bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Characterization of the Covalent Binding of Keap1 and Thiol‐containing Small Molecules
Authors: Pyeatt Gwendolyn , Muellers Samantha , Whitty Adrian , Allen Karen N. 
Published: April 2020

Oxidative stress is involved in a wide range of human diseases such as cancer, diabetes, and Alzheimer’s disease. High levels of reactive oxygen species (ROS) cause oxidative damage to DNA and other molecules vital to cell function, such as membrane lipids and proteins. Kelch‐like ECH‐associated protein 1 (Keap1) is a primary regulator of the body’s natural defense system against oxidative stress. In the absence of oxidative stress, Keap1 forms a homodimer that binds a single molecule of transcription factor nuclear factor‐like 2 (Nrf2), causing Nrf2 ubiquitination and degradation. Oxidative stress causes Keap1 to release Nrf2, allowing it to translocate to the nucleus, bind to Maf protein, and activate the production of antioxidant enzymes by binding to the antioxidant response element (ARE). Blocking the protein‐protein interaction (PPI) between Keap1 and Nrf2 could have great therapeutic value. The antioxidant enzymes upregulated by free Nrf2 provide an efficient, catalytic detoxification of ROS. However, as PPIs frequently take place between large, flat interfaces on the two participating proteins, these interactions can be difficult to block. A previous study by our collaborators at UCSF identified 43 thiol‐containing compounds (monophores) that can be covalently tethered to Keap1 at a cysteine near the protein interface. The goal of this project is to characterize the interaction between Keap1 and the monophores using X‐ray crystallography to obtain structures of the protein‐monophore complexes, or adducts. This structural data will allow us to judge which compounds are suitable for scaffold optimization, a technique in which a larger molecule is built from an initial smaller compound to improve its potency and selectivity. Information about the binding modes of the monophores could also be incorporated into the design of other types of drugs that have proven effective in blocking PPIs, such as macrocycles. To identify adduct crystallization conditions, a DTNB assay, which measures the number of reactive sulfhydryl groups in a protein, was used to screen buffer conditions without dithiothreitol (DTT) present. A high‐throughput version of this assay was developed and utilized, in combination with differential scanning fluorimetry (DSF), to identify stabilizing conditions in which adduct can form. This assay was validated with a monophore known to have a stabilizing effect on Keap1, and a new buffer condition (50 mM EPPS, 50 mM NaCl, pH 8.0) was identified in which adduct crystallization attempts can be made.Support or Funding InformationThis work was supported by the Arnold and Mabel Beckman Foundation.
The Dynamic Proteome and Lipidome of Plastoglobule Lipid Droplets of Plant Plastids
Authors: Lundquist Peter Knut , Espinoza-Corral Roberto 
Published: April 2020

Plastoglobules are ubiquitous lipid droplets of plant plastids that consist of a lipidic core that is enclosed by a monolayer membrane surface studded with structural and enzymatic protein. The dramatic changes in plastoglobule morphology in response to environmental stresses, including more than 10‐fold increases in droplet diameter, suggest a crucial role in plant stress tolerance. However, in the absence of an understanding of protein or lipid changes of the plastoglobule under stress, it is difficult to connect the plastoglobule to specific functions. We have used proteomic and lipidomic profiling to determine the quantitative protein and lipid compositions, and oligomeric state of isolated plastoglobules from Arabidopsis thaliana under unstressed and high light treatment conditions. Our analysis has revealed evidence of multiple high molecular weight complexes at the plastoglobule and changes to the prenyl‐lipid composition. Building on our results, we propose a model of plastoglobule function related to mediating membrane remodeling processes in response to stress adaptation.Support or Funding InformationMichigan State University, MSU AgBioResearch
Comparison of Sweat versus Serum IL‐1α and IL‐1β Concentrations during Exercise
Authors: Brown Shyretha D. , Barnes Kelly A. , Reimel Adam J. , Nuccio Ryan P. , Ungaro Corey T. , Baker Lindsay B. 
Published: April 2020

Interleukin (IL) 1 cytokines, IL‐1α and IL‐1β, are well known for their immunological responses as blood biomarkers; however, it is unclear if sweat cytokine concentrations can be used to predict that of blood. The purpose of this study was to determine the correlations between sweat and serum for IL‐1α and IL‐1β concentrations measured during exercise. Nine moderately‐trained recreational athletes (35±7 y, 73.9±14.4 kg, VO2max 46.2±8.0 ml/kg/min) completed 90 min of cycling at 80±5% HRmax in the heat (31°C, 50% RH). Preceding exercise, left ventral forearm (LVF) was cleaned with alcohol and deionized water, and an absorbent patch (10 cm2 absorbent pad, 3M Tegaderm™ + Pad) was applied 10 min into exercise. The patch was removed at the end of 90 min of exercise alongside a synchronous blood draw. Sweat and serum IL‐1α and IL‐1β were measured using Multiplex (EMD Millipore, MagPix) IL‐1α and IL‐1β kits. Spearman correlation was performed to determine the relation between sweat and serum for IL‐1α and IL‐1β. Non parametric sign test was used to determine mean differences between sweat and serum. Cytokine measurements are shown as mean ± SD. There were no significant correlations between sweat and serum for IL‐1α (r=−0.19, p=0.61) or IL‐1β (r=0.20, p=0.61). There were significant mean differences in IL‐1α expression between sweat (1004.5±613.8 pg/mL) and serum (24.9±58.1 pg/mL) (p=0.004), and non‐significant mean differences in IL‐1β between sweat (2.3±1.9 pg/mL) and serum (1.3±1.3 pg/mL) (p=0.45). These results suggest that there were no significant correlations between sweat and serum IL‐1 cytokine concentrations. Unlike IL‐1β, IL‐1α expression in sweat was significantly different between sweat and serum. Therefore, it seems that IL‐1 expression in sweat may have limited utility in predicting blood IL‐1 concentrations.Support or Funding InformationThis study was funded by the Gatorade Sports Science Institute, a division of PepsiCo, Inc. The views expressed in this abstract are those of the authors and do not necessarily reflect the position or policy of PepsiCo, Inc.
Unraveling the Role of the Gut Microbiome in Iron‐Deficiency Anemia During Pregnancy
Authors: Celis Arianna , Relman David 
Published: April 2020

Iron‐deficiency anemia (IDA) during pregnancy is linked to preeclampsia, premature birth, and irreversible impairment to the neurocognitive development of the nascent child. IDA affects approximately 20% of all pregnancies, yet it is still underdiagnosed and the mechanisms for its cause are not fully understood. It has been documented that the gut microbiome can positively affect the availability of micronutrients, including iron, to the host, however, the specific microbes involved and the underlying mechanisms of how this occurs have not been elucidated. We are analyzing the composition of gut microbial communities in healthy and anemic pregnant women. Using shotgun metagenomic sequencing, strain isolation, characterization, intracellular iron quantification and proteomics, we aim to determine the proportions of iron scavengers and iron adaptable species. The bacterial species and gut microbiome compositions that could be associated with the development of IDA during pregnancy and the clues that these results provide for new preventative measures and more efficient treatment for IDA during pregnancy will be discussed.Support or Funding InformationMarch of Dimes, Stanford Microbiome Therapies Initiative
Relative Profibrotic Gene Expression in Cardiac Fibroblasts from Low Aerobic Capacity “Disease Prone” Rats Following Ischemic Reperfusion
Authors: Alsahly Musaad B. , Zakari Madaniah O. , Koch Lauren G. , Britton Steven L. , Nguyen Hilary M. , Fisher-Wellman Kelsey , Katwa Laxmansa C. , Lust Robert M. 
Published: April 2020

BackgroundIschemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic after myocardial infarction, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction, but little is known about the effect of intrinsic aerobic capacity on early fibroblast gene expression following ischemia reperfusion.MethodsOn each study day, one HCR/LCR pair (n = 8 each) was anesthetized and hearts were rapidly excised. The hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice for subsequent isolation of fibroblasts. Cardiac fibroblasts from the LV of HCRs and LCR rats were isolated, cultured and characterized. Immunohistochemistry verified fibroblasts/myofibroblasts markers. RNA was isolated from the cultured fibroblasts, and RT‐PCR was used to determine baseline gene expression for Collagen I and III, MMP2, TIMP1, BMPR2, ATR1, SMAD5, and Bag3 in each phenotype.ResultsImmunocytochemistry successfully confirmed the presence of both fibroblasts and myofibroblasts markers. In these post IR fibroblast/myofibroblasts, LCRs showed significantly higher gene expression (49%) in the collagen I/collagen III ratio but lower expression of extracellular matrix regulatory genes including MMP2 (20%), TIMP1 (17%), and SMAD5 (15%) compared to HCRs counterparts. BMPR2 (50%), Agtr1a (25%), also were both decreased, and the anti‐apoptotic gene, Bag3, was significantly (67%) higher in LCRs.ConclusionThese results indicate that the LCR phenotype is predisposed to expressing a more fibrotic character in fibroblasts harvested immediately following IR, and may indicate poorer long term outcome/earlier onset heart failure following acute ischemic injury to the heart. While the phenotype produced no differences in the acute injury, there may be phenotypic differences that develop later during resolution of the injury.
An Innovative Approach to Accommodate Different Learning Needs in Medical Histology course
Authors: Xu Lu , Wang Andrew , Sultan Angela , Krakosky Danielle 
Published: April 2020

BackgroundMedical Histology teaching has changed drastically over the last decade from traditional microscopy to virtual microscopy. In this digital age, the way that students learn has changed as well. Many students prefer independent study and preparation over traditional mandatory lectures and laboratory sessions. However, due to the hands‐on nature of Histology lab, not all the students were able to learn how to recognize tissues by themselves. We sought to develop an approach that would accommodate students’ different learning needs without compromising the practical elements of Histology.DescriptionTulane Histology course has transitioned to team‐based learning (TBL) in the 2018–2019 school year. The preparation materials for TBL were provided online including the Histology lab guide. Students can choose to do the Histology lab independently at home. Option lab sessions associated with TBL preparation materials were offered to the students who preferred learning under faculty supervision. A survey was administered to 191 students from 2018–2019 who experienced the TBL with optional lab approach.ResultsStudent satisfaction was significantly higher after implementing TBL with optional lab approach (p< 0.05; 3.79 > 3.34). Most students reported the new method to be helpful for their Histology learning. With the same number of faculty involved in the Histology lab, the approach led to an increase of a faculty to student ratio. Faculty reported an improved quality of discussion with students and less time spent in the optional lab compared with the traditional laboratory teaching.ConclusionUsing an innovative approach of combining TBL with an optional lab, we manage to accommodate students with different learning needs, maximize students’ learning efficiency, increase the faculty to student ratio during the contact time, improve the interaction between faculty and students, decrease faculty total time spent in the lab and increase faculty satisfaction.
A Crosstalk Between Glucosylceramide Synthase and Long‐Chain Fatty Acid Elongase is Critical for Regulating Cyst Production in an Intestinal Protozoan, Giardia lamblia
Authors: Enriquez Vanessa , Ellis Cameron , Das Siddhartha 
Published: April 2020

The waterborne parasite, Giardia lamblia, is a major cause of intestinal illness (giardiasis) worldwide. Giardia is classified as a model organism to study evolution because of its compact genome, reduced metabolic pathways, and lack of emblematic eukaryotic organelles. The infection by Giardia is transmitted by osmotically‐resistant cysts in contaminated food, water, and feces. It has been shown that giardial glucosylceramidesynthase (gGlcT1), an important enzyme of the sphingolipid metabolic pathway, plays a critical role in inducing encystation and cyst production. Modulation of gGlcT1 expression by overexpression interferes with cyst production and cyst viability in culture. In addition, our laboratory has reported that gGlcT1 is a dual‐substrate enzyme, with separate catalytic domains that facilitate the synthesis of both glucosylceramide and galactosylceramide, and its overexpression increases the uptake of membrane lipids and cholesterol significantly. Interestingly, although this parasite has a limited lipid synthesis ability, it expresses a long chain fatty acid elongase (gFAELO) gene, which is mostly expressed in cysts. Because FAELO enzymes are involved in remodeling of fatty acyl chain of membrane lipids and gGlcT1 overexpression increases lipid uptake by Giardia, we investigated whether the interplay between gGlcT1 and gFAELO is important for maintaining the lipid homeostasis in this parasite and whether that drives the process of cyst production. Five clones were developed to track gGlcT1 and/or gFAELO expression. Under different promoters, it was found that the enzyme expression of both enzymes and the enzymatic activity of gGlcT1 was modulated depending on life stage and co‐expression with gFAELO. The production and morphology of cysts was also affected as evaluated by encystation and confocal microscopy. These results suggest that the coordinated temporal expression of gGlcT1 and gFAELO alters lipid homeostasis in Giardia, which could be critical for infective cyst production.Support or Funding InformationThis work was supported by grant R01AI095667 from the NIH (NIAID). Vanessa was supported by the NIH RISE Grant 5R25GM069621 from NIGMS. The biochemical, molecular, and confocal microscopy experiments were carried out at the Genomic Analysis, Cytometry, Screening, and Imaging Core Facilities at the Border Biomedical Research Center (UTEP), supported by grant G12MD007592 from NIMHD (NIH).
Endoplasmic Reticulum (ER) stress‐generated microparticles self‐perpetuate ER stress and mediate endothelial cell dysfunction independently of cell survival
Authors: Agouni Abdelali , Osman Aisha , El Gamal Heba , Pasha Mazhar 
Published: April 2020

Microparticles (MPs) are nanosized membrane‐shed vesicles released from activated cells. MPs can induce endothelial dysfunction, the first step in atherosclerosis, through endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release and what biological messages these MPs would carry. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial function.Human umbilical vein endothelial cells (HUVECs) were treated for 24h with ER stress inducer, thapsigargin, in the presence or absence of 4‐Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat naïve HUVECs (20 g/mL) for 24‐48h before assessing ER‐stress response, autophagy and apoptosis.MPs generated under ER stress conditions did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. The treatment of naïve HUVECs with ER stress‐generated MPs (48h) increased mRNA and protein expression of key ER stress markers [activating transcription factor (ATF)‐4, and Binding immunoglobulin protein (BiP)], indicating a vicious circle between MPs production and ER stress activation. ER stress‐generated MPs impaired the angiogenic capacity of HUVECs to form tube‐like structures, indicating an impaired endothelial function. However, ER stress‐generated MPs did not affect autophagy (no changes in protein expression of LC3‐II) or apoptosis in HUVECs.Our data show that ER stress activation caused the shedding of MPs, which were capable of activating ER stress in HUVECs in a vicious circle and impaired angiogenic capacity of these cells. These MPs impaired endothelial function in a mechanism independent from cell death and autophagy.Support or Funding InformationThis work was supported by awards from Qatar University (QUUG‐CPH‐CPH‐15/16‐6; QUST‐2‐CPH‐2017‐19; QUST‐1‐CPH‐2018‐1) to Dr Abdelali Agouni. The findings achieved herein are solely the responsibility of the authors.
Applying the Brakes: Understanding the Conformational Changes in Kinesin‐5
Authors: Griffin Jessica P. , DeVry Jenelle , Robinson Kingston , Chaney Joseph 
Published: April 2020

Human Kinesin‐5 (Eg5), an anticancer drug target, is key to the assembly of the bipolar spindle during mitosis. Its promise as a clinical target comes from the fact that it is involved only in metaphase and not interphase like some of the leading clinical drugs; however, the overall mechanism of inhibition needs further exploration. Inspection of the crystal structures of Kinesin‐1 dimers reveal the addition of strands to the central β‐sheet is found in one head and absent in the other; this previously unrecognized structural asymmetry between the two heads may suggest a regulatory mechanism involving the N‐terminal Neck‐Linker (NL) and the C‐Terminal Cover‐Neck. These structural changes in the neck‐linker and cover‐neck of Kinesin‐5 have not been reported. Thus, the importance of establishing this conformational switch in Kinesin‐5 requires additional experiments for understanding and molecular validation. However, we must first understand how changes in the neck‐linker modulate kinesin function. We have made mutations at the start of the neck‐linker of dimeric Kinesin‐5 (1‐513) and KHC (1‐560). Our results indicate these neck‐linker mutants alter kinesin ATPase activity. This indicates that the neck‐linker participates in allosteric regulation of kinesin. Our future goal is to test the response to inhibition by current anticancer inhibitors such as S‐trityl‐L‐cysteine.Support or Funding InformationBUILD Technicians supported under grant #RL5GM118966 and BUILD undergraduate students under grant #TL4GM118968. An Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #5 P20 GM103424‐15 and 3 P20 GM103424‐15S1. The Louisiana Cancer Research Consortium (LCRC) and National Center for Research Resources RCMI program Grant #8 G12MD007595 and the Louisiana Biomedical Research Network Grant #8P20GM103424 The National Institute of Minority Health and Health Disparities of the National Institutes of Health under award #G12MD007595. Students are supported by GRAD Star Scholar Mini Grants through Center for Undergraduate Research and Graduate Opportunity (CURGO).
Oroxylum indicum extract does not induce hepatotoxicity in mice
Authors: Salamat Julia M. , Abbott Kodye L. , Majrashi Mohammed , Almaghrabi Mohammed , Govindarajulu Manoj , Ramesh Sindhu , Gill Kristina , Fahoury Eddie , Narayanan Natasha , Desai Darshini , Nadar Rishi , McElroy Edwin , Moore Timothy , Nagabhushanam Kalyanam , Majeed Muhammed , Pondugula Satyanarayana , Dhanasekaran Muralikrishnan 
Published: April 2020

While natural products exhibit several beneficial health effects, they can induce unintended hepatotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) has several health benefits such as anti‐inflammatory, diuretic, astringent, expectorant, anti‐arthritic, antipyretic, aphrodisiac antifungal, antibacterial and neuroprotective effects. However, it is unknown whether OIE induces hepatotoxicity. We sought to determine whether OIE administration in mice results in altered levels of serum biomarkers of hepatotoxicity and changes in liver histology. Male C57BL/6J mice were fed with powdered rodent food or powdered rodent food mixed with OIE (500mg/kg) daily for 4 weeks. After the treatment period, liver histology and serum biomarkers of hepatotoxicity were assessed. OIE did not affect the total body weight of the mice after 4 weeks of treatment period. OIE treatment did not alter the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, albumin, globulin and total protein. Similarly, OIE treatment did not result in a noticeable change in the liver histology. Taken together, these results suggest that OIE, at maximum concentration used for beneficial effects, may not induce hepatotoxicity in mice. However, dose‐dependent and time‐dependent future studies are warranted to confirm that OIE does not induce hepatotoxicity.Support or Funding InformationOIE (Sabroxy®) was provided by Sabinsa Corporation. This project was supported by Sabinsa Corporation, Animal Health and Disease Research Grant and Auburn University Research Initiative in Cancer Grant.