bluuebunny/crossref_metadata_2025 huggingface dataset:
3D Embryonic Heart Goes Mobile: Efficacy of Mobile Application in Congenital Cardiac Embryology Education
Authors: Gustilo Katherine S. , Stratford Jennifer , Hight Marissa , Lee Lisa M.
Published: May 2022

Mastery of embryonic heart development is challenging but critical for medical professionals due to the frequency and variety of congenital cardiac defects that arise when the process goes awry. Unfortunately, teaching and learning cardiac embryology is difficult due to a lack of accurate and effective visual resources that depict this complex spatiotemporal event. To that end, an interactive mobile application named Embryonic Virtual Heart Application (EVHA) was developed as an accessible and interactive learner platform featuring a set of anatomically accurate 3D embryonic heart models with and without congenital cardiac defects (Figure 1). The project aims to assess whether the EVHA usage leads to measurable learning outcomes for first‐year medical students in the absence of a traditional lecture on cardiac embryology and congenital cardiac defects.

In a COMIRB exempt study (#21‐4827), as a part of an optional pre‐work activity prior to the cardiac embryology class, 172 first‐year medical students were given access to a pre‐quiz (Q1) on cardiac embryology before interacting with the EVHA. After the EVHA usage, a post‐quiz (Q2) comprised of a set of matched, but different, questions than the Q1 was provided. During the class, students’ retention on this topic was assessed by a set of clinically oriented questions (Q3). Lastly, students completed an optional survey on their perceived value of the EVHA after the class. The learning outcomes from the EVHA usage were assessed by a one‐way ANOVA to compare Q1, Q2, and Q3 performances. To analyze the survey results, the frequency of each Likert scale score was averaged while thematic analysis was performed on the comments.

The learning outcomes analysis revealed a significant increase in quiz performance between Q1 (22% ± 19%) and Q2 (65% ± 24%) with a 43% increase in the score (p<0.001; d=0.7; n=21). In‐class performance (Q3) average (61%; n=61) for all participants was not significantly different from Q2 (p>0.05), however those students who completed all optional prework (Q1, EVHA, Q2) had a significantly higher Q3 performance (79%; p <0.05; n=9). Survey analysis indicates that students perceive EVHA as an effective learning tool with its greatest strength being the interactive 3D virtual models of the congenital cardiac defects.

Despite the limitations and confounds in this project, these results indicate that the EVHA may be an effective tool contributing to significant learning outcomes independent from a formal class and can further enhance learning in the classroom and on higher‐order assessments. The impact of this project is its contribution of an accessible, portable and visually‐oriented learning resource on cardiac embryology with clinical emphasis. Based on the learning outcomes data, the project also demonstrates the potential of educational mobile applications in medical curricula in which contact hours are limited.
A Systematic Review of Assessment Practices for Self‐Directed Learning in Health Professions Education
Authors: Lerchenfeldt Sarah , Kemp Kyeorda , Mi Misa , Taylor Tracey A.
Published: May 2022

Introduction

This systematic review aimed to describe the assessment practices related to self‐directed learning (SDL) in undergraduate health professions education. There were three main objectives of the review: 1) evaluating the different types of assessment used to determine the effectiveness of SDL; 2) assessing the effectiveness of SDL methods on student learning; and 3) exploring how readiness for SDL is assessed.

Methods

The PRISMA standards were followed for this systematic review. A total of seven electronic databases were searched: CINAHL, Embase, ERIC, PsycINFO, PubMed, Scopus, and Web of Science. Articles published in English between 2015 and March 2020 were retrieved for further evaluation. Two independent investigators screened abstracts and full‐text articles using the inclusion and exclusion criteria. The same investigators independently extracted data from included articles: the type of participant, sample size, study design, SDL activities, and assessment methods used to evaluate effectiveness of SDL. In addition, two independent investigators evaluated study quality using the validated Medical Education Research Study Quality Instrument (MERSQI). A third investigator resolved discrepancies and disagreements.

Results

A total of 65 studies from nineteen different countries were included in the analysis. Most study participants were medical (N=33) or nursing (N=20) students. Overall, there were wide variations in the definition of SDL, SDL activities used, and in the assessments developed to evaluate the effectiveness of SDL activities. The most common type of assessment used was a student perception survey, unrelated to a SDL readiness assessment. Internally‐developed knowledge exams were also commonly used. A total of 38 studies concluded that the SDL had a positive outcome, measured by the assessment instrument, whereas 21 studies reported a mixed or neutral outcome and six studies reported a negative outcome.

Conclusions

This systematic review demonstrates the wide variations in both the SDL activities used as well as the assessment methods developed to determine SDL effectiveness. Since SDL is a necessary skill for lifelong learning and success as a healthcare professional, it must be used and assessed appropriately. It may be valuable to standardize SDL activities and assessment methods across healthcare professions education. In addition, future research should be conducted to help establish the most accurate and effective assessment methods for SDL.
High‐polyphenol sorghum bran extracts decrease glutathione peroxidase expression and activity in colon cancer celle
Authors: Jupinka Alexandria , Singh Ashish , Kamagate Zeguela , Smolensky Dmitriy , Tsuji Petra
Published: May 2022

Polyphenols are secondary plant metabolites found in many fruits, vegetables, and also grains. Sorghum bicoloris a cereal grain that contains varying amounts of polyphenols. Many polyphenols have been implicated in the regulation of colon cancer through modulating cellular antioxidant defense enzymes, which includes the glutathione peroxidases (GPX). These redox‐active enzymes protect cells from oxidative stress and are therefore involved in the regulation of tumorigenesis. Because cancer cells are thought to be undergoing oxidative stress, antioxidant defense enzymes, including but not limited to GPX, are often upregulated in those cells, contributing to their survival. The goal of our study is to investigate the impact of high‐polyphenol sorghum extracts on the expression and activity of these antioxidant enzymes. Our hypothesis is that glutathione peroxidase expression and catalytic activity will decrease in colon cancer cells treated with high‐polyphenol Sorghum extracts, thus potentially presenting a dietary chemoprevention strategy. The USDA ARS Kansas provided us with extracts from high‐polyphenol Sorghum bran, labeled ‘HP’ or ‘SC’. Human colon cancer cells (HCT116) were incubated with 1.25 mg extract per mL medium for 24hr hours. Total RNA was isolated with the Trizol method, and reverse transcribed to cDNA. mRNA expression was subsequently quantified using qPCR and normalized to the housekeeping gene GAPDH. Catalytic activity of GPX was determined with enzyme specific commercially‐available assays that measure the conversion of 1.0 µmol of NADPH to NADP+. Our preliminary results show that, compared to the solvent control, extract ‘HP’ slightly and extract ‘SC’ significantly (p<0.05) decreased mRNA expression of GPX 1 in HTC116 cells. However, GPX2 mRNA was modestly (p>0.05) increased in cells exposed to SC extract. Overall GPX activity, which measures catalytic activity of all present cellular GPXs, was significantly decreased by extracts ‘SC’ and ‘HP’. These results suggest that high polyphenol sorghum extracts may influence gene expression and catalytic activity of glutathione peroxidases involved in cellular antioxidant defense, and that GPX activity primarily reflects GPX1 as expected. Further investigation using other cell lines and antioxidant systems may help explain whether and how Sorghum extracts may modulate tumor prevention and promotion.
The stretch region within the TTD domain of UHRF2 is a disordered region
Authors: Mellado Fritz Carlos A. , Ginnard Shane , Trievel Ray , Albaugh Brittany
Published: May 2022

UHRF1 and UHRF2 are multiple‐domain epigenetic proteins that share a high degree of sequence similarity. These proteins play a very important role in histone modification and DNA methylation. Although both proteins contain a TTD and a PHD domain, the TTD domain of UHRF2 contains a unique ~35 amino acid long region that is highly basic called the “stretch”. This region was analyzed for the possibility of being a disordered region using the Sypro Orange Thermofluor assay. We found that UHRF2 TTD‐PHD has a lower melting temperature than UHRF2 TTD‐PHD without the stretch. It was also found that at higher salt concentrations, the melting temperature of UHRF2 TTD‐PHD increases, likely due to the disordered “stretch” region becoming stabilized. This data provides support towards the model that the stretch region is disordered and regulates protein stability.
Interleukin‐18 Enhances Contractile Responses to Phenylephrine in Pudendal Artery from Wistar Rats
Authors: Arishe Olufunke O. , Priviero Fernanda , Wilczynski Stephanie , Webb Clinton
Published: May 2022

Background

Hypertension is a major risk factor for the development of cardiovascular diseases and is a leading cause of mortality. The etiology of essential hypertension remains unclear, and its mechanisms are complex. Inflammation is a complex phenomenon, comprises the first response of the immune system in the presence of harmful stimuli and is considered to be a major cause of increased blood pressure. Indeed, low‐grade inflammation and activation of the immune system have been implicated in hypertension. Cytokine levels are elevated in hypertension and have been reported to be implicated in the associated disturbances in vascular reactivity reported in this disease. Clinical and population studies have consistently found increased circulating levels of interleukin 18 (IL‐18) in patients with hypertension.

Hypothesis

This study tests the hypothesis that IL‐18 will enhance contractile responses to phenylephrine in isolated arterial rings from rats.

Methods

Vascular function was performed on isolated pudendal arteries from male Wistar rats using wire myographs, kept in Krebs solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration‐response curves to phenylephrine (PE; 10‐9‐3x10‐5M), Acetylcholine (ACH; 10‐9 – 3x10‐5M) were performed in the absence or in the presence of IL‐18 (20ng/ml) for 1 hour. 10 minutes prior to the incubation with IL‐18, some arterial rings were incubated separately with the following agents: NADPH oxidase 2 inhibitor, gp91 ds‐tat (10‐5M); NADPH oxidase 1 and 4 inhibitor, GKT137831(10‐6M); and mitogen‐activated protein kinase (MKK / MEK) inhibitor, PD98059 (10‐5M) Concentration‐response curves were analyzed using non‐linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data was analyzed with GraphPad Prism 9.2.0.

Results

In the Wistar pudendal arterial rings, the potency of PE was increased in the presence of IL‐18 (pEC506.510 ± 0.3987 vs 6.022 ± 0.2738 for control; p = 0.0268) A, IL‐18 plus gp91 ds‐tat (pEC506.643 ± 0.2215 vs 6.264 ± 0.2471 for control; p = 0.0058) B and IL‐18 plus GKT137831 (pEC506.700 ± 0.2937 vs 6.272 ± 0.2963 for control; p = 0.0003) C. Incubation with IL‐18 plus PD98059 restored the contraction to PE to control levels. (pEC506.105 ± 0.4007 vs 6.170 ± 0.2847 for control; p = 0.7120) D. The relaxation response to ACh was not changed by IL‐18 after incubation for 1 hr.

Conclusion

Our results show that IL‐18 enhances the contractile responses to PE in pudendal arteries from Wistar rats by activation of the Mitogen‐Activated Protein Kinase/Extracellular Signal‐Regulated Kinase Pathway.
Obese adipose tissue extracellular vesicles inhibit recovery from dextran sulphate sodium (DSS)‐induced colitis
Authors: Delgado‐Deida Yaritza , Coughlan Christina , Theiss Arianne
Published: May 2022

Similar to the general American population, approximately 1/3rd of inflammatory bowel disease (IBD) patients are overweight and an additional 1/3rd are obese. Obesity in IBD patients is associated with increased difficulty in achieving remission, higher risk of disease relapse, higher hospitalization costs, and difficult surgical management. Adipose tissue is the largest endocrine organ producing and secreting cytokines, hormones, growth factors, and extracellular vesicles. Adipose tissue cellular composition, function, and secretion is strikingly altered during obesity. Alteration of adipose tissue‐derived EVs, including exosomes, during obesity is less understood. EVs carry molecular cargo that is actively taken up by other cell types and mediate phenotypic changes in recipient cells, resulting in intra‐organ communication. Since obesity is associated with abnormalities in gastrointestinal epithelial turnover, increased gut permeability, enhanced stemness, and decreased goblet cells, further studies are needed to elucidate the influence of obesity on pathomechanisms of underlying IBD. Here, we aim to investigate whether obese adipose tissue EVs alter acute disease or recovery in a model of induced colitis. As EV donor mice, C57BL/6 mice were fed from 4‐20 weeks of age a purified high fat diet (HFD) containing 45% kcal fat, a well‐established model of obesity, or purified matched diet (MD) containing 10% kcal fat. Visceral adipose tissue was cultured ex vivo, secreted EVs were isolated by ultracentrifugation, and concentration was determined by Nanoparticle Tracking Analysis. As EV recipient mice, a separate set of C57BL/6 mice were treated with 2.5% (wt/vol) dextran sodium sulfate (DSS) in drinking water for 6 days to induce acute colitis and were i.p. injected with MD EVs, HFD EVs, or vehicle (1xPBS) at a concentration of 10^8 EVs/g body weight every 2 days of DSS treatment. To test recovery from colitis, C57BL/6 mice were treated with 2.5% DSS for 6 days, were allowed to recover for an additional 6 days by removing DSS, and were treated with EVs every 2 days beginning on day 4 of DSS. Body weight and disease activity index (DAI) scores were measured daily. Colon was collected and swiss‐rolled for histology scoring (inflammation, extent, crypt damage, percent involvement) or RNA isolation for quantitative real‐time PCR analysis of Lcn2 as an indicator of inflammation. During acute DSS, body weights, DAI, colon length, and histology scoring did not change between mice injected with HFD EVs or MD EVs compared to vehicle. However, HFD EV treated mice exhibited delayed recovery from DSS colitis as evidenced by significantly higher DAI scores, colon shortening, and elevated levels of Lcn2 mRNA following recovery from DSS. These results suggest that EVs released by obese adipose tissue inhibit colitis recovery and provide insight on adipose‐to‐intestinal tissue communication elicited by EVs.
Changes in Icelandic soil microbiomes from the forefield of retreating glaciers revealed by Illumina and MinION sequencing
Authors: Smith Emmett , Jackobs Faith , Peck Charlie
Published: May 2022

Climate change is affecting the sub‐Arctic landscape more than mid‐latitude locations on Earth. This is especially evident in Iceland where glaciers are now receding at a rapid pace. As glaciers retreat, newly exposed land undergoes primary succession. The soil microbiome is a critical component of succession; microbial communities often act as first colonizers and are key players in soil neogenesis. Warming‐related changes in the composition of soil microbes may increase the amount of gasses such as methane and CO2 produced by soil bacteria, as well as expose potentially new pathogens. Therefore, it is important to collect long‐term data sets from diverse locations throughout the Arctic and sub‐Arctic to form a clearer picture of how climate change affects these areas. We are developing a chronosequence of soil microbiome data and extent mapping at two outlet glaciers on the South coast of Iceland (Sólheimajökull and Kvíárjökull). During our work, we have optimized a field‐based soil extraction protocol for remote DNA extraction. We compare microbiome communities between multiple sampling locations to understand how the organismal composition changes as the land recovers. We hypothesize that microbial populations will change in composition as we move farther away from the glacier’s margin. We also compare Illumina and MinION sequencing results on the same samples. We expect that MinION sequencing will be able to produce species‐level identification but may produce a different yet overlapping population set when compared with Illumina sequencing data. In addition to population‐level changes (such as nitrogen pathway development) we are searching the data to identify antibiotic resistances. The data gathered in this first‐pass analysis expands our current knowledge of soil microbiomes in the sub‐Arctic and provides a foundation for a long‐term study of the region.
Lysine acetylation during oxidative stress response in the halophilic archaeon Haloferax volcanii
Authors: Couto‐Rodriguez Ricardo L. , Maupin‐Furlow Julie
Published: May 2022

Stress adaptation strategies have been a central topic of research in halophilic organisms due to biotechnological and astrobiological applications. This study is focused on how the halophilic archaeon Haloferax volcaniimay use lysine acetylation to regulate the oxidative stress response. Previous studies have shown that a) HOCl stress causes the lysine acetyltransferases Pat2:Pat1 ratios to increase and b) mutation of the lysine deacetylase sir2 gene renders cells hypertolerant to oxidative stress, suggesting this post translational modification system has a role during oxidative stress response. Immunoblotting experiments carried out in this study show an increase in lysine acetylation when glycerol‐grown cells are exposed to sodium hypochlorite. Mass spectrometry analysis of the lysine acetylome shows central metabolism pathways are modified under non‐treatment conditions. Of note during oxidative stress, a 2Fe‐2S ferredoxin (HVO_2995) was shown to be reduced in lysine acetylation levels suggesting an important role during oxidative stress response in the organism. These results now pave the way for a comprehensive quantitative mass spectrometry study to assess the global role of lysine acetylation during this oxidative stress response in the organism. Understanding this system can give us new answers into mechanisms of stress response in halophilic archaea. Expansion of this knowledge can provide a balanced evolutionary perspective of post translational modifications in living organisms.
Characterizing the Drosophila Orthologs of Phosphoglycolate Phosphatase and Pyridoxal Phosphate Phosphatase
Authors: Liu Junjie , Kennell Jennifer A.
Published: May 2022

Phosphoglycolate phosphatase (PGP) and pyridoxal phosphate phosphatase (PDXP) are members of the haloacid dehalogenase (HAD) family of non‐protein phosphatases. PGP plays an important role in central carbon metabolism by regulating glycolysis, the pentose phosphate pathway, and lipid metabolism. PGP also acts as a metabolite repair enzyme by cleaning up toxic side products from glyceraldehyde 3‐phosphate dehydrogenase, pyruvate kinase, and DNA repair. PDXP regulates pyridoxal phosphate (PLP; active vitamin B6), a cofactor for 140+ enzymes. Although previous studies have characterized PGP and PDXP in mammals, their fly orthologs remain unidentified. Here, we use mutagenesis and transgenic tools to investigate the putative PGP and PDXP orthologs in the fruit fly D. melanogaster, CG5567 and CG5577. We found that CG5567 mutants survive to adulthood, unlike mice lacking PGP, but they are sensitive to ethylene glycol and have increased triglyceride levels, consistent with previous studies showing that mammalian PGP de‐phosphorylates phosphoglycolate and glycerol‐3‐phosphate. CG5567 mutants are also more resistant to starvation. CG5577 mutants also survive to adulthood but do not share these phenotypes. However, flies overexpressing CG5577 die as late third instar larvae. We are currently testing if we can rescue the CG5577 overexpression phenotype by increasing PLP levels. Additionally, we are using mass spectrometry to analyze substrate and product levels to confirm that CG5567 and CG5577 are functioning as PGP and PDXP orthologs. Our results demonstrate that D. melanogaster is a useful model for investigating the function and evolution of PGP and PDXP.
IL‐19 Regulates Atherosclerotic Plaque Progression via Lymphangiogenesis
Authors: Peluzzo Amanda , Preston Kyle , Leigh Tani , Kelemen Sheri , Autieri Michael
Published: May 2022

Introduction

Cardiovascular diseases are the leading cause of deaths globally, representing approximately 31% of deaths per year, 85% of which are due to myocardial infarction (MI) or stroke. This does not account for the millions of patients suffering daily with current therapies that are not efficacious in treating the root cause of MI and stroke, atherosclerosis, by reducing inflammation or increasing reverse cholesterol transport (RCT). Atherosclerosis is a vascular inflammatory disease characterized by the formation of fatty plaques which can rupture, leading vessel occlusion. Lymphangiogenesis is the process whereby lymphatic vessels are generated, but its role in atherogenesis is currently under‐characterized and controversial. It has been suggested that lymphangiogenesis is either protective by promoting plaque regression through RCT or it is deleterious by promoting plaque instability through white blood cell trafficking. Our lab has previously reported that Interleukin‐19 (IL‐19), an anti‐inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro‐angiogenic. This drives our hypothesis that one mechanism whereby IL‐19 is atheroprotective is by driving lymphangiogenesis, allowing attenuation of plaque progression.

Methods & Results

Several approaches were performed to test the hypothesis that IL‐19 is lymphangiogenic. IL‐19 can significantly induce primary human lymphatic endothelial cell (LEC) proliferation, migration, and tube formation, all angiogenic assays, to the same degree as VEGF‐C, a known mediator of lymphangiogenesis. RNA sequencing (RNAseq) analysis indicates that IL‐19 induces chemokine expression as well as an angiogenic transcriptional program in LECs. Notably it induces expression of Prox1, a master transcription factor of lymphangiogenesis, 6.2‐fold. The increases of chemokine and transcription factor expression from RNAseq were verified through qPCR analysis. To further analyze the translational effects of IL‐19, we utilized an LDL Receptor Knockout (LDLR‐/‐) mouse model fed a high fat diet for 16‐weeks with daily injections of either recombinant mouse IL‐19 or saline. Preliminary results suggest an increase in lymphatic vessel presence associated with the known decrease IL‐19 causes in atherosclerotic plaque progression.

Conclusion & Summary

These data suggest that IL‐19 increases lymphatic vessel formation, potentially allowing RCT to take place to decrease plaque burden in atherosclerosis. Further studies will confirm IL‐19’s mechanistic pathway in LECs. We will also determine RCT and inflammatory cell trafficking by intravital microscopy. These results have the potential to shift our understanding toward a positive view of the association between the lymphatic system and atherosclerosis.
Preventing Iatrogenic Injury to the Dorsomedial Cutaneous Nerve of the Hallux
Authors: Dumford Emily R. , Hess Allison J. , Gumble Zachary , Pocwierz Alexander J. , Quinn Makaela , Zdilla Matthew J. , Lambert H. Wayne
Published: May 2022

The dorsomedial cutaneous nerve of the hallux (DMCN) is a sensory nerve that crosses the extensor hallucis longus (EHL) tendon to innervate the great toe and the first metatarsophalangeal joint. The DMCN is vulnerable to injury during operative procedures including hallux valgus or hallux rigidus correction, bunionectomy, and cheilectomy, as well as EHL tendon transfer surgeries. Intraoperative injury to the DMCN may cause pain that is intractable without subsequent surgical intervention. Accordingly, understanding anatomical variation of the DMCN is of the utmost importance. Therefore, this study assessed 23 cadaveric legs and feet to identify morphological variations of the DMCN. Observed variations included supernumerary branches crossing the EHL tendon and atypical superficial fibular nerve branching that affected the anatomical relationship between the DMCN and the EHL tendon. This study provides details of atypical variations of the DMCN and the resulting data, when used in conjunction with current imaging techniques (ultrasonography), may help to prevent unwarranted iatrogenic nerve injury.
Minocycline Blunts Isoproterenol‐Induced Cardiac Hypertrophy
Authors: Russell Jacob , Bailey Chastidy , Brown Scott , DeMarco Vincent G. , Mummidi Srinivas , Bender Shawn B. , Chandrasekar Bysani
Published: May 2022

Introduction

Excess and chronic activation of the sympathetic nervous system often results in progressive adverse remodeling and heart failure development. Here we investigated whether chronic administration of isoproterenol (Iso), a β‐AR agonist, would promote hypertrophy, fibrosis, and dysfunction, and whether these maladaptive changes are prevented by minocycline, an FDA‐approved antibiotic, that has been shown to have cardioprotective effects outside of its antibacterial properties.

Methods

Wild‐type male C57BL/6J mice were subcutaneously implanted with osmotic minipumps, delivering either 30 mg/kg body weight per day of isoproterenol, or vehicle control. Following pump implantation, mice received daily intraperitoneal (I.P.) injections of either sterile saline or 50 mg/kg of minocycline. After 21 days, cardiac function was assessed by echocardiography, and hearts were collected for further analysis. A transverse slice of the ventricles was fixed for histology, and a portion of the left ventricle was used for RNA isolation, and mRNA and miRNA sequencing.

Results

Echocardiography revealed that isoproterenol increased heart rate, heart weight (hypertrophy), ejection fraction and fractional shortening, without leading to significant changes in fibrosis. Minocycline prevented the hypertrophic remodeling and changes in systolic function without affecting heart rate. While mRNA sequencing revealed that isoproterenol enriched gene networks associated with changes in inflammation, the miRNA sequencing showed that minocycline enhanced miR‐26b expression, which is reported to suppress GATA4‐mediated hypertrophy.

Conclusion

Minocycline mitigates isoproterenol‐induced cardiac hypertrophy while preserving contractility, as well as induces changes in RNA and miRNA pathways involved with inflammation and cardiac remodeling
Spinal Afferent Innervation of the Rat Stomach: Anterograde Tracing
Authors: Ma Jichao , Nguyen Duyen , Madas Jazune , Kwiat Andrew , Mistareehi Anas , Kogut Nicole , Chen Jin , Powley Terry , Cheng Zixi J.
Published: May 2022

Nociceptive afferents from spinal dorsal root ganglia (DRG) and vagal nodose ganglia innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. In our other two posters (Mistareehi et al. 2022, Nguyen et al. 2022), nociceptive afferents are detected with SP and CGRP markers. To specifically study spinal afferent innervation of the stomach, we injected tracer Dextran Biotin (DB) into the left DRG (T7‐T11; 1.5 µl/each ganglion). 14 days after tracer injection, rats were perfused through the left ventricle with 40℃ PBS (1M phosphate‐buffered saline, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1 M PBS (4℃, pH 7.4). After the perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was removed. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser and greater curvatures, thus yielding two flat‐mounts per animal. The external muscle wall of the stomach was then isolated as a whole mount by removing the gastric mucosa and submucosa. Ventral stomach flat‐mounts were examined using the Neurolucida system. We found that Tracer DB‐labeled axons formed serval categories of the terminal structures. The simple type: long varicose axons without branching; The branching type: axons with multiple branches running in parallel with either circular or longitudinal muscles; The complex type: single axons with multiple branches running in various directions in both circular and longitudinal muscle layers; The ganglionic type:axons innervating myenteric ganglia; The blood vessel type: axons innervating blood vessels. Our work will provide a foundation for specific labeling of spinal afferent innervation of the stomach. In the future, we will use tracer injection of DRG and nociceptive markers (e.g., SP, CGRP, TRPV1) to examine topographical innervation of spinal nociceptive afferents in the stomach. The first two authors contributed equally to this work.
Angiotensin AT2 Receptor Activation during Ischemic Injury Protects against High Salt‐Induced Hypertension and Proteinuria in the Long‐term: Role of IL‐17A Cells
Authors: Ali Riyasat , Patel Sanket , Hussain Tahir
Published: May 2022

Short time renal ischemia/reperfusion (I/R) causes reversible acute kidney injury (AKI) that often recovers within 5‐7 days. However, a single event of AKI predisposes and makes kidneys susceptible to develop chronic kidney disease (CKD) in conditions such as obesity and hypertension. Recently we have shown that angiotensin type 2 receptor (AT2R) activation by the agonist compound C21 (C21) protects against I/R‐induced AKI in the early injury phase in terms of suppressing pro‐inflammatory cytokines and improving functional injury markers such as reducing proteinuria and blood urea nitrogen (BUN). The late recovery phase (day 5) in the I/R and I/R treated with C21 remained same i.e., the C21 treated and un‐treated animals fully recovered functionally to the same extent but with higher anti‐inflammatory IL‐10 producing and protective Treg cell (FoxP3+) in I/R+C21 compared I/R groups. However, the long‐term protective effect of the AT2R agonist C21 on these recovered kidneys against chronic injury is unknown. To address this question, male Sprague‐Dawley (SD) rats were administered C21 (0.3 mg/kg, i.p.) prior to 30‐minute bilateral renal ischemia and allowed to recover for 5 days, with daily C21 treatment (I/R+ C21), without treatment (I/R) and sham control. On 7th day, all the three groups were placed on high salt (1% NaCl) in drinking water for two weeks but without C21 treatment. On day 6 (prior to initiating high salt treatment), week 1 and 2 of the high salt treatment, renal function parameters (proteinuria), and T cells were quantitated in the blood and kidney (only at 2 weeks). On 6th day, all the three groups showed similar level of proteinuria, suggesting same renal function. However, at week 1 and 2, I/R group had significantly greater proteinuria, suggesting a function injury. Similarly, I/R group showed higher mean arterial blood pressure at two weeks compared with the I/R+C21 and sham control. Consistent to the higher proteinuria and blood pressure in I/R group, there were higher infiltrating proinflammatory (IL‐17A) cells in the kidney in I/R group compared with sham control and I/R+C21 groups. Also, kidney infiltrating CD45 cells, T cells and CD4 T cells were found to be similar among all the groups. Overall, the data indicate that I/R pre‐disposes the kidneys to high salt‐induced chronic injury which is completely prevented by AT2R agonist treatment during AKI phase. Moreover, AT2R’s protective effect could be related to preventing the T cell modulation into proinflammatory IL‐17A cells.
Progress Towards Expression and Purification of Moonlighting Proteins for Directed Evolution of Novel Therapeutics
Authors: Patel Jade E. , Beyers Ryan T. , Streu Craig N.
Published: May 2022

Antibiotic resistance is a significant threat to human health in all parts of the world. New resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases and necessitating new strategies for developing antimicrobial compounds. Moonlighting proteins are a key contributor to bacterial virulence. These proteins are multifunctional proteins in which a single protein performs multiple independent functions in different cell compartments, often making use of different conformations to do so. Moonlighting proteins that act as virulence factors tend to play key roles in conserved metabolic processes and elicit relatively muted responses from the immune system. Their evolutionary conservation, evasion of immune response and critical role in pathogenesis suggest that moonlighting proteins may make excellent targets for therapeutic development. One way to target these moonlighting proteins is with nanobodies. Nanobodies are single‐domain fragments generated from camelid antibodies that combine the beneficial properties of small molecules and monoclonal antibodies, thus making them an appealing agent for creating new antibiotics. Nanobodies are easier to produce cheaply in bulk than antibodies while maintaining excellent thermal and pH stability. Just as importantly, nanobodies may be developed using a combination of surface display and directed evolution methods allowing for expedited drug discovery in response to the development of resistance. This poster outlines progress toward the expression and purification of moonlighting proteins from S. aureusand S. epidermidisfor use in the directed evolution of next‐generation antibiotics.
Affinity Maturation of an Ovarian Cancer‐Targeting Peptide
Authors: Asar Mallika C. , Deutscher Susan L. , Soendergaard Mette
Published: May 2022

Ovarian cancer is among the leading causes of cancer deaths in women. The low survival rate is in part due to inadequate means of detection, which emphasizes the need to develop effective screening techniques. Peptide J18 was previously discovered using phage display technology and was found to specifically target and image human ovarian tumors in a SKOV‐3 xenografted mouse model.

Here, affinity maturation of J18 was carried out to improve its binding affinity by conducting an alanine scan of the peptide. A cell‐based modified ELISA using SKOV‐3 cells was carried out and dose‐response curves were modelled using non‐linear regression. The results showed that substitution of serine‐5 (S5) and aspartic acid‐6 (D6) with alanine decreased the EC50 value of the original peptide J18 from 45.02 µM (39.15‐52.94 95% confidence interval; CI) to 19.54 (9.70‐38‐51 95% CI) and 6.92 (5.2‐9.80 95% CI), respectively. Next, a double alanine substitution of S5 and D6 (J18‐S5A‐D6A) was investigated to further increase the binding affinity. The EC50 value was determined to be 4.22 (2.16‐16.14 95% CI), thus improving the binding affinity approximately 10‐fold.

To ensure that J18‐S5A‐D6A maintained specificity for ovarian cancer cells, the peptide was evaluated against ovarian cancer SKOV‐3, pancreatic cancer Mia‐Paca‐2, prostate cancer LNCaP, and embryonic kidney HEK 293 cells using a modified ELISA and fluorescent microscopy. Both assays showed significantly increased binding of J18‐S5A‐D6A to SKOV‐3 cells compared to the other cell lines. Based on these results, peptide J18‐S5A‐D6A may be used as an improved imaging agent of ovarian cancer.
Lipocalin‐2 Stimulates Proliferation and a Profibrotic Cholangiocyte Phenotype during Cholestasis
Authors: O'Brien April , Stiles Danaleigh , White Tori , Lopez Matthew , Little Ashleigh , Childs Jonathan , Douthitt Ashley , Phan Thien , Medford Abigail , Chen Lixian , Francis Heather , Alpini Gianfranco , Chakraborty Sanjukta , Glaser Shannon
Published: May 2022

Lipocalin‐2 (LCN2), also known as neutrophil gelatinase‐associated lipocalin (NGAL), is a secreted glycoprotein that has been shown to regulate cell proliferation, innate immunity, metabolism, and tumor metastasis in several cell types. LCN2 expression is elevated in hepatic tissues in experimental liver injury models and patients with alcoholic hepatitis. LCN2 has been shown to stimulate proliferation and metastasis of cholangiocarcinoma. However, the role of cholangiocyte LCN2 and LCN2 receptors (NGALR/24p3‐R) during cholestasis and primary sclerosing cholangitis (PSC) has not been evaluated. Therefore, we aimed to assess the role of LCN2 and its receptor, 24p3‐R, in acute and chronic cholestatic models.

Methods

LCN2 and 24p3‐R were evaluated in liver sections, total liver, and isolated cholangiocytes from wild‐type (WT; C57) normal and BDL (7 days) mice and WT (FVB/NJ) and Mdr2‐/‐ mice (model of primary sclerosing cholangitis, PSC) by immunohistochemistry and qPCR, respectively. LCN2 levels were evaluated by ELISA in cholangiocyte supernatants (6 hrs) collected from animal models and serum from BDL and Mdr2‐/‐ and WT controls and late‐stage PSC patients and healthy human controls. Serum bile acid (BA) levels were measured using a commercial BA assay kit in BDL, Mdr2‐/‐, and control mice. In vitro, both human (H69) and mouse pooled (MPC) cholangiocytes were treated with either 50 ng/ml LCN2 or 100 mM taurocholic acid (TCA) for 24 hrs after which the expression of proliferation, fibrosis, LCN2, and 24p3‐R genes was determined via qPCR.

Results

There were significant increases in LCN2 and 24p3‐R immunostaining in BDL mice compared to controls. By qPCR significant increases were observed for both LCN2 and 24p3‐R in total liver and isolated cholangiocytes from BDL and Mdr2‐/‐ mice compared to WT. Increased levels of LCN2 were found in serum from PSC patients and serum and supernatants from isolated cholangiocytes from BDL and Mdr2‐/‐ mice compared to control. In vitro, MPC cells treated with LCN2 had increases in fibrotic marker Col1a1, proliferation marker PCNA, and inflammatory markers IL‐1b and IL‐6. Also, significant increases were seen in fibrotic markers (aSMA, FN‐1, and TGF‐b1) and LCN2 receptor 24p3‐R in H69 cells treated with LCN2 compared to untreated cells. As expected, increased serum BA levels were observed in both BDL and Mdr2‐/‐ mice compared to controls. H69 cells treated with TCA also had significant increases in FN‐1, TGF‐b1, and the LCN2 receptor 24p3‐R, indicating that BA may regulate 24p3‐R expression.

Conclusion

LCN2 and 24p3‐R expression is elevated in cholestatic models and stimulates a profibrotic cholangiocyte phenotype. These findings suggest that LCN2 may play a crucial role in the pathogenesis of cholestatic liver injury.
Impact of Interindividual Variability in CYP3A Activity on Ibrutinib and Venetoclax Metabolism In Vitro
Authors: Lee Jonghwa , Jackson Klarissa D.
Published: May 2022

Ibrutinib and venetoclax are orally administered active agents approved for the treatment of chronic lymphocytic leukemia (CLL), the most common form of adult leukemia in the Western world. Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), whereas venetoclax is a B‐cell lymphoma 2 (BCL2) inhibitor. Although these drugs are effective in CLL treatment, managing life‐threatening side effects remains a clinical challenge. Previous studies have shown that ibrutinib and venetoclax are primarily metabolized by cytochrome P450 (CYP) 3A4. However, the influence of interindividual variation in CYP3A activity on the metabolism of ibrutinib and venetoclax has not been addressed. The objectives of this study were to 1) examine ibrutinib and venetoclax metabolism in single‐donor human liver microsomes and cryopreserved human hepatocytes, and 2) assess the correlation between metabolite formation and CYP3A activity markers. Ibrutinib (5 μM) or venetoclax (2.5 μM) were incubated with human liver microsomes from 20 individual donors. Relative levels of ibrutinib metabolites (PCI45227 and PCI45752) and venetoclax metabolites (M5, M4, and M3) were measured by LC‐MS/MS analysis. Formation of metabolites varied widely between the donors. Metabolite formation was highly correlated with CYP3A activity, as measured by midazolam 1’‐hydroxylation. Pearson correlation coefficients (r) for PCI45227, PCI45752, M5, M4, and M3 were 0.97, 0.85, 0.92, 0.93, and 0.95, respectively. These findings indicate that CYP3A phenotype may have a significant influence on the pharmacokinetics of ibrutinib and venetoclax in CLL treatment. Future studies will be conducted in human hepatocytes to evaluate the effect of CYP3A activity and protein levels on ibrutinib and venetoclax metabolism.
Neonatal ventilatory changes in the control of breathing in a hyperoxia‐induced mouse model of bronchopulmonary dysplasia
Authors: Mouradian Gary , Rohde Neil , Hodges Matt
Published: May 2022

While hyperoxia is needed for survival of very premature infants (<28 weeks gestation) due to immature lungs and immature neural control of breathing, it contributes to bronchopulmonary dysplasia (BPD), a chronic lung disease associated with heighted pediatric morbidity and mortality. Given the plasticity of the neural control of breathing, this study seeks to address whether the neural control of breathing adapts to lung disease and the roles of clusters of pulmonary neuroendocrine cells called neuroepithelial bodies (NEBs) in mediating breathing adaptations (or maladaptations). NEBs line the airways, are O2 and CO2 sensitive, are hypertrophied/hyperplastic in human BPD, and are innervated by vagal fibers that inhibit breathing when activated. Herein, we test the hypotheses that chronic neonatal hyperoxia causes age‐dependent changes to cardiorespiratory control and these changes are in part mediated by NEBs. Mice expressing eGFP in all NEBs were exposed to hyperoxia (FIO2: 0.7 from 0‐14 days of life; P0‐14) had reduced alveolar density (P<0.001) and fewer alveoli (P<0.001) compared with normoxic controls (n=4) at P22 (by 2‐tailed t‐Test), consistent with clinical BPD. Whole‐body flow through plethysmography did not reveal baseline breathing differences between normoxic (n=10) and hyperoxic (n=10) mice at any of the ages studied (P14‐22), though there is a trend (n=3/group) for a greater respiratory exchange ratio (RER) measured by respirometry in hyperoxic mice. There was also a significant main effect of hyperoxia (n=7) reducing the hypoxic and hypercapnic ventilatory responses compared to normoxic mice (n=10; two‐way mixed effect model with Sidak post‐hoc test). Ongoing studies aim to lesion NEBs using Saporin toxin conjugated to an NEB specific membrane protein in a subset of mice to address the functional role of NEBs in breathing in normoxic and hyperoxic treated mice.
Kinetic Characterization of a Crystallized Putative Hydroxybutyrate Dehydrogenase from the Opportunistic Pathogen Burkholderia cenocepacia
Authors: Capalbo Kathryn I. , Beadle Azzeiza S. , Palenchar Jennifer B.
Published: May 2022

Burkholderia cenocepaciais an opportunistic pathogen posing risk among individuals with cystic fibrosis or who are immune compromised. The crystal structure of a putative hydroxybutyrate dehydrogenase (HBDH), based on sequence analysis, from Burkholderia cenocepaciahas been solved by the Seattle Structural Genomics Center for Infectious Disease and deposited in the Protein Database (PDB ID 4TRR). We sought to determine if the protein is an HBDH and to determine the kinetic parameters of the enzyme. HBDHs catalyze the reversible conversion of acetoacetate to hydroxybutyrate utilizing the cofactor NAD(H). The SSGCID kindly provided us the construct encoding a His‐tagged BcHBDH. The protein was overexpressed in E. coli, purified to approximate homogeneity using nickel affinity chromatography, and activity assayed spectrophotometrically. The putative BcHBDH is confirmed to have HBDH activity and the kinetic parameters for the enzyme were determined in both the direction of acetoacetate and hydroxybutyrate formation. BcHBDH has a catalytic efficiency of 2.85x104 mM‐1 sec‐1 and 1.21x105mM‐1 sec‐1 in the direction of hydroxybutyrate formation for acetoacetate and NADH, respectively. In the direction of acetoacetate formation, the enzyme has a catalytic efficiency of 4.1x103 mM‐1 sec‐1 and 2.86x104 mM‐1 sec‐1 for hydroxybutyrate and NAD+, respectively. By comparing both the structure and kinetic parameters to known kinetically characterized and crystallized HBDHs, we conclude the protein from Burkholderia cenocepacia is an HBDH.
Empagliflozin Accelerates Escape from Vasopressin in Rats
Authors: Eubanks Elena E. , Rodriguez Eva L. , Klein Janet D. , Sands Jeff M.
Published: May 2022

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) consists of unregulated, elevated vasopressin levels and can lead to hyponatremia. Vasopressin escape is the body’s defense mechanism to limit hyponatremia. The sodium‐glucose transporter (SGLT‐2) inhibitor, empagliflozin, was identified as a treatment for hyponatremia; however, the mechanism of empagliflozin’s effect is unclear. This project investigates empagliflozin’s impact on vasopressin escape in rats with the hypothesis that empagliflozin accelerates escape via key transport protein alteration. Rats were implanted with vasopressin mini‐pumps to mimic SIADH. The first cohort consisted of male rats and a second cohort compared responses in female rats. On day 2, half the rats were given empagliflozin (~15 mg/kg) via diet. Rats were sacrificed on days 7 or 14. Blood and kidney tissue were collected. Total Aquaporin‐2 (AQP2), pSer‐Aquaporin‐2, and NKCC2 were analyzed by western blot. At day 7, serum sodium levels for the female rats were: empagliflozin 110.1+1.7 mmol/L vs 93.3+1.1 mmol/L for the controls (n=5, p<0.001). Serum sodium levels for the male rats were: empagliflozin 136.0+4.8 mmol/L vs 121.4+3.0 mmol/L for the controls (n=4, p<0.05). NKCC2 abundance in the empagliflozin group was 60% lower than control on day 7 (n=5, p<0.05) for female rats, and 77% lower than control in male rats (n =4, p<0.001). Because changes in the AQP2 levels were similar between the sexes, these data were combined. Total AQP2 abundance was 2.3 fold increased in the empagliflozin group on day 7 and 8 fold greater in the empagliflozin group at day 14 (n=9) compared to the control group. Both pSer256‐AQP2 and pSer261‐AQP2 were increased in the empagliflozin groups at both 7 and 14 days vs their respective control groups. pSer256‐AQP2 increased 2.1 (day 7) and 8.5 fold (day 14); pSer261‐AQP2 increased 3.2 fold (day 7) and 5.0 fold (day 14) (n=9/group). We conclude that empagliflozin accelerates vasopressin escape during SIADH primarily through a decrease in NKCC2 abundance in both male and female rats. This will hasten recovery from hyponatremia in SIADH.