� ;�UD\ DEVRUSWLRQ VSHFWURVFRS\ �;$6� LV DQ H[SHULPHQWDO WHFKQLTXH XVHG WR SUREH WKH DWRPLF SURSHUWLHV RI PDWHULDOV� ;$6 PHDVXUHPHQWV SHUIRUPHG LQ IOXRUHVFHQFH PRGH DUH VXEMHFW WR VR�FDOOHG ³VHOI�DEVRUSWLRQ´ GLVWRUWLRQ� HVSHFLDOO\ ZKHQ WKH VDPSOH XQGHU LQYHVWLJDWLRQ LV WKLFN RU FRQFHQWUDWHG ZLWK UHVSHFW WR WKH DWRP RI LQWHUHVW� +HUH ZH LQYHVWLJDWH WKH EHKDYLRU RI 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VLQ(") ) T VLQ(!) ] ���� $33(1',; %� '(5,9$7,21 2) 6$' 6$' " µn ! If I0 N ���� :H WKHQ FRQVLGHU ���� DQG ����� \LHOGLQJ� If I0 N = µnµe0+ µnµe0++$ [1! e!(µnµe0++$)L] µe0+ µe0++$ [1! e!(µe0++$)L] = (µe0+ + &)[1! e!(µnµe0++$)L] (µnµe0+ + &)[1! e!(µe0++$)L] & 6$' = µn[1! (µe0+ + &)[1! e!(µnµe0++$)L] (µnµe0+ + &)[1! e!(µe0++$)L] ] ���� �� 20 Protein-Polymer Complex Coacervates as Synthetic Mem- brane-less Organelles Hansen Tjo1*, Nicholas Zervoudis2, Allie C. Obermeyer2 1Department of Chemical Engineering, University of Massachusetts Amherst 2Department of Chemical Engineering, Columbia University in the City of New York COMPLEX COACERVATION, PROTEIN ENGINEERING, SOFT MATTER ABSTRACT: In solution, oppositely-charged macromolecules undergo charge-mediated liquid-liquid phase separation into a complex coacervate phase – a dense, macromolecule-rich liquid. In nature, the basis for intracellular compartmentalization in the formation of membrane-less organelles has been shown to follow similar complexation principles, where charged proteins represent the ionic species. We seek to capture the spatiotemporal tunability properties of such organelles for enzymatic reac- tions in vitro. However, the in vitro formation and deformation of protein-based coacervate microenvironments as a nanoreactor is a limiting factor. Here, we prescribe high-precision turbidimetry coupled with optical microscopy, to characterize the phase behavior of binary protein-polymer complexes between the weak anionic enzyme Glucose Oxidase (GOx) and four different synthetic polycations as functions of composition and ionic strength. Establishment of conditions ideal for coacervate formation in each protein-polymer system informed pH titration experiments on characterizing self-assembly regulation. The results from this study will help inform the design of novel coacervate microenvironments for industrial enzyme cascades and elucidate the role of associative phase separation in cellular evolution. INTRODUCTION Selectivity and specificity are an enzyme’s key characteris- tics [1, 3, 4]. As biological catalysts, enzymes increase rates of reaction such that greater amounts of biological product may be obtained under less time. They also have been used to improve chemical processes in industries from food, agriculture, and petroleum in addition to reducing en- ergy costs and operation time [4]. It is difficult to synthetically match the efficacy and specificity of enzymes as biochemical systems have had millions of years to evolve [4, 16]. While advents in di- rected evolution and protein engineering attempt to circum- vent the time required for natural evolution, amongst other approaches to improve enzyme activity, there is also partic- ular interest to achieve similar goals by optimizing an en- zyme’s surroundings [1, 4, 16]. This strategy is inspired by metabolic reactions, such as those that make up cellular respiration, where the spatiotemporal efficacy of enzymes is enhanced by its surrounding biological environment [1, 4]. With recent reports highlighting the complex functions of biological condensates in vivo (signaling, reaction net- works etc.), we took a biomimetic approach in constructing a stable and responsive enzymatic compartment [1-5]. Us- ing de novo liquid-liquid phase separated synthetic orga- nelles, these microenvironments may be the bridge to ad- vance how enzymatic power is harnessed industrially. Complex coacervation, an example of associative liq- uid-liquid phase separation, describes how oppositely charged polyelectrolytes phase separate into a coacervate phase – a dense, polyelectrolyte-rich phase with potential applications in biomolecular encapsulation – and a dilute phase, the supernatant [1]. Because the coacervate phase compartmentalizes both enzymes and substrates within the same microenvironment, enzymes can perform their cata- lytic functions with greater spatiotemporal efficacy [1-4]. These de novo systems are especially advantageous due to their tuneability by a variety of parameters: pH, charge stoichiometry, ionic strength, mixing order, and others [1, 2]. The use of proteins or other charged biomacromolecules as coacervating macro-ions allows further structural modu- lation through ionic tagging and supercharging [6, 7, 9]. Complex coacervate systems typically involve binary mixtures of oppositely charged components. To simplify coacervate formation, a net charged enzyme is chosen such that it constitutes one of the electrostatic components for complexation [10, 11]. In particular, the weakly anionic en- zyme Glucose Oxidase (GOx) was used in conjunction with four structurally distinct polycations: poly(4-vinyl N- methyl pyridinium iodide) (qP4VP), poly(allylamine hy- drochloride) (PAH), poly(ethyleneimine) (PEI), and poly(1-vinyl imidazole methyl iodide) (PVI). In sum, we sought to elucidate the effects of mixing order, salt concen- tration, and finally pH on the phase behavior of four differ- ent GOx-polycation systems: GOx-qP4VP, GOx-PAH, GOx-PEI, GOx-PVI. 21 METHODS The phase behavior of four sets of binary GOx-polycation systems were studied. The four sets of polymer-protein mix- tures consisted of poly(4-vinyl N-methyl pyridinium iodide) (qP4VP), poly(allylamine hydrochloride) (PAH), polyethyl- enimine (PEI), and poly(1-vinylimidazole methyl iodide) (PVI) as the polycations, with Glucose Oxidase (GOx) as the anionic charged protein. Additionally, we investigated the effects of salt on coacervate formation via the addition of sodium chloride (NaCl). Finally, we utilized pH titrations to explore how complex coacervation can be regulated by so- lution pH. Sample Preparation: Glucose Oxidase from Aspergillus Ni- ger was purchased from Sigma Aldrich (G2133). A stock so- lution of 2 mg/mL GOx was prepared in 10 mM Tris at pH 7.4. Application of Beer’s Law was used to determine true concentrations of GOx via its absorbance at 280 nm in a 4 mL quartz cuvette. Polymer solutions were diluted from liq- uid stocks of 5 mg/mL; relevant stoichiometric calculations using the molar equivalency equation were used to deter- mine the requisite volumes of 10 mM Tris needed to dilute appropriated samples to a mass concentration of 2 mg/mL. Each polycation solution was subsequently adjusted to a pH of 7.4. Mixing Ratios: Four mixing ratios of GOx/polymer were in- vestigated: 88% GOx/12% polymer, 84% GOx/16% poly- mer, 80% GOx/20% polymer, 76% GOx/24% polymer. Such values were determined from preliminary data indicat- ing an optimum mixing ratio range for GOx at roughly 80%. Data results for variations in mixing ratios were obtained through turbidimetry analysis and optical microscopy. To exclude external ionic strength contributions in studying mixing ratio effects, salt species were absent in all mixtures. Salt Effects: The effects of added salt on system phase behavior were examined via turbidimetry analyses and opti- cal microscopy. Sodium chloride (NaCl) concentration was varied from 25 mM to 50 mM on all polymer/protein sys- tems. This salt range was predicted to be conducive for liq- uid-liquid phase separation in GOx-polymer systems based on preliminary work. For the GOx-PEI system, no form of phase separation was observed at all salt concentrations. Thus, further planned investigations on the GOx-PEI system with salts were abandoned. pH Titration: The effects of pH on phase behavior reversi- bility was examined via turbidimetry analysis, based on ab- sorbance readings from an UV-Vis Spectrophotometer, and a pH probe at constant ambient temperature (25 ℃). 1 M Hydrochloric Acid (HCl) was used as the titrant; a 1 cm stir- bar at roughly 500 rpm was used to ensure consistent solu- tion mixing throughout the procedure. Each GOx-polycation system was set at its experimentally-determined optimum mixing ratio and salt concentration: 88% mixing ratio, and 50 mM NaCl for all systems with the exception of GOx- PAH, which was set at 175 mM NaCl. Turbidimetry Analyses: Turbidimetry analyses were done to investigate the effects of mixing ratios and salt concentra- tion. Each sample was prepared in triplicate in tissue culture- treated polystyrene 96-well half-area plates (Corning), fol- lowed by incubation at room temperature for 3 h. Using a plate reader (Tecan Infinite M200 Pro), the absorbance of the mixture was taken at a wavelength (λ = 600 nm) to mon- itor scattering of the phase separated mixture. Each sample had an invariant volume of 50 µL allowing for appropriate absorbance measurements and physical mixing by a Tecan Infinite M200 Pro plate reader (10 s of orbital shaking). Fi- nally, the Absorbance was converted into Turbidity using the following set of relationships: 𝜏 = 100 −%𝑇 (1) %𝑇 = 10(./0) (2) Where: • 𝜏 is the Turbidity of the solution and an indicator of the extent of phase separation present within the sample. • 𝑇 is the Transmittance of the solution as a function of mixture Absorbance. • 𝐴 is the measured Absorbance of the mixture. Optical Microscopy: All four protein-polymer samples were prepared in triplicate, followed by individual well examina- tion with optical microscopy using an EVOS FL Auto 2 in- verted fluorescence microscope (Invitrogen). Each sample, controlled at a volume of 50 μL, were formulated in an opti- cally clear 384-well plate (Nunc) and then underwent 3 h in- cubation period at room temperature to maximize the degree of liquid-liquid phase separation taking place in the wells (preliminary data indicated that samples tended to favor Figure 1. Chemical structures of the four cationic polymers used in this work. Structures were drawn using the ChemDraw Prime software. 22 precipitation following initial mixing before eventual transi- tion into a coacervate phase). All optical microscopy images were taken under 20X objectives with transmitted light. RESULTS AND DISCUSSION Initial experiments investigated the effects of mixing ratio of protein to polymer using GOx and four different polycations (qP4VP, PAH, PEI, PVI). Preliminary data suggested each binary systems’ tendency to phase separate at all four pre- selected mixing ratios; however, certain mixing ratios re- sulted in greater extents of phase separation as indicated by their relative turbidity magnitudes. Nevertheless, this initial assumption did not hold as no phase separation was observed with PEI at all mixing ratios. However, we hypothesized that additions of salt may facilitate phase separation through ex- ternal charge compensations and increasing the total possi- ble conformations of electrostatic associations given the presence of non-polyelectrolyte ions [1, 2, 6]. Thus, PEI was kept for subsequent experiments on the effects of salt addi- tion. Turbidity data as a function of mixing ratio was plotted in such a way that optimum mixing ratios for each GOx-pol- ycation system can be determined. Although ranging in value from 0 – 100%, turbidity is typically employed as a qualitative indicator in characterizing phase behavior: for example, turbidity values above 20% usually suggest (but do not guarantee) the presence of phase separation whereas val- ues nearing 0% imply the absence of phase separation. How- ever, the presence and exact nature of phase separation, be it liquid-liquid or liquid-solid phase separation, can only be verified through optical microscopy. Nevertheless, turbidity is useful as a continuous measure for when a system exhibits the same morphology under different conditions, and there- fore can indicate the conditions most conducive to the de- sired phase behavior. In this paper, for example, turbidity is used to determine mixing ratios that best drives phase sepa- ration. Out of the four mixing ratios investigated, the two mixing ratios most favoring liquid-liquid phase separation will be selected for subsequent investigations involving salt and pH. It was expected that maximum complexation, as deter- mined by peaks in relative turbidity, for a given GOx-poly- cation system would lie around mixing ratios of 84% and 88% (although turbidity values were generally higher at a mixing ratio of 92%, optical microscopy showed greater tendencies for precipitation). Indeed, all GOx-polycation systems except for GOx-PEI exhibited coacervate formation at such mixing ratios as determined via optical microscopy (data not shown). Phase separation absence in GOx-PEI also explains the low turbidity magnitudes (below 20% at all mixing ratios). We predicted the absence of phase separation in the GOx-PEI system to be due to the lack of entropic gains from bound counter-ion release in electrostatic interactions within the system as a result of PEI being too weakly charged. The larger relative sample standard deviations with GOx-PVI and GOx-PEI suggests possible systematic inac- curacies: e.g., bubble formation interference at select mixing ratios, but the consistency of turbidity maximums coupled with optical microscopy images (data not shown) at mixing ratios of 84% and 88% supported the selection of these par- ticular mixing ratios for subsequent experiments. While the quantitative limitations of turbidity must be considered, this may also suggest the dominance of (charged) protein-poly- mers system’s electrostatic interactions driving macro-phase separation on turbidity readings as opposed to the strengths of individual components. Salt Effects Based on the two selected mixing ratios (88% and 84%) from the previous experimental section, the effects of salt (NaCl) concentration on GOx-polycation phase behavior were investigated (Fig. 3). Fig. 3c illustrates the characteristic binodal curve phase diagram in phase separating polyelectrolyte systems [1, 8, 12, 14]. In this investigation, however, we are more con- cerned with determining salt concentrations that promoted liquid-liquid phase separation for the two mixing ratios: 84% and 88%. Turbidimetry analysis was used to provide a rela- tive measure for the degree of phase separation across all GOx-polycation systems and facilitate comparison across the two different salt concentrations. The two chosen salt concentrations were 25 mM and 50 mM based on prelimi- nary data suggesting that this resulted in liquid-liquid phase separation in the GOx-polycation systems being studied. At both mixing ratios of 84% and 88%, all GOx-poly- cations that exhibited liquid-liquid phase separation in the absence of salt (Fig. 2) continued to do so with increasing salt concentration. Although GOx-PVI and GOx-qP4VP systems underwent a decrease in turbidity with the addition of salt from 25 mM to 50 mM at constant mixing ratio of 84% (Fig. 3b), optical microscopy still showed liquid-like morphologies. Given that there were still no indicators of phase separation in GOx-PEI with the addition of salt, as suggested by low turbidity magnitudes below 20% (Figs. 3a, Figure 2. Complex coacervation of Glucose Oxidase (GOx) enzyme with a palette of synthetic polycations. Mixtures were prepared across a select range of macromol- ecule mixing ratios informed from a more thorough inves- tigation of the protein’s phase behavior. Error bars describe the standard deviation of each triplicated data point (n = 3). As a control, all turbidity values shown have had 10 mM Tris turbidity reference values subtracted. 23 3b), and confirmed by optical microscopy, the system was excluded from subsequent experiments on the effects of pH on coacervate formation. As an exception amongst the phase-separating systems, PAH did not demonstrate the desired formation of biomolec- ular condensates at both salt concentrations of 25 mM and 50 mM based on optical microscopy (data not shown). In- stead, its turbidity values consistently above 20% for 25 mM and 50 mM salt concentrations (Figs. 3a, 3b) were shown to be a result of precipitate formation, which furthers evidence for the GOx-PAH system’s greater relative propensity for liquid-solid phase separation at the current salt concentration range. For GOx-PAH, low concentrations of salt ions may not provide sufficient charge compensations to favor liquid- liquid phase separation, so much as it is strengthening elec- trostatic interactions via increasing possible Coulombic- driven conformations. Conversely, at higher salt concentra- tions, its charge screening effects would work towards dampening such electrostatic interactions to reduce the en- tropic gains from bound counter-ion release and drive coac- ervation [2, 6, 12]. An alternative explanation as to the GOx- PAH system’s propensity for liquid-solid phase separation at low salt concentrations could be due to kinetic trapping effects as is prevalent in solid phases [2, 8, 12]. Next, experiments with the GOx-PAH system involving higher salt concentrations were necessary to identify a “min- imum” salt concentration at which the system undergoes complex coacervation instead of precipitation. Thus, we conducted an additional salt titration exploring GOx-PAH phase behavior at higher salt concentrations: 150 mM to 200 mM NaCl at 25 mM intervals. Samples were analyzed via turbidimetry (data not shown) and optical microscopy (Fig. 3d). While the possibility to utilize a different salt as per the Hofmeister series e.g. KBr was considered, ultimately liq- uid-liquid phase separation was observed at a NaCl concen- tration of 150 mM, with the optimum salt concentration for GOx-PAH determined to be 175 mM (Fig. 3d), thereby ne- gating the need to use a different salt species and maintain- ing consistency across all GOx-polycation systems. pH Effects Building on preceding work determining ideal mixing ratio and salt compositions conducive to coacervate formation, the goal of this section is to effectively explore coacervate Figure 3. The phase behaviors of multiple GOx-polycation mixtures as functions of salt (NaCl) concentration. GOx- polycation mixtures were prepared using the two optimum mixing ratios for complex coacervation from Fig. 2 (84% and 88%). Both turbidity (λ = 600 nm) and optical microscopy were used to confirm liquid-liquid phase separation. Error bar values represent sample standard deviation; n = 3 for all data points. a, Turbidity versus salt concentration at constant mixing ratio of 88%. b, Turbidity versus salt concentration at constant mixing ratio of 84%. All turbidity values shown in Figs. 3a and 3b have had 10 mM Tris turbidity reference values subtracted; results from Fig. 2 were also included as no-salt controls. c, Binodal phase boundary of complex coacervate systems where both charged polymers and charged proteins facilitate phase separation; Φ denotes ‘phase’. Arrow points to increasing two-phase region with increasing macromolecular charge density or patterning, demonstrating phasic tuneability with charge-associated parameters. d, Optical microscopy images of GOx-PAH phase behav- ior (88% mixing ratio) provide sufficient qualitative evidence of liquid-liquid phase separation at salt concentration ranges beyond those in Figs. 3a and 3b. 24 self-regulation by pH. All GOx-polycation systems were set at an initial pH of ~9.5 in addition to their optimum compositions (specified in the Experimental Section), and a fixed volume of acid was titrated into the solution to slowly decrease the solution pH (Fig. 4b). Reductions in turbidity as pH decreases marks the dissolution of the coacervate mi- croenvironment. Since GOx is characterized as a weak poly- electrolyte and has an isoelectric point (PI) of roughly ~4.2, protonation of GOx below its PI reduces its net negative charge. Thus, the coacervate microenvironment formed by GOx and a polycation would deform as liquid-liquid phase separation dissipates from weakening electrostatic interac- tions. This is evident from the turbidity of each system being lowest at pH values below 4.2, the isoelectric point of GOx (Fig. 4b). However, the periodicity of the trends, notably with the GOx-PAH and GOx-PVI systems, was unexpected (Fig. 4b). We hypothesize possible induced charging effects on prolonging liquid-liquid phase separation given the une- ven anisotropy and charge patchiness of GOx [5, 8]. It is possible the coacervate microenvironment may persist at some of the lower pH ranges due to such effects, which may explain why each system undergoes a transition towards a turbidity maximum at the same pH value of 6 (indicating GOx as the limiting factor) following a local minimum at pH 8~ due to the initial decrease in pH. Ultimately, we hope to demonstrate pH-mediated regu- lation of our coacervate microenvironments. Throughout the experimental section, we have shown how coacervate micro- compartments can be formed with polymer-enzyme com- plexes. Further, we can tune the interaction strength and morphology via mixing ratio, salt, and pH. Due to many en- zymatic reactions being affected by system pH, demonstrat- ing the microenvironment’s ability to form and dissolve re- versibly in response to pH represents the primary goal of our work on protein-polymer synthetic nanoreactors [4, 16]. It is important to stress that these results are building blocks to- wards this objective. CONCLUSION An enzyme-polymer complex coacervate system was inves- tigated on the basis of its ability to capture the complexities seen in biological condensates. The phase behaviors of mul- tiple GOx-polycation systems as functions of mixing ratios and ionic strength were investigated. We were interested in each GOx-polycation system’s propensity to undergo liquid- liquid phase separation, and the ionic stability of their formed coacervate microenvironments. GOx phase sepa- rated upon mixing with the polycations qP4VP, PAH, and PVI; but did not phase separate with PEI. For the GOx-PAH system, the addition of salt was needed to screen existing charge such that the entropic gains favoring liquid-solid phase separation may be suppressed. The GOx-PEI system did not undergo any type of phase separation both with and without the addition of salt. It is also worth remarking that the same species of salt, NaCl, may be used to induce liquid- liquid phase separation for all phase separating GOx-poly- cation systems, suggesting the salt species’ versatility for driving complexation. pH titrations were used to investigate the dynamics of the formed coacervate microenvironments for each GOx-polycation system; turbidimetry suggests that all GOx-polycation systems that phase separated at a pH of ~9.5 no longer phase separated upon reaching pH < PI of GOx. We suggested the possibility of induced-charging ef- fects as an explanation for the periodicity in phase behavior of GOx-PVI as pH decreases, where the anisotropy of GOx and presence of charge patches prolonged phase separation despite non-ideal pH [5, 10, 11]. Nevertheless, while the ex- act phase behavior of GOx-polycation systems at low pH were not determined as with optical microscopy, due to methodology limitations, the global minimums in turbidity at low pH compared to relative maximums at high pH strongly suggested dissolution of coacervates and dissipa- tion of liquid-liquid phase separation. This crucial finding will inform future work on demonstrating the reversibility of coacervate formation in GOx-polycation systems such that these microenvironments can be made smart and self-regu- lated to mimic the complexity of condensates found in cells. The establishment of ideal parameters at which various GOx-polycation systems undergo liquid-liquid phase sepa- ration provides a foundation for coacervate microenviron- ment formation for use in advanced synthetic nanoreactor design and elucidation of cellular compartmentalization phe- nomena. Figure 4. The effects of pH on complex coacervation in multiple GOx-polycation systems. Each GOx-polycation mixture was set at pH of roughly 9.5, followed by titration of hydrochloric acid (HCl) until dissolution of complexation. Turbidity (λ = 600 nm) was used to indicate absence of phase separation upon convergence towards a minimum value after multiple pH titrates. a, Schematic depicting pH-induced dissolution of complex coacervation via protonation of GOx, which reduces its net negative charge. b, Measured turbidity of GOx-polycation systems as a function of pH. 5th order polynomials were plotted to visualize turbidity trends with pH and to guide the eye (a 5th order polynomial represented the lowest-degree polynomial containing the local extremas that track the data) 25 AUTHOR INFORMATION Corresponding Author *htjo@umass.edu Author Contributions Hansen Tjo performed the experiments, analyzed data, and produced the figures and the manuscript. Nick Zervoudis devised the experimental plan and reviewed the manu- script. Dr. Allie Obermeyer reviewed the manuscript. Funding Sources This work was supported by the Fu Foundation School of Engineering and Applied Sciences at Columbia University and funding from the Columbia Chemical Engineering REU program. ACKNOWLEDGMENTS The authors would like to acknowledge members of the Obermeyer Group for helpful discussions. ABBREVIATIONS GOx – Glucose Oxidase HCl – Hydrochloric Acid NaCl – Sodium Chloride PAH – Poly(allylamine hydrocholoride) PEI – Poly(ethylenimine) PVI – Poly(1-vinyl imidazole methyl iodide) PI – Isoelectric Point qP4VP – Poly(4-vinyl N-methyl pyridinium iodide) REFERENCES [1] L.P. Bergeron-Sandoval, N. Safaee, S.W. Michnick, Mechanisms and Consequences of Macromolecular Phase Separation. Cell 165(5),1067–1079 (2016). [2] W.C. Blocher, S.L. Perry, Complex coacervate-based materials for biomedicine. Wiley Interdisciplinary Re- views: Nanomedicine and Nanobiotechnology 9(4), doi:10.1002/wnan.1442 (2016). [3] M. Castellana, M. Z. Wilson, Y. Xu, P. Joshi, I. M. Cristea, J. D. Rabinowitz, Z. 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Priftis, N. Laugel, M. Tirrell, Thermodynamic Char- acterization of Polypeptide Complex Coacervation. Langmuir 28(45), 15947–15957 (2012). [16] E. Ricca, B. Brucher, J.H. Schrittwieser, Multi-Enzy- matic Cascade Reactions: Overview and Perspectives. Advanced Synthesis & Catalysis 353(13), 2239–2262 (2011). 26 I’m Upset, not Ashamed: An Investigation into Adolescent Shame Acknowledgement Olivia Putnam1 , Keely Lake2 1Department of Psychology, Northwestern University, Evanston, IL 2Wayland Academy, Beaver Dam, WI SHAME, EMOTION, PSYCHOLOGY, NEGATIVE AFFECT, MOOD DISORDER, SELF-CONSCIOUS EMOTION ABSTRACT: Shame is a powerful and acutely painful “master emotion” that is strongly correlated with maladaptive behav- iors and a host of psychological symptoms. Concerningly, the affect remains under-researched and difficult to identify or address in a clinical setting. This may be caused, at least in part, by shame’s intrinsically hidden nature, which drives people to deny the emotion and express it through other means. This study aimed to understand the degree to which people fail to acknowledge their own shame and the psychological and behavioral implications of this shame. Participants completed both a self-report measure of shame and an empirical assessment of internalized shame, as well as measures of shame coping methods and emotional regulation. As expected, results showed no significant correlation between participants’ self-rated shame and measured shame. We also saw a significant correlation between assessed internalized shame and use of shame- coping methods as well as difficulty in emotion regulation – specifically, difficulty with clarity of emotion, acceptance of emotions, and strategies for coping with emotions. These findings indicate that people struggle to acknowledge their own shame and also speak to the maladaptive, dysregulated ways people manage their shame. Recognizing shame as a powerful emotion with implications in psychiatric disorders and understanding the factors that prevent people from acknowledging their own shame may help improve treatment for those who struggle with the emotion and reduce the likelihood that they will engage in maladaptive coping behaviors. INTRODUCTION Shame is one of the most under-researched emotions. Whereas emotions like sadness, anger, and nervousness have undergone decades of psychological research and have come to be seen as the underlying feelings behind widespread clinical diagnoses like Anxiety or Depression, shame research within the field of psychology was virtu- ally nonexistent until the last two decades and remains remarkably limited today. This lack of research is particu- larly troubling when it comes to shame because the affect is highly maladaptive. Unsurprisingly, as a result, it is highly correlated with addiction, depression, violence, aggression, bullying, suicide, and eating disorders. In recent years, it has also become an emerging component of PTSD (Taylor, 2015). A Brief History of Shame Conceptualization One especially influential early premise for defining shame, and differentiating it from guilt, is early anthro- pologists’ focus on public vs. private transgressions (e.g., Benedict, 1946). Anthropologists commonly distin- guished shame based on the situations they believed elic- ited it. More specifically, shame was conceived as a "pub- lic" emotion, arising from public exposure and disapprov- al of some transgression in societal rules and norms. Guilt, on the other hand, was described as a more "pri- vate" experience arising from self-inflicted criticism and regret. However, more recent empirical research has failed to support this public/private distinction (Tangney, Marschall, Rosenberg, Barlow & Wagner, 1994; Tangney, Miller, Flicker & Barlow, 1996). One such ex- ample is a study conducted in 1992 asking participants to describe three guilt-inducing events and three shame- inducing events. A systematic analysis of the social con- text of these events found that shame and guilt are equally likely to be experienced in the presence of others (Tangney, et al., 1992). "Solitary" shame experiences were equally as common as "solitary" guilt experiences. Even more to the point, “the frequency with which others were aware of the respondents' behavior did not vary as a function of shame and guilt” (Tracy, 2011). This led to a new conceptualization of shame which remains today: shame as holistic negative self-concept. Helen Block Lewis, renowned psychologist and pioneer of this shame-understanding, asserted that while guilt involves a negative evaluation of a specific behavior, shame involves a negative evaluation of the global self ("I did something bad" vs. "I am bad") (1971). Though the distinction may appear inconsequential, this contrasting emphasis on the self “sets the stage for very different emotional experiences and very different patterns of motivation `and subsequent behavior” (Tracy, 2011). The two emotions, for instance, produce distinct * 27 “action tendencies.” Shame is commonly accompanied by attempts to deny, hide from, or escape the experiences that elicit shame, while guilt typically leads to “reparative action” – confessing, apologizing, undoing. This difference in internal conceptualization of the self and subsequent “action tendencies” is part of what ultimately makes shame maladaptive. While guilt can be painful and overwhelming, it is generally limited to the guilt-inducing action or experience. Shame consumes the entire self, leaving experiencers with a globally-negative self-conception (“I am a terrible person”). This negative self-concept is not only painful and distressing, but it also feels irreparable. A person can correct a behavior, but one’s fundamental essence seems permanent. This sense of futility drives much of shame’s maladaptivity. Rather than embracing adaptive behaviors like apologies or changes in behavior, which increase psychosocial suc- cess, shame-experiencers tend to recede and hide from the shame-inducing event. Often, this leads people to isolate themselves socially, withdraw from activities that poten- tially remind them of the shame, and engage in anhedon- ic-behaviors. In other cases, this avoidance manifests as anger or hostility, as experiencers attempt to “turn the tables” on others to avoid their own shame, or as risky behavior (i.e. substance abuse) which many use a distrac- tion from their shame (Ellison, 2006). In instances where shame-experiencers have in fact committed some wrongdoing (the determination of which is of course subjective) the failure to take the expected “reparative action” can lead to social conflict (Tangney, Stuewig & Mashek, 2007). For those who experience shame about events for which reparative action is not typically expected (i.e. being the victim of sexual assault, mental illness, minor mistakes or failures), shame can cause dissociation, debilitate people from talking about their experience, and limit much-needed processing of their own emotions and/or trauma (Taylor, 2015). Either way, these maladaptive responses to shame led research- ers to “consistently report a positive relationship between proneness to shame and a host of psychological symp- toms, including depression, generalized anxiety and social anxiety, low self-esteem, PTSD, eating disorder symp- toms, Cluster C personality disorders, suicidal behavior and self-injurious behavior, and substance abuse” (Tracy, 2011). Shame Acknowledgement In the context of shame’s maladaptive consequences, the lack of research into the affect becomes dangerous. With- out comprehensive research into shame and its implica- tions in psychological disorders, we cannot develop evi- dence-based treatments for shame-related disorders or, more importantly, adjust treatments for preexisting men- tal health disorders in which shame plays a more im- portant role than previously-realized. One roadblock that commonly hinders the develop- ment of these treatments or the confrontation of shame within a therapy setting is the fact that shame often goes unacknowledged by the experiencer (McGonigal, 2016). As Terry F. Taylor Ph.D. writes in a review article of peritraumatic shame, “Shame...is a virtually invisible, ubiquitous part of everyday life. Because the experience of shame is often considered to be painful and disempow- ering, and because recognition of shame in itself can be felt as shameful… shame remains unacknowledged and is expressed as avoidant behavior” (2015). This instinct to hide one’s shame “makes it difficult to recognize internal- ly when it happens,” let alone acknowledge out loud (Luoma, 2012). Concerningly, this tendency among peo- ple not to acknowledge their own shame also makes it difficult to study the affect, as it renders self-report measures unreliable. Present Study The consensus that shame characteristically goes unrec- ognized has never been scientifically reviewed. Further, researchers have not studied whether the degree to which people report their own shame correlates with their men- tal health in other capacities. Our research attempts to fill this gap. Like most shame-studies before it, we utilize assessments intended to empirically measure participants’ levels of shame. In addition to these assessments, howev- er, we also use an assessment of emotional affect that asks participants to self-rate the frequency with which they experience different emotions, “ashamed” being one them. Comparisons of participants’ scores on the shame assessments and their self-reported level of shame will act as quantified measurements of how well they acknowledge their own shame. This research will not only test the assumption of shame’s unidentified nature but may also provide some insight into the prevalence of unacknowledged shame and how it affects people’s mental health. METHODS Participants and Procedure The assessments were administered to a sample of 54 adolescent students between the ages of 14 and 17 (58% female, 42% male, M = 16.3 years old, SD = 1.3, 55% American). Participants were recruited from the student population at Wayland Academy, a small boarding high school with international students. As an incentive for participation, students were offered a small amount of extra credit in their science and math classes. Participants were each given a battery of psycho- logical assessments including the Positive and Negative Affect Scale (PANAS), the Internalized Shame Scale (ISS), the Compass of Shame Scale (CSS), and the Diffi- culty in Emotion Regulation Scale (DERS). Participants were assured that their responses would be anonymous and confidential. Assessments were given in a quiet, dis- traction free room. Assessments Positive and Negative Affect Scale: (PANAS; Watson and Clarke, 1998) The most commonly used measure of affect in scholarly research, the PANAS is comprised of 10 negative affects (afraid, upset, distressed, jittery, nerv- ous, ashamed, guilty, irritable, hostile) and 10 positive affects (enthusiastic, interested, determined, excited, in- 28 spired, alert, active, strong, proud, attentive). Participants use a 0-4 Likert scale to rate the frequency with which they tend to experience each affect. For the purposes of this research, the “ashamed” item was used to measure self-rated shame because, unlike empirical assessments of shame, it requires participants to explicitly endorse the word “ashamed.” Internalized Shame Scale: (ISS; Rosario and White, 2006) The Internalized Shame Scale, a 30-item question- naire, is the most widely used empirical measure of shame across psychology and sociology research. The assessment has two subscales that are intended to be re- ported separately: a 24-item shame scale and a 6-item self-esteem scale. The shame scale attempts to tease apart different experiences of shame to create a holistic meas- ure of the affect. Importantly, the ISS does not actually use the word shame, because shame can itself be a shame- ful thing to admit. There are four identified cutoffs: a score of 50 or higher indicates problematic levels of shame, a score of 60 or higher indicates possible depres- sion and/or other emotional or behavioral problems, a score of 70 or higher indicates a high probability of de- pression and/or other emotional or behavioral problems. Compass of Shame Scale: (CSS; Ellison, 2006) Because shame is an emotion that commonly goes unacknowl- edged both internally and outwardly, it is often expressed through other emotions or behaviors. The Compass of Shame Scale recognizes this tendency and assesses the maladaptive ways people cope with shame. The four cop- ing methods that it identifies are “Attack Self,” “Attack Others,” “Withdraw,” and “Avoid.” This scale is of par- ticular importance because it acknowledges shame as a fundamental source for many other maladaptive, un- healthy behaviors and is the first of its kind to assess and quantify these shame-based behaviors. Additionally, the test questions are situational rather than experiential. Par- ticipants cannot always recognize feelings as shame, but they can often identify situations that produce those shame feelings. Difficulty in Emotional Regulation Scale: (Gratz, 2004) This assessment represents one of the most popular, com- prehensive and well-established measures of emotion regulation and is widely used in both clinical and nonclin- ical settings. The questionnaire assesses five primary components of emotional regulation: emotional aware- ness, emotional clarity, emotional acceptance, impulse control, ability to engage in goal-directed behavior while experiencing negative emotions, and ability to use situa- tionally appropriate emotion regulation strategies flexibly to modulate emotional responses as desired. RESULTS Self-Rated Shame vs. Measured Shame To investigate the relationship between self-rated shame and measured shame, Pearson correlations were comput- ed between ratings from the PANAS item “ashamed” and scores from the Internalized Shame Scale. As expected, there was no significant correlation between self-rated shame and assessed “true” shame. The correlation and significance are displayed in Figure 1. Figure 1. Pearson correlation between PANAS “ashamed” score and Internalized Shame Score. This graph shows the Pearson correlation between PANAS “ashamed” rating and ISS scores, r = 0.0806, p > 0.05 To visualize how shame acknowledgment relates to shame’s maladaptivity, an ANOVA test for the signifi- cance of differences in mean PANAS “ashamed” ratings among internalized shame cutoff groups – shame w/in normal limits, problematic levels of shame, possible indi- cator of mental health disorders, and likely indicator of depression/mental health disorder -- was performed and is displayed in Figure 2. An ANOVA test for the differences in PANAS “ashamed” ratings between ISS cutoff groups was com- puted and is displayed in Figure 2. Figure 2. Analysis of Variance test of PANAS “ashamed” score by ISS cutoff groups. This graph shows the mean PANAS ashamed ratings reported by individuals in different ISS cutoff groups, f ratio = 0.903, p > 0.05 As expected, and in line with our other findings, there was no significant difference in self-rated shame (PANAS “ashamed”) between among ISS cutoff groupings (i.e. all participants reported similar levels of sham, no matter how much shame an individual was actually experienc- ing). 29 Identifying Shame as Negative Affect In order to explore whether participants may identify their internalized shame as other negative emotions, Pearson correlations were computed between PANAS Negative Affect item scores and Internalized Shame scale scores. Both are displayed in table 1. Table 1. Pearson correlations between PANAS nega- tive affect subscores and Internalized Shame Scale score. This table shows the results of Pearson correla- tions between various PANAS negative affect subscores and ISS score. There was also a significant positive correlation be- tween PANAS Negative Affect Subscale total score and Internalized Shame score (r = 0.534, p < 0.01). Implications of Shame To investigate shame’s relationship with other emotional and behavioral problems, Pearson correlations were com- puted between ISS scores and scores on the individual Compass of Shame scales and the Difficulty in Emotion Regulation Scale (Figure 3). Strong correlations between the ISS and the With- drawal and Attack Self scales were expected and ob- tained. Both correlations were significantly stronger than the ISS correlations with the Avoidance and Attack Other scales. All correlations were significant. Category Differences Male/Female, Age, and Continent of Origin differences were assessed for both the Internalized Shame Scale and the PANAS “ashamed” rating. Women tended to have higher Internalized Shame scores than men (t = 1.76, p < 0.05). There was no significant difference, however, be- tween men and women for the PANAS “ashamed” rating. There was also no significant correlation between age and Internalized Shame Score or the PANAS “ashamed” rat- ing. Continent of origin did not produce any significant differences in ISS score or PANAS “ashamed.” Figure 3. Pearson correlations between CSS sub- scores and ISS Scores. a, Pearson correlation between CSS attack self subscore and ISS score, r = 0 .75, p < 0.001 b, Pearson correlation between CSS withdraw subscore and ISS score, r = 0 .72, p < 0.001 c, Pearson correlation between CSS avoidance subscore and ISS score, r = 0.41, p < 0.01 d, Pearson correlation between CSS attack others subscore and ISS score, r = 0.27, p < 0.05 30 DISCUSSION The lack of correlation between ISS score and PANAS “ashamed” rating, as displayed in Figure 1, indicates that there is no relationship between self-rated shame and em- pirically-measured “true” shame. This is further demon- strated by the analysis of variance displayed in Figure 2, which shows that there is no significant difference in self- rated shame between cutoff categories of “true” ISS shame. Essentially, even those who experience shame at an intensity high enough to indicate depression or other mental health disorders tended to describe their experi- ence of shame as “rare.” A paired t-test comparing aver- age ISS shame and PANAS shame affirmed this underre- porting phenomenon in individuals, with nearly 74 per- cent of participants reporting lower self-rated PANAS shame than “true” ISS shame. Importantly, there was a significant positive correla- tion between ISS score and PANAS Negative Affect sub- scale score, suggesting that while people struggle to accu- rately identify their shame, they may describe it broadly as negative affect. This point is reiterated by the fact that, with the exception of “distressed,” the individual PANAS negative affect items had weak or insignificant correla- tions with ISS score and none had stronger correlations than the Negative Affect subscale score. This indicates that people are not calling shame by another name, but instead use negative umbrella terms, or a variety of dif- ferent emotion words, to imprecisely describe the feeling. These results both confirm a common understanding that shame often goes unacknowledged and also underline the difficulty of identifying and properly addressing shame. This pattern becomes especially meaningful in the context of internalized shame’s negative implications. ISS score was found to have positive correlations with all CSS scales: attack self, withdraw, avoid, and attack oth- ers. Given their naturally internalized nature, the “with- draw” and “attack self” coping mechanisms had a strong- er relationship with internalized shame than “avoid” or “attack other.” Internalized shame also showed a strong positive relationship with DERS score and with the lack- of-clarity, non-acceptance, and strategies subscales spe- cifically. This fits well with the findings about unacknowledged shame, as clarity and acceptance are both components of emotional acknowledgment, and are necessary for strategic management of one’s emotions. The male/female differences on the ISS reflect those of previous studies, with women tending to experience more internalized shame than men. Also similar to adult studies, age was not a significant factor in ISS score, sug- gesting that experience of shame does not change signifi- cantly during adolescent development. Given that this comes from a cross-sectional review rather than a longi- tudinal one, however, the accuracy of this conclusion is limited. Continent of origin, which has never before been studied in relationship to internalized shame, produced no significant difference. The accuracy of this conclusion, however, may be compromised by the small and varying number of participants within each continent group. CONCLUSION The comparisons of self-rated shame and empirically measured “real” shame confirm both our hypothesis and a larger long-held public understanding that people hesitate to acknowledge their shame. This finding is important not only because it is the first of its kind to quantitatively validate that informal understanding, but, more signifi- cantly, because it speaks to the extent of the disparity. More than one third of participants experienced internal- ized-shame with a frequency associated with depression and other clinical disorders, yet the great majority of these participants rated their own experience of shame as rare. This is problematic because, if people cannot acknowledge shame as a component of their emotional distress or mental illness, then these problems become much more difficult to address within a clinical or in- trapersonal context. This lack of acknowledgment be- comes especially concerning if the shame centers around a specific, potentially-traumatic event (i.e. sexual assault). If shame prevents a person from speaking up about and working through such an experience, then symptoms can worsen dramatically. As time goes on, this can also be- come a self-fulfilling prophecy of sorts because the longer one avoids their shame the more internalized it becomes and, consequently, the more inhibiting it becomes. Given that shame is a powerful emotion, however, it cannot be entirely ignored or suppressed. In fact, this study’s findings indicate that people may be able to rec- ognize the emotion broadly as emotional distress. How- ever, without specific identification and management, shame is often expressed through maladaptive coping mechanisms. The correlation between internalized shame and each pole of the shame-coping scale suggests that, rather than addressing shame head on, shame- experiencers tend to avoid the emotion through problem- atic behaviors such as: risk-taking and distraction, which can develop into substance abuse and have been shown to be severely maladaptive; withdrawal from social interac- tion, often a symptom of depression; excessive self- criticism, also related to mental illness; or attacking oth- ers, an instinct associated with aggression and potential violence. Though they vary in commonality, each pole is maladaptive in its own right—an attempt to ignore, hide from, wallow in, or push back one’s shame without ever truly acknowledging it. It is also important to note that these individuals coping mechanisms are not orthogonal and in fact tend to converge. This tendency to cope with shame is similarly reflect- ed by the significant correlation between internalized shame and difficulty in emotional regulation. Even more to the point, the DERS subscales that had the strongest relationships with shame were lack-of-clarity, non- acceptance, and strategies. This indicates that people with high levels of internalized shame have significant trouble identifying their emotions, acknowledging their emotions without guilt or embarrassment, and coping with their emotions effectively. This not only speaks to the trou- bling ways people manage their shame but also to the dysregulatory nature of shame itself, which can make it 31 more difficult for a person to manage any of their emo- tions. The implication of these findings is three-fold: tools for shame recognition must be better integrated into both clinical and intrapersonal settings, shame must be better accounted for within diagnostic criteria, and more re- search into the affect must be conducted. It is imperative that mental health practitioners recognize that patients are unlikely to forthrightly acknowledge or report their shame and that these practitioners are trained in how to identify this underlying shame and address it without causing the patient to shut down or react with anger. It is also an un- fortunate truth that, because the perception of shame in others “can also evoke a discomforting emotion in the observer,” it may fail to be addressed in therapy, with the therapist remaining in an “unconscious collusion with the patient” to ignore the shame (Taylor, 2015). Additional shame-specific training and wider acknowledgement of the emotion’s role in psychopathology may help mediate this problem An important step in helping clinicians (and, in fact, any individual) better identify and work with shame is better accounting for the emotion in diagnostic criteria. Despite the fact that the emotion is strongly correlated with many different mental illnesses and social-emotional problems, shame is rarely listed as a symptom in the DSM IV and is generally relegated to the “associated fea- tures” of a disorder. Not only does this lack of representa- tion reinforce shame’s hidden nature, but it also fails to account for the way shame’s role in a disorder can change the way it must be treated. Perhaps most importantly, more scientific research must be conducted into shame. Since shame’s psycholog- ical conceptualization as holistic self-blame was estab- lished, only a handful of scientific studies into the affect have been conducted and, even fewer yet have investigat- ed shame’s role in psychiatric disorders. If we are to help people recognize and cope with their internalized shame, we must first understand what prevents that acknowledg- ment and which tools are the most effective in fostering it. AUTHOR INFORMATION Department of Psychology, Northwestern University Evanston, IL, USA REFERENCES [1] H. L. Bash, A. Papa, Shame and PTSD symp- toms. Psychological Trauma: Theory, Research, Practice, and Policy. 6(2), 159–166 (2014). [2] R. 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