bluuebunny/crossref_metadata_2025 huggingface dataset:
Beneficial effects of imidazoline compounds in a rat model of type 2 diabetes (1142.1)
Authors: Edwards Lincoln , Manaenko Anatol , Crane Louis , Baziard‐Mouysset Genevieve , Vann Nicholas , Corbin Kathryn , Nunemaker Craig
Published: June 2021

Therapeutic agents capable of reducing both hyperglycemia and hypertension would prove beneficial to type 2 diabetics. The objective of this study is to highlight the beneficial effects of imidazoline compounds S43126 and moxonidine (mox) in the Zucker diabetic fatty rats (ZDR). Control Zucker lean rats (ZLR) and ZDR were treated with S43126 or mox for 2 weeks, and then plasma glucose and blood pressure were measured. The effect of S43126 on insulin release was measured in mouse islets. Time‐course and dose‐response studies were also carried out in cells, followed by Western blotting. S43126 and mox lowered blood glucose in ZDR by 69% and 52% relative to control, but had no significant effect in ZLR. Both drugs significantly reduce blood pressure in ZDR only. S43126 caused a dose‐dependent insulin release of up to 4 fold, only under conditions of high glucose. Mox did not increase insulin release. Cells treated with mox showed an increased phosphorylation of protein kinase B (PKB) by greater than 3 fold, and this effect could be blocked by efaroxan. Cells overexpressing the imidazoline receptor anti‐sera (IRAS) protein showed greater phosphorylation of PKB compared to control, following mox treatment. These data suggest that imidazoline compounds decrease blood glucose by a mechanism involving insulin release, sensitization, or both. These compounds should be developed as agents to treat diabetics with hypertension.

Grant Funding Source: Supported by NIH1R15DK084468‐01 A1
Lipid emulsion rescue therapy blocks AMPK activation and Akt inhibition induced by bupivcaine cardiotoxicity (1154.9)
Authors: Fettiplace Michael , Ripper Richard , Bonini Marcelo , Minshall Richard , Weinberg Guy
Published: June 2021

OBJECTIVE: To assess changes in energy sensing and cell survival pathways both during bupivacaine‐induced cardiac pharmacotoxicity and during the accelerated recovery caused by lipid‐emulsion.

METHODS: Male Sprague‐Dawley rats were anesthetized and instrumented to monitor arterial pressures, carotid flows and to deliver drugs to both jugular veins. Animals were separated into 4‐conditions: no treatment (control), 10mL/kg lipid emulsion (30% Intralipid, Frensius Kabi, Uppsala, Sweden) over 1 minute, 10mg/kg Bupivacaine over 20s, or 10mL/kg Bupivacaine followed 10 seconds later by 10mL/kg lipid emulsion. Animals were sacrificed at four time‐points 0 minutes (for control), 1, 5 & 10 minutes following infusions. Hearts were flash‐frozen and proteins extracted in an anti‐protease, anti‐phosphatase buffer. Following protein quantified for equal loading, the samples were run on a 4‐15% Bis‐Tris gel, transferred to nitrocellulose paper, stained with phosphorylation‐site‐specific antibodies in the AMPK and Akt pathways, exposed to ECL and imaged using light‐sensitive film.

RESULTS: Treatment with bupivacaine caused a rapid activation of low energy sensing pathways and deactivation of cell survival pathways including phosphorylation of AMPK, dephosphorylation of Akt, GSK3β & p70‐s6k. Treatment with lipid emulsion reversed this phosphorylation patterns with dephosphorylation of AMPK, and rephosphorylation of Akt, GSK3b and p70‐s6k. Additionally, by 10‐minutes lipid emulsion treatment inactivated Acetyl‐CoA‐carboxylase via phosphorylation.

CONCLUSIONS: Bupivacaine toxicity rapidly changes energy modulation and proliferation pathways in the heart. Lipid emulsion treatment reverses these changes and restores cell survival pathways in the context of cardiac pharmacotoxicty. These results imply the importance of energy sensing pathways and energy balance during resuscitation.

Grant Funding Source: Supported by American Heart Association Predoctoral Fellowship 13PRE16810063
YAP and TAZ drive matrix stiffness‐dependent fibroblast activation (1180.6)
Authors: Tschumperlin Daniel , Varelas Xaralabos , Liu Fei
Published: June 2021

Fibroblasts actively respond to changes in extracellular matrix mechanical properties, and positive feedback between matrix stiffness and fibroblast activation may play a pivotal role in fibrosis. Microarray analysis of matrix‐stiffness regulated genes in human lung fibroblasts identified significant overrepresentation of transcriptional targets of YAP (yes‐associated protein) and TAZ (transcriptional coactivator with PDZ‐binding motif), consistent with a prominent role for these factors in fibroblast mechanoresponsiveness. Immunofluorescence imaging demonstrated that YAP and TAZ transitioned from primarily nuclear localization on relatively stiff matrices (25.6 kPa) to cytoplasmic on soft matrices (0.4 kPa). RNAi‐mediated knockdown of YAP/TAZ significantly reduced the cell spreading, focal adhesion length, traction forces, proliferation, pro‐fibrotic gene expression and collagen synthesis observed on stiff matrices, but exerted negligible effects on soft matrices. Conditional overexpression of YAP overcame soft matrix limitations on proliferation, while overexpression of either YAP or TAZ promoted pro‐fibrotic gene expression on both soft and stiff matrices. Taken together, these results support a central role for YAP and TAZ in matrix stiffness‐dependent activation of fibroblasts to a proliferative, contractile and matrix synthetic state implicated in the pathogenesis of fibrosis.

Grant Funding Source: Supported by NIH RO1 HL092961
Zinc supplementation improves outcomes associated with traumatic brain injury but does not overcome risks associated with combined ethanol intake (122.4)
Authors: Morris Deborah , Levenson Cathy
Published: June 2021

In addition to the known behavioral and cognitive impairments, including memory deficits, depression, and anxiety associated with traumatic brain injury (TBI), there is an increased risk for new onset heavy weekly drinking, binge drinking, and alcohol‐related problems. Our previously published work has shown that zinc supplementation robustly reduces behavioral deficits associated with TBI. Our objective was to examine the behavioral and cellular outcomes associated with TBI that are complicated by ethanol consumption, as well as the effect of zinc supplementation on these outcomes. Adult male rats were fed a zinc supplemented (180 ppm) or zinc adequate (30 ppm) diet for 4 weeks followed by a moderately‐severe TBI using controlled cortical impact. After injury, rats were given 3 g/kg of ethanol daily for 7 days via gavage. Ethanol intake exacerbated TBI‐induced anxiety‐like and depression‐like behaviors and significantly impaired the efficacy of zinc supplementation. While ethanol did not worsen learning and memory, ethanol intake prevented zinc supplementation from improving Morris water maze performance. Furthermore, the world’s highest field strength magnet (21.1 Tesla) for in vivo MRI, diffusion mapping, and spectroscopy, is being used to examine the mechanisms by which ethanol prevents the efficacy of zinc after TBI.

Grant Funding Source: Supported by U.S. Army MRMC and NIH grant GM081382
Developing research questions and designing studies for your scholarly activities in medical education (14.2)
Authors: Borges Nicole
Published: June 2021

Attendees will be introduced to medical education research and will learn and apply important concepts regarding designing research studies. Attendees will work to identify their scholarship interests, transform everyday work into scholarship, and develop robust research questions by following strategies and principles.

Specific Objectives: At the end of the session, participants will have: 1. Identified their research and scholarship interests 2. Formulated meaningful and feasible questions within those areas
Unraveling the mechanism on how Entamoeba histolytica evokes MUC2 exocytosis (152.3)
Authors: Cornick Steve , Moraeu France , Chadee Kris
Published: June 2021

MUC2 is the major secreted gel‐forming mucin produced by goblet cells that form the protective mucus blanket covering the colonic epithelium. This mucus barrier confers the first line of innate host defence against invasion by the protozoan parasite Entamoeba histolytica (Eh). During Eh infection, a subset of individuals develop infectious colitis when the parasite degrades and/or deplete mucin stores. In this study we quantified the exocytosis SNARE complex that regulated mucin secretion in vitro in response to Eh using human colonic LS174T goblet cells. Unlike MUC2, SNARE components were not transcriptionally regulated or specifically degraded in response to live Eh. Rather, mucin exocytosis occurred through a protein kinase C and calcium dependent manner. Furthermore, we have characterized the Eh surface virulent factor, cysteine protease 5, as the predominant component that mediated mucin secretion. Interestingly, only viable Eh but not heat‐killed and/or glutaraldehyde fixed Eh evoked mucin exocytosis upon contact with colonic cells. These studies were confirmed using live cell confocal microscopy imaging with fluorescent SNAREs and Eh. Predictably, shRNA silencing of a putative SNARE, SNAP23, markedly decreased mucin exocytosis and rendered the colonic monolayer susceptible to Eh degradation. This work sheds light into the pathogenesis of Eh and the regulatory mechanisms of the host which act to subvert infection.

Grant Funding Source: Supported by CIHR and NSERC CREATE HPI
Flipping the neuroanatomy labs: how the production of high quality video and interactive modules changed our approach to teaching (211.3)
Authors: Krebs Claudia , Holman Parker , Bodnar Tamara , Weinberg Joanne , Vogl Wayne
Published: June 2021

Neuroanatomy is often approached with apprehension, often described as “neurophobia”. The result has been a triage approach by students: memorizing as much information as possible to pass the exam, and relegating a deep understanding of CNS systems as they relate to the clinical reality to clinical experiences.

Aware of this reality, we wanted to create content that is accessible and engaging; moreover, we wanted to “flip” the classroom so that students could begin to use class time for knowledge application instead of memorization.

The theory behind a flipped classroom approach is to provide resources to the students to prepare with prior to coming to class, and then use the classroom time for the application of this knowledge to clinical cases and in‐depth discussions about CNS systems.

We created eight highly produced mini‐documentaries to provide conceptual overviews of key brain systems, and 20 interactive modules for more in‐depth didactic content as well as formative assessment for the students. All of these resources are posted online under a Creative Commons license.

A Readiness Assessment Test (RAT) at the beginning of the session gauges student understanding of the material; lab time is then used to address areas of weakness as well as to apply knowledge to clinical cases ‐ a core focus of each lab.

Evidence suggests that this approach can make the classroom experience more engaging for both faculty and students.

Grant Funding Source: Supported by: UBC Flexible Learning Initiative
Antigenic variation by malaria parasites: noncoding RNAs, histone modifiers and RNA pol II (232.2)
Authors: Deitsch Kirk , Ukaegbu Uchechi , Kishore Sandeep
Published: June 2021

P. falciparum malaria parasites invade and ultimately destroy circulating red blood cells (RBCs) causing anemia and disrupted blood flow. As they develop, they place on the RBC surface the primary antigenic determinant, a protein referred to as PfEMP1. The var gene family is a large repertoire of genes encoding different variants of PfEMP1. Parasites only expresses a single var gene at a time while maintaining the remaining members of the family in a transcriptionally silent state. Switches in gene expression result in antigenic variation and allow the parasites to avoid the antibody response. var gene regulation is known to be dependent on specific histone modifications, however how various histone modifiers are specifically recruited to var loci remains a mystery. Our most recent work has focused on the histone modifier PfSET2. This protein is recruited to var loci and deposits the histone mark H3K36me3 at both silent and active var genes. PfSET2 binds directly to the C‐terminal domain of RNA pol II. RNA pol II is known to transcribe non‐coding RNAs from both active and silent var genes, providing a possible mechanism for recruitment of PfSET2 to var loci. Over‐expression of a dominant negative form of PfSET2 designed specifically to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment.
Inositol phosphate regulation of variant surface glycoprotein expression in Trypanosoma brucei (232.3)
Authors: Cestari Igor
Published: June 2021

Trypanosoma brucei evades the host immune system by antigenic variation, expressing only one of >1,000 variant surface glycoprotein (VSG) genes through transcription at one of ~20 subtelomeric expression sites (ESs), thereby producing a homogenous surface coat. These coats vary periodically via VSG gene recombination or transcriptional ES switching, and the mechanisms regulating these processes are unknown. T. brucei has an inositol phosphate pathway encompassing 26 genes for inositol phosphate (IP) and phosphatidylinositol (PI) kinases, phosphatases, and metabolic enzymes. We generated T. brucei bloodstream form (BF) conditional null lines for genes in this pathway and found that PI(4) 5‐kinase and IP3 5‐phosphatase are essential for growth and infection of mice. Conditional repression of PI(4) 5‐kinase or IP3 5‐phosphatase, or overexpression of phospholipase C, resulted in expression of VSGs from multiple ESs, including BF and metacyclic ESs. Expression of procyclins or other genes encoded by RNA polymerase I, II or III were not affected, and repression of other IP pathway genes did not induce VSG expression. Repression of PI(4)5‐kinase or IP3(5)‐phosphatase also caused telomere repositioning in the nucleus; re‐expression of these enzymes generated cells that switched VSGs. Thus, T. brucei possesses an IP pathway which appears to be involved in regulation of VSG expression and antigenic switching.
Genome‐wide approaches to dissecting Leishmania differentiation (232.4)
Authors: Myler Peter , Zilberstein Dan
Published: June 2021

Leishmania donovani are intracellular parasitic protozoa that cause visceral leishmaniasis in humans. During their lifecycle these organisms cycle between the midgut of female sand flies, where they reside as extracellular flagellated promastigotes, and the phagolysosomes of macrophages, where they live as aflagellated amastigotes. Promastigote‐to‐amastigote differentiation can be induced in axenic parasites by exposure to the high temperature and acidity (37°C, pH5.5, 5% CO2) typically found in the phagolysosome. Analyses using high‐coverage transcriptomic and proteomic approaches showed that L. donovani differentiation is well‐regulated, with ordered and coordinated changes in mRNA and protein abundance that result in the parasites retooling their metabolic pathways for life in the new environment. We recently described stage‐specific protein phosphorylation patterns, and have now completed an extensive examination of phosphorylation changes during differentiation. In addition to identifying global changes in protein translation between promastigotes and amastigotes, we are now using the new technology of ribosome profiling to identify gene‐specific changes in translation efficiency between the lifecycle stages. The results of these analyses have begun to reveal the molecular basis of differentiation, including a role for protein kinase A (PKA) in its regulation.
Translesion DNA synthesis: structure and specificity (236.3)
Authors: Aggarwal Aneel
Published: June 2021

The survival of all organisms depends critically on the ability to faithfully replicate DNA. However, cellular DNA is continually subjected to damaging agents such as sunlight and chemical pollutants externally, and oxidation and hydrolysis internally. The replicative DNA polymerases (Pols) are unable to replicate through these lesions, resulting in the stalling of the replication fork. Whether cells can replicate through DNA lesions has been a key question in the areas of DNA replication, mutagenesis, and carcinogenesis. The answer to this longstanding puzzle has come relatively recently with the discovery of a number of translesion DNA synthesis (TLS) polymerases that are related and belong to the Y‐superfamily. I will present some of our structural studies on TLS DNA polymerases, addressing the basis of their selectivity and ability to bypass to specific DNA lesions.
β‐Hydroxy‐β‐methylbutyrate extends lifespan and attenuates age‐dependent loss of flight ability in Drosophila melanogaster (246.2)
Authors: Beattie Alexandra , Garvey Sean , Vigoreaux Jim
Published: June 2021

β‐Hydroxy‐β‐Methylbutyrate (HMB) extends lifespan and attenuates age‐dependent loss of flight ability in Drosophila melanogasterAlexandra Beattie1, Sean M. Garvey2 and Jim O. Vigoreaux11Department of Biology, University of Vermont, Burlington, VT 054052Strategic R&D, Abbott Nutrition, Columbus, OH 43219 Muscle function deteriorates with age. HMB enhances muscle strength in humans and attenuates disease‐induced and disuse atrophy in rodents. We investigated the feasibility of utilizing Drosophila as a model organism to study the biological effects of HMB in muscle. Using flight ability as an index of muscle function, we found that HMB attenuates the age‐dependent decline in muscle function. Male flies fed a diet supplemented with 10 mg/ml HMB (MB10) had higher flight scores than control flies fed a normal diet (MB00) after median age (weeks 4 and 6; p<0.01). Similar results were observed in female flies (weeks 4 and 5; p<0.01), and in the total population of flies (weeks 4 and 6; p<0.01). Significant differences in flight ability were found when comparing male flies fed 5mg/ml HMB (MB05) v 1 mg/ml HMB (MB01) (week 6; p<0.01) and total fly population (week 6; p<0.01). MB10 increased maximum lifespan by ~15% (75 ± 2 v 65 ± 2 days for MB00). The life extension effect was seen in females (12%) and males (17%). MB10 increased the median age of males (42±1 v 38±1 days for MB00, p<0.05) but not of females. A similar lifespan extension effect was seen when comparing MB05 (58±1 days) to MB01 (53 ±1 days; p<0.01); however, there was no difference in the median age between the two groups. We propose that HMB attenuates the age‐dependent decline in flight ability and prolongs lifespan by influencing muscle health. This research was funded by Abbot Laboratories, Inc.
Dietary flavonoid intake and breast cancer risk by hormone receptor status among postmenopausal women in a prospective U.S. cohort (267.7)
Authors: Wang Ying , Gapstur Susan , Gaudet Mia , Peterson Julia , Dwyer Johanna , McCullough Marjorie
Published: June 2021

Flavonoids, concentrated in plant‐based diets, are suggested to lower breast cancer risk in pre‐clinical studies. However epidemiological studies that examined associations of individual flavonoid intakes and breast cancer are limited. We examined seven subclasses of dietary flavonoids with overall breast cancer risk and by estrogen receptor (ER) status in a prospective cohort of postmenopausal women. A total of 56,627 postmenopausal women completed extensive self‐administered questionnaires in 1999‐2000 and were followed through June 30, 2009. During a mean follow‐up period of 8.5 years, 2,114 invasive breast cancers, including 1,498 ER‐positive (ER+) cases and 218 ER‐negative (ER‐) cases were ascertained. Cox proportional hazards regression was used to calculate multivariable‐adjusted relative risks (RRs) and 95% confidence intervals (CIs). Total flavonoid intake was not significantly associated with any breast cancer risk. However, inverse associations were observed for flavones and overall breast cancer risk (P‐trend=0.04), as well as for an increment of 47 milligram of flavan‐3‐ols per day (difference between medians of first and fourth quartile) with ER‐ breast cancer risk (RR: 0.77; 95% CI: 0.62, 0.97). The inverse association for flavan‐3‐ols with ER‐ but not ER+ breast cancer was consistent with research that suggests a beneficial role of plant‐based diets on ER‐ breast cancer risk.
Development of No Más Hambre (No More Hunger), an innovative promoter‐driven nutrition education and skill‐building curriculum to reduce the risk of hunger in Mexican‐origin older adults in Texas border colonias (273.3)
Authors: Bustillos Brenda , Sharkey Joseph , Gomez Melissa
Published: June 2021

Mexican‐origin older adults who live along the Texas‐Mexico border experience high rates of poverty, food insecurity and hunger, and health disparities. Among this hard‐to‐reach population, promotoras de salud (community health workers) serve as a cultural bridge, provide outreach, and are recognized as respected health advocates and peer health educators. The purpose of this study was to design, develop, and pilot test curricula and instructional materials that provide promotoras with knowledge and skill‐building training to target seniors at risk for hunger through tailored and culturally appropriate education and skill building on food acquisition and preparation using community food resources. 95 Mexican‐origin seniors from four Texas border areas and 25 promotoras participated in focus groups to determine educational and skill needs. Emergent themes included desire for home‐based nutrition education and guidance regarding food acquisition and food preparation, given limited resources. No Más Hambre (No More Hunger) consists of seven modules of instruction that engage and empower promotoras to deliver evidence‐based education and skills. This Spanish‐language curriculum was developed and delivered by employing Adult Learning Theory and applying strategies and techniques such as interactive instructional modules and case‐based discussions to guide resource‐dependent education. The curriculum includes strategies to overcome barriers to food insecurity and encourages seniors to engage in collaborative networks to locate resources within the community. Pre‐ and post‐tests comparisons were assessed for each module. This project constitutes a major new effort to enhance nutrition knowledge and skills among Mexican‐origin older adults and to effectively apply the science of human nutrition to meet the needs of this vulnerable and food insecure population.

Grant Funding Source: Supported by AARP Foundation and CDC's PRC Healthy Aging Research Network
The SDF‐1/CXCR4 axis utilizes mTOR signal transduction to promote angiogenesis (278.8)
Authors: Ziegler Mary , Hughes Christopher
Published: June 2021

Pathological angiogenesis, which occurs during chronic inflammation or tumor growth, results when positive and negative angiogenic regulators are disrupted. The chemokine stromal‐cell derived factor‐1 (SDF‐1) and its receptor CXCR4 promote endothelial cell (EC) migration and tube formation. PI3K is a key downstream regulator of CXCR4‐mediated chemotaxis and mammalian target of rapamycin (mTOR) is at the center of PI3K‐mediated signal transduction. We have hypothesized that the SDF‐1/CXCR4/mTOR signaling axis contributes to the molecular mechanism of angiogenesis. This study illustrates that SDF‐1 stimulation activates mTOR signal transduction in EC grown in monolayer cell culture. The pharmacological inhibition of CXCR4 or mTOR blocks sprouting in an in vitro 3D angiogenesis model, and also abolishes EC migration in a 3D angiogenic invasion assay. Specific inhibition of mTOR complex 1 or mTOR complex 2 (mTORC2) reveals that SDF‐1‐induced angiogenesis occurs via mTORC2 signal transduction. An analysis of targets downstream of mTOR indicates that 6‐phosphofructo‐2‐kinase (PFKFB3), which is a key regulator of glycolysis, is downregulated in EC in which mTORC2 is inhibited. Taken together these data indicate that the SDF‐1/CXCR4 axis activates angiogenesis by utilizing an mTORC2 signal transduction pathway. In addition, these data present a potential link between mTORC2, metabolism and angiogenesis, which deserves further exploration.

Grant Funding Source: The project described was supported by Grant Number T32CA009054 from the National Cancer Institute.
How to develop students’ metacognitive skills: lessons learned from an upper level undergraduate anatomy course (336.1)
Authors: O'Loughlin Valerie
Published: June 2021

Metacognition is a term to describe when a person learns how to learn, and becomes more perceptive of one’s learning. Metacognitive techniques may be incorporated in the classroom to help students develop a better perception of what they know. In this session, Valerie O’Loughlin will describe how reflective writing (through the use of online journals or ‘blogs’) in an upper level undergraduate anatomy course helped students refine their metacognitive skills. She will provide examples of how reflective writing may be incorporated in other anatomy courses so students may better gauge their understanding of the complex material.
Using the ubiquitin‐modified proteome to monitor protein synthesis fidelity (353.1)
Authors: Bennett Eric , Webb Kristofor , Zuzow Nathan , Gendron Joshua
Published: June 2021

The ubiquitin‐dependent destruction of misfolded, misassembled, or improperly translated proteins serves as a defense strategy to protect against the deleterious accumulation of defective translation products (DTPs). DTP destruction represents a potential significant burden on protein homeostasis function. Previous studies estimate that between 10‐30% of all newly synthesized proteins undergo UPS‐dependent degradation. The mechanisms that govern mammalian DTP recognition and destruction by the UPS are not well‐characterized. Further, ubiquitin‐targeting of endogenous DTPs has not been directly examined. This is mostly due to difficulties in detection and quantification of low abundance DTPs amongst the comparatively large pool of properly synthesized and folded proteins. We have previously established an immunoaffinity method that specifically captures peptides containing diGlycine (diGly)‐conjugated lysine residues that result from trypsinolysis of ubiquitylated proteins. We used this approach to demonstrate that over 70% of all identified ubiquitylation events require continued protein translation for detection. We have applied these proteomic techniques to directly examine and identify ribosome‐associated ubiquitylation substrates. We have characterized how ubiquitin‐targeting of potential DTPs is altered in response to translational and protein homeostasis stress.
Astaxanthin, a xanthophyll carotenoid, inhibited and reversed the activation of mouse primary hepatic stellate cells via the modulation of histone deacetylases (39.1)
Authors: Yang Yue , Lee Ji‐young
Published: June 2021

Activation of hepatic stellate cells (HSC) is a major event in the development of liver fibrosis. Previously, we have shown that astaxanthin (ASTX), a xanthophyll carotenoid, has a potent anti‐fibrogenic effect in LX‐2 cells, a human HSC cell line. In this study, we investigated if ASTX prevents the activation of quiescent HSC and also facilitates the deactivation of activated, myofibroblast‐like HSC using mouse primary HSC. Incubation of ASTX with quiescent HSC inhibited their activation as evidenced by decreased loss of intracellular lipid droplets, and repressed expression of HSC activation markers, such as α smooth muscle actin (αSMA), at mRNA and protein levels. Moreover, in activated HSC, the addition of ASTX for 2 or 4 d resulted in reappearance of lipid droplets and decreased αSMA expression, suggesting that ASTX reversed HSC activation. As histone deacetylases (HDAC) have a potential role in HSC activation, we screened the effect of ASTX on the expression of all 11 classical HDAC isoforms. Notably, HDAC9 expression was significantly elevated during HSC activation, which was markedly attenuated by ASTX. Taken together, ASTX not only prevented the activation of quiescent HSC but also reversed morphological changes and repressed αSMA expression in activated HSC, possibly via the modulation of HDAC9 expression. Therefore, ASTX may be used for the prevention of liver fibrosis.

Grant Funding Source: USDA AFRI 2012‐67018‐19290
Regulation of the immune response by carbohydrates (469.2)
Authors: Cobb Brian
Published: June 2021

Over the past several decades, Western society has witnessed a marked increase in allergy and autoimmune diseases. This trend has been thought to be caused by a greater number of vaccines coupled with increasingly sterile environments. The hygiene hypothesis posits that these changes limit microbial exposure to a degree that is ultimately pathologic in terms of the development and regulation of the immune system. Within that context, our research has focused on the commensal bacterium Bacteroides fragilis and its capsular polysaccharide PSA. This and other “glycoantigens” are gaining recognition as potent immunomodulatory factors that help to not only direct the maturation of the immune system, but also to limit pathologic inflammation associated with disease. Our findings demonstrate that these molecules stimulate a protective T cell‐dependent response in the gut that can influence peripheral tissues, and that the ability to activate these protective T cells depends upon the regulation of host glycosylation machinery in antigen presenting cells. This presentation will highlight our published and unpublished data that collectively establishes the relationship between host glycosylation and commensal polysaccharide antigens as a critical regulatory axis underlying the hygiene hypothesis and the associated increase in allergy and autoimmunity.
Development and assessment of group problem solving exercises to advance student reasoning skills (531.17)
Authors: Beedle Aaron
Published: June 2021

The development and application of reasoning skills in scientific disciplines is important for student success in undergraduate education. To promote these skills in undergraduates of the new Bachelor in Pharmaceutical Sciences program at the University of Georgia, the required didactic course in human physiology was designed to incorporate six small group problem solving sessions dispersed throughout the course. Sessions were designed for groups of four to five students and used current course topics as a basis for critical thinking exercises. Students were assigned to random groups to work through a series of quests in content areas such as muscle metabolism, the baroreceptor reflex, drug absorption in the GI tract, and autoimmune endocrine disorders. Quests were designed to address skills such as identifying a problem/question, evaluating sources of medical information (bias), understanding limitations of individual studies, and logical application of homeostasis and negative feedback. Group interactions were reinforced by providing unique materials or tasks to individual group members that were necessary for completion of the exercise. Concepts from group sessions were reinforced with individual reflective homework assignments. The efficacy of group sessions was evaluated with pre‐ and post‐course assessments of reasoning skills in the inaugural class of fifteen students. Overall, student responses to problem solving sessions were positive; however, application of the exercises in a larger student population is necessary to better determine their impact on reasoning skills.

Grant Funding Source: Supported by the Lilly Teaching Fellow program, Univ. of Georgia Center for Teaching and Learning
The dynamic chromatin configuration during pituitary differentiation (562.1)
Authors: Ho Yugong , Cooke Nancy , Liebhaber Stephen
Published: June 2021

Pit‐1, a pituitary specific POU homeodomain transcription factor, plays an essential role in the process of differentiation of anterior pituitary. Abolishing the expression of Pit‐1 results in the combined block in the differentiation of somatotropes, lactotropes, and thyrotropes in anterior pituitary. Prior studies have revealed that the initial activation of Pit‐1 at embryonic day 14 (E14) in the developing mouse pituitary. This activation is mediated by the binding of transcription factor Atbf1 at the ‘early enhancer’ of Pit‐1, located ‐5.8 kb upstream of the Pit‐1 promoter, and by concurrent binding of Prop‐1 at the promoter. The maintenance phase of Pit‐1 expression is initiated at E16.5 by an auto‐regulatory mechanism in which Pit‐1 binds to its own promoter and to a distal enhancer (‘late enhancer’) located 10 kb upstream of the Pit‐1 promoter. Despite the identification of these regulatory elements, the accompanying changes in chromatin architecture at the Pit‐1 locus during the pituitary development remains undefined. In the current study, we investigate the chromatin configuration of the Pit‐1 locus at different developmental stages. We detect acetylation that is restricted to the early enhancer and the promoter at E14.5 while acetylation spreads throughout the entire locus at the adult stage. Our 3C assays reveal that the early enhancer is positioned proximal to the Pit‐1 promoter while in the maintenance phase is accompanied by a switch in the locus conformation, with positioning of the distal enhancer in proximity to the promoter. Furthermore, our studies suggest that the interaction between the distal enhancer and the promoter is mediated by Pit‐1 and CBP in differentiated pituitary. Taken together, our studies demonstrate that the regulation of Pit‐1 expression is accompanied by the dynamic alternation of the chromatin conformation during the pituitary differentiation and the chromatin architectures play important roles in activation and maintenance of gene expression.

Grant Funding Source: Supported by University Research foundation of University of Pennsylvania
Biogenesis of mitochondrial metabolite carrier proteins in Trypanosoma brucei (574.1)
Authors: Smith Joseph , Chaudhuri Minu
Published: June 2021

Trypanosoma brucei, a parasitic protozoan and the causative agent of human African trypanosomiasis, possesses a single mitochondrion in which many essential metabolic processes occur. These biological processes require various metabolites to be fluxed in and out of the mitochondrial matrix by specialized transporters called metabolite carrier proteins (MCPs). Metabolite carriers are encoded in the nuclear genome and post‐translationally imported into mitochondria. In yeast and humans, there is a dedicated inner membrane protein translocase, TIM22, and soluble small Tim proteins of the intermembrane space, which are involved in MCP biogenesis. Trypanosoma brucei possesses a large family of MCPs; however, the parasite appears to lack the TIM22 translocase in its entirety. A few putative homologs of soluble Tims have been identified in this parasite, although functions of these proteins are not elucidated. Here, we begin to characterize the T. brucei homologs of the small Tim proteins (TbTim9, TbTim10, and TbTim13/8 that shows similarity to both Tim8 and Tim13 in higher eukaryotes) and their possible involvement in metabolite carrier import. We found that TbTim9, TbTim10, and TbTim13/8 are expressed and localized in mitochondria in T. brucei. Knockdown of TbTim9 and TbTim10 individually decreased cell growth, while knockdown of TbTim13/8 ceased cell growth within 5 days. Reducing the expression of these proteins also reduced the steady‐state levels of the metabolite carrier protein, TbMCP5, suggesting that these TbTims are likely involved in MCP biogenesis. Further experiments are now ongoing to understand the roles of TbTims in the import of TbMCP5 into the mitochondrial inner membrane and also to identify the inner membrane translocase involved in this process.

Grant Funding Source: Supported by 2SC1GM081146 and 2R25GM059994
The study of the impact of genetic polymorphism of aromatase (CYP19) enzyme and the susceptibility to polycystic ovary syndrome (575.5)
Authors: Hemimi Neveen , Shaafie Ishtiyaq , Alshawa Haya
Published: June 2021

Abstract
Background: Aromatase is a key steroidogenic enzyme that catalyzes the conversion of androgens to estrogen. Several studies reported the association of the SNP 50 (rs2414096) in aromatase enzyme with hyperandrogenism.

Aim: To investigate the association of genetic polymorphism of aromatase enzyme with polycystic ovary syndrome (PCOS).

Material and Method: The study consisted of 62 women diagnosed with PCOS and 60 healthy women as a control group. Individuals were genotyped for rs2414096 of aromatase enzyme by using polymerase chain reaction‐ restriction fragment length polymorphism. Statistical analysis was done by SPSS program.

Results: Frequency of (GG) and (GA&AA) genotypes were 64.5% and 35.5% in PCOS group compared to 76.7% and 23.3% in control group. Statistical analysis demonstrated that carriers of (GA&AA) genotype were at higher risk for PCOS compared to carriers of (GG) genotype (OR= 1.81, 95%CI= 0.8‐3.99). Carrier of A allele were also at higher risk of PCOS compared to carrier of G allele (OR=1.3, 95% CI= 0.7‐ 2.3).

Conclusion: Polymorphism of rs 2414096 in CYP19 is associated with the pathogenesis of PCOS. Key word: Aromatase, Polymorphism, PCOS.
Single molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae (602.1)
Authors: Gambin Yann , Sierecki Emma , Polinkovsky Mark , Alexandrov Kirill , Parton Robert
Published: June 2021

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae.

Here we have developed new methods that allow reconstitution of the cavin complex and performed a quantitative assessment of cavin complex formation. We use single molecule fluorescence for its proven ability to directly observe multiple populations and quantify interactions in complex mixtures. These techniques are especially well suited for the study of coat proteins and have been used to study the mechanisms of clathrin assembly/disassembly. Those studies required labeling of recombinantly expressed purified proteins, which in the case of the cavin complex is difficult. We have taken an alternative approach to obtain the stoichiometry of the cavin complex and the interactions between the members of the cavin family (cavin1, cavin2, cavin3) directly from cell extracts. Instead of relying on organic dyes, we could perform the full quantitative analysis using genetically encoded fluorophores, shortcutting purification steps.

We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. We could observe fascinating behavior of the cavin members, with exquisite segregation of interactors and defined stoichiometries in mixed oligomers. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1‐cavin2 and cavin1‐cavin3 subcomplexes. Each of these subcomplexes contain 9±2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.
Arf guanine nucleotide‐exchange factors BIG1 and BIG2 regulate β‐catenin signaling via direct interaction and cAMP/PKA pathway (604.1)
Authors: Le Kang , Li Chun‐Chun , Moss Joel , Vaughan Martha
Published: June 2021

BIG1 and BIG2, brefeldin A‐inhibited guanine nucleotide‐exchange factors (GEFs) that activate ADP‐ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP, contain A kinase‐anchoring protein (AKAP) sequences that act as scaffolds for multimolecular complexes to facilitate and limit intracellular cAMP signaling in both time and space. β‐Catenin, with its critical roles in cell‐cell adhesion and Wnt signaling, was among HeLa cell proteins co‐immunoprecipitated by antibodies against BIG1 or BIG2. Direct interaction of BIG1 N‐terminal sequence with β‐catenin, found in yeast two‐hybrid studies, was confirmed using in vitro‐synthesized proteins. Depletion of BIG1 and/or BIG2 or over‐expression of a GEF‐inactive mutant, but not wild‐type, BIG1 or BIG2 resulted in β‐catenin accumulation in perinuclear Golgi structures. As association of protein kinase A (PKA) with β‐catenin, BIG1, and BIG2 in HeLa cells, and PKA‐catalyzed phosphorylation of β‐catenin S675 were diminished after depletion of BIG1 or BIG2, we infer that function of the AKAP sequences in BIG1 and/or BIG2 may be required for PKA‐catalyzed β‐catenin modification. In addition, transcriptional activity of β‐catenin was lower in HeLa cells after BIG1 and/or BIG2 depletion, further consistent with their roles in regulating β‐catenin action via S675 phosphorylation.
Gtr1‐Gtr2, Ego1‐Ego3 and Vam6‐independent cytoplasmic Gln3 sequestration in conditions of nitrogen excess (609.17)
Authors: Cooper Terrance , Tate Jennifer , Rai Rajendra , Georis Isabelle , Dubois Evelyne
Published: June 2021

The Gtr1/2, Ego1/3 complexes and Vam6 have been reported to be required for TorC1 kinase activation. In turn, activated TorC1 is accepted to be required for cytoplasmic Gln3 sequestration based on the observations that: (i) a Tor1‐Gln3 interaction is required for high level cytoplasmic Gln3 sequestration in nitrogen excess and (ii) rapamycin treatment elicits cytoplasmic to nuclear Gln3 relocation. These correlations lead to the a priori prediction that Gtr1/2, Ego1/3 and Vam6 should also be similarly required for downstream cytoplasmic Gln3 sequestration when cells are cultured in excess nitrogen. To test this hypothesis, we individually deleted the genes encoding each of the above proteins and determined whether the Gtr‐Ego complexes were required for cytoplasmic Gln3 sequestration in nitrogen rich medium. In every case cytoplasmic Gln3 sequestration occurred to the same degree in the deletion mutants as in wild type. These data indicate that either cytoplasmic Gln3 sequestration occurs in response to a TorC1‐independent regulatory pathway and/or alternatively that TorC1 activation can occur via both Gtr1/2‐Ego1/3‐Vam6‐dependent and ‐independent regulatory pathways.

Grant Funding Source: NIH GM‐35642; FRFC 2.4547.11
Control of bacterial chemotaxis by heme nitric oxide/oxygen proteins (614.2)
Authors: Hespen Charles , Marletta Michael
Published: June 2021

Hemoprotein gas sensors are integral components in the cellular machinery to recognize and rapidly respond to dynamic changes in redox state and the concentration of reactive gases. Heme‐Nitric oxide/Oxygen sensing (H‐NOX) proteins, found in both eukaryotes and bacteria, respond to NO in facultative aerobic bacteria, and regulate biofilm formation through the control of a complex two‐component signaling network. H‐NOX proteins are also found in obligate anaerobic bacteria, predominantly Clostridia, and are fused to Methyl‐accepting Chemotaxis Proteins (MCPs). MCP domains control the chemotaxis‐signaling pathway, which guides bacteria toward beneficial molecules (attractants) and away from harmful molecules (repellents). Obligate anaerobe H‐NOXs contain a conserved H‐bonding network associated with the formation of a stable O2 complex. Because NO and O2 are both toxic to obligate anaerobes, the H‐NOX‐MCP likely senses one or both of these gases and activates a repellent response in bacteria. A gas sensing chemoreceptor has not yet been identified in Clostridia, a class of organisms whose ability to rapidly avoid increasing concentrations of O2 is vital for survival. To characterize how gases affect the H‐NOX‐MCP's control of chemotaxis, the chemotaxis pathway of Clostridium sporogenes has been reconstituted in vitro. Changes in activity of the chemotaxis histidine kinase, CheA, were measured in different H‐NOX‐MCP heme ligation states.

Grant Funding Source: Supported by NIH Ruth L. Kirchstein NRSA
Functional characterization of cytochrome P450 1A1 Ala62Pro variant by recombinant protein expression (615.3)
Authors: Lee Seung Heon , Kang Sukmo , Lee Seungwoo , Ryu Doug‐Young
Published: June 2021

Cytochrome p450 1A1 is an extra‐hepatic mono‐oxygenase involved in the activation of pro‐carcinogenic xenobiotics. G184C polymorphism of CYP1A1, resulting in the mutation of an alanine residue at position 62 to proline, has been identified in Korean population. In order to functionally characterize the A62P substitution, the A62P variant and wild‐type were expressed in E.coli. Although the A62P variant showed a similar level of apoprotein expression compared to the wild‐type, E.coli expressing the A62P variant showed significantly decreased CYP content level in spheroplast and membrane fraction compared to E.coli expressing the wild‐type. In agreement with those findings, heme content of A62P variant in E.coli membrane fraction was lower than that of the wild‐type. A crystal structure of CYP1A1 demonstrates that residues located at A’/A and K helices as well as β1‐4, β4‐1 and β4‐2 sheet regions are involved in heme binding. Substitution of residues in those regions, including A62 in the A helix, into glycine affected heme binding in various degrees. In addition, the A62P variant in membrane fraction prepared from E.coli and mammalian CHO cells showed lower catalytic activity toward ethoxyresorufin compared to the wild‐type. These findings suggest that CYP1A1 A62P variation causes functional alteration of CYP1A1 due to structural changes such as heme incorporation.
Cloning and expression of cryptochrome proteins from Arabidopsis thaliana and sunflower (617.3)
Authors: Dyer James , Li Mengqi
Published: June 2021

Two cryptochrome proteins, CRY1 and CRY2, occur in plants and animals. CRY1 regulates the circadian clock in a light‐dependent fashion in insects and plants. Also in plants, blue light photoreception can be used to cue developmental signals. Despite much research on the topic, the specific way that the pterin and flavin chromophores function in these proteins in Arabidopsis thaliana and other plants is still poorly understood. The goal of this research was to clone and express the Arabidopsis and Sunflower CRY1 and CRY2 proteins in an E. coli expression system. Consensus sequence primers flanking the complete coding regions of the two cryptochromes were made based on known sequences of other dicot plant cryptochromes. These primers will be used in a 5’and 3’Rapid Amplification of cDNA Ends protocol, and the complete CRY sequences will cloned into a pBAD‐His vector and purified using Ni‐NTA affinity chromatography. Purified proteins will be used for structural and functional analysis.
Identifying and refining threshold concepts for biochemistry using a community of faculty and students (618.1)
Authors: Loertscher Jennifer , Minderhout Vicky , Green David , Lewis Jennifer
Published: June 2021

Threshold concepts are concepts that when mastered, represent a transformed understanding of a discipline, without which the learner cannot progress. We have undertaken a process involving over 75 faculty members and 40 undergraduate students to identify a working list of threshold concepts for biochemistry. The process of identifying threshold concepts for biochemistry was modeled on extensive work related to threshold concepts across a range of disciplines. Three major sources of information were used to identify threshold concepts for biochemistry: faculty workshops, student interviews, and existing data from the literature and other sources. We will report on use of an iterative process to identify three to five concepts to be the focus of future curriculum and assessment materials development. The concepts are likely to relate to foundational ideas from chemistry yet be situated in essential biochemical contexts. Candidate concepts relate to equilibrium, inter/intramolecular interactions, and individual versus populations of molecules. The finalized working list is not intended to be exhaustive, but rather is meant to identify a subset of threshold concepts for biochemistry for which instructional and assessment tools for undergraduate biochemistry will be developed.

Grant Funding Source: NSF‐DUE 1224868