bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of in‐vitro exercise‐induced endothelial shear stress on adhesion molecule gene expression
Authors: Conde Daniel , Gomez Manuel , Garcia Mario , Morales-Acuna Francisco , Gurovich Alvaro
Published: April 2020

Background

Exercise‐Induced Shear Stress (ESS) is characterized as a mechanical stimulus effecting cellular morphology and physiology. Varying ESS intensity elicit different response mechanisms through cellular signaling pathways. Higher intensity ESS enhances the expression of genes involved in protection against atherosclerosis. Adhesion molecules expression is common in atherosclerosis. Therefore, the purpose of this study was to determine the effects of different intensities of pulsatile ESS on vascular cell adhesion molecule‐1 (VCAM1) and intercellular adhesion molecule (ICAM1) gene expression.

Methods

Commercially available human carotid artery endothelial cells (Sigma Aldrich) were cultured until 95–100% confluence and then exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour low‐intensity ESS (35 dynes/cm2), or high‐intensity ESS (80 dynes/cm2) on parallel fluidic units (Ibidi, USA). Ibidi Pump Control Software (version 1.5.4, Ibidi, USA) was used to control shear stress intensity. mRNA was extracted and expression of VCAM1 and ICAM1 was assessed using qRT‐PCR. Samples were run in Changes in gene expression were calculated using the ΔΔ‐CT method, using GAPDH as the normalizing control gene. One‐way ANOVA was used to asses statistically significant differences (p < 0.05) using Prism version 8 (Graphpad).

Results

The mRNA expression of VCAM1 and ICAM1 significantly increased (p <0.01). 6 and 8 folds respectively after exposure to low‐intensity ESS. Furthermore, high‐intensity ESS did not significantly increased the expression of VCAM1 and ICAM1.

Conclusion

Low‐intensity pulsatile exercise‐induced shear stress improves endothelial function in‐vitro through the increase in adhesion molecule’s gene expression.
Collaborative Exams to Promote Significant Learning in University Students in the Subjects of Cell Biology and Biochemistry
Authors: Arroyo Ana Gabriela Campos , Chávez Ricardo Adolfo Manivel , Flores Benjamin Rogelio Hernandez , Matías Flora María Cabrera
Published: April 2020

Some teachers and researchers share the concern that one of the main obstacles involved in learning theoretical subjects, such as Mathematics, Physics, Chemistry or Biological Sciences, is the little practical sense that the student sees in them. Also, at the time of evaluation, there is a great concern about the amount of information that must be understood or retained generating anxiety, stress, confusion, among other factors. Therefore, it is important to create collaborative learning strategies that encourage student interactions, promoting peer education to achieve meaningful learning.

Objective

Know if collaborative exams promote significant learning in the subjects of Cell Biology and Biochemistry.

Materials and methods

107 university students from two biology and two biochemistry groups were considered. During a semester the subjects were taught following the programmatic content of each one, the subjects were biased, and exercises were applied between pairs of review, in addition 3 exams were applied collaboratively (in pairs or teams of 3). At the end of the semester, an adapted survey by Stokes, J. (2019) was applied, with questions aimed at assessing perceived learning, knowledge retention, peer‐to‐peer teaching experience and feelings towards group work.

Results

94.9% of the students stated that they like to take collaborative exams, 91% consider collaborative exams to be a valuable learning activity and 92.3% mention that they helped them retain knowledge due to the interaction generated in the Time to decide the correct answer.

Conclusions

Collaborative exams are a peer learning strategy that can lead to meaningful learning, as well as helping to retain knowledge in subjects that are theoretical or even theoretical practical, improving their academic performance.

Support or Funding Information

Research supported and endorsed by the Michoacana University of San Nicolás of Hidalgo
Heparan Sulfate Modulates Heart Vascular Remodeling Induced by S. Aureus Sepsis
Authors: Morris Claire L. , Golden Greg , Toledo Alejandro Gomez , Esko Jeffrey D. , Nizet Victor
Published: April 2020

Sepsis is a life‐threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction and organ failure. Infection by Staphylococcus aureus infections can result in sepsis, which is correlated with remodeling of the vascular glycocalyx. Heparan sulfate (HS), a major component of the vascular glycocalyx, undergoes shedding during sepsis, but the impact of vascular HS and shedding on sepsis outcomes is unknown. In this study, we modified the composition of HS in endothelia cells in mice by crossbreeding animals bearing a conditional allele in N‐acetylglucosamine N‐deacetylase‐N‐sulfotransferase‐1 (Ndst1f/f) with mice bearing the Cre recombinase driven by the Tie2 promoter (Tie2‐Cre). Ndst1f/fTie2Cre mice exhibit HS with overall reduced sulfation in endothelial and myeloid cells. Ndst1f/fTie2Cre mice succumbed earlier to sepsis induced by S. aureus. Application of an in vivo vascular tagging method revealed drastic proteomic changes in the hearts of Ndst1f/fTie2Cre mice, including increased markers of cardiac damage. These studies provide a link between vascular heparan sulfate and early death due to S. aureus infection. Additional measures of characterizing cardiac damage will further explore the heart proteomics signature in Ndst1f/fTie2Cre mice to increased S. aureus susceptibility.
Regenerative properties of the humerus bone in the absence of Stat3
Authors: Lopez Noah , Rigueur Diana , Lyons Karen
Published: April 2020

The Interleukin‐6/STAT3 (IL‐6/STAT3) signaling pathway is essential for proper immune response to invading pathogens and viral infections. This signaling pathway has also been greatly investigated in cancer. Moreover, STAT3 has high implications as a therapeutic target for rheumatoid and osteo‐arthritis. Recent literature has found that the IL‐6/STAT3 signaling pathway plays an imperative role in the development of cartilage and bone. However, the direct role and the downstream effectors of STAT3 signaling have not been clearly delineated. Through skeletal preparations, X‐ray micrographs, and histology, our data show that conditional loss of Stat3 in mouse cartilage, via the Col2‐Cre driver, is essential for proper growth plate formation in the forelimbs of the skeleton. Furthermore, loss of STAT3 results in spontaneous bone fractures in the humerus. Interestingly, these severe fractures repair in adulthood, indicating an untapped mechanism for regeneration in bone. These studies will elucidate the imperative role the Stat3‐mediated pathway plays in modulating regeneration of the humerus during endogenous development and repair.

Support or Funding Information

NIH R01 80094
Antioxidant and Anti‐inflammatory Effects of the Methanolic Extract of Potentilla indica Fruits
Authors: Sereno-Villaseñor Luis , Hernández-García Alejandra , Torres-Martínez Rafael , Meléndez-Herrera Esperanza , Manzo-Avalos Salvador , Martínez-Flores Héctor Eduardo , Saavedra-Molina Alfredo , Salgado-Garciglia Rafael
Published: April 2020

Potentilla indica (Jacks.) Th. Wolf (Rosaceae) also known as mock strawberry, to which medicinal properties are attributed related to oxidative stress and chronic inflammation. A methanolic extract was obtained from the mature fruits of P. indica, and methanolic and chloroformic fractions of the extract were obtained by liquid‐liquid fractionation. The chloroformic fraction (1 mg/mL) presented the highest antioxidant activity by in vitro methods, 67.90% (TAC), 67.25% (DPPH) and 44.51% (ABTS). The anti‐inflammatory activity was tested using the carrageenan‐induced paw edema model in male Wistar albino rats (400 g). Ibuprofen was used as standard drug at concentration of 25 mg/kg of body weight. The methanolic extract and chloroformic fraction (dissolved in DMSO 5%) were administered topically and orally (by 28 days) at doses of 0.1, 1 and 10 mg/kg of body weight (50 μL). Control animals received an equal volume of vehicle. Edema was induced with 1% carrageenan to all the groups after four hour of treatments, and paw thickness was checked at 1, 2, 3, 4, and 8 h. Topical administration of the chloroformic fraction (1 mg/kg) decreased by 77.7% in paw thickness, and with oral administration decreased by 72% in paw thickness. The chloroformic fraction of methanolic extract of P. indica fruits had a high antioxidant activity and significantly decreased the inflammatory reactions induced by carrageenan.

Support or Funding Information

Support grant from CIC‐UMSNH (2.10 to RSG).
Chromatin Immunoprecipitation in Native Budding Yeast (Saccharomyces cerevisiae) Using Photo‐Crosslinking Unnatural Amino Acids
Authors: Brown Alon , Wilkins Bryan
Published: April 2020

Exploiting an expanded genetic code in yeast, we can site‐specifically encode unnatural amino acids into the native chromatin landscape of the living cell. Utilizing the UV‐activated photo‐crosslinking amino acid, p‐benzoylphenylalanine (pBPA), we can covalently trap protein‐protein interactions when contacts between proteins are made within binding distances of ~ 0.4 nm. We have mapped the RSC remodeler complex protein, Sth1, to the nucleosome and shown that its binding is regulated by posttranslational modifications, including acetylation and SUMOylation. RSC has been linked to promoter sites and we want to further our binding map with an understanding of its occupancy across the genome. To map the loci of binding sites we use a double IP chromatin immunoprecipitation (ChIP) technique that utilizes the power of in vivo protein‐protein crosslinking from histones. Histones are well associated with DNA and efficient targets for ChIP analysis; therefore, we propose that if the histone protein is first crosslinked to a nucleosomal protein target, that the histone can be initially precipitated in tandem with its crosslinked protein and associated DNA fragments. The target protein can then be further precipitated to purify DNA, via its interaction with the histone. Our first goal was to establish histone occupancy of pBPA‐containing histones. Using ChIP, we were able to isolate DNA from both wild type and mutant histones where we can achieve a quantitate analysis of the difference with qPCR. Building on this we will move to a double IP system to determine Sth1 occupancy. This work aims to establish a novel approach in ChIP targeting of proteins that are difficult to analyze due to more transient interactions at the nucleosomal core.

Support or Funding Information

NIH R15 grant
The Effects of Prolonged Mechanical Ventilation on Structural and Material Properties of Diaphragm Arterioles
Authors: Horn Andrew Gary , Kunkel Olivia Nicole , Baumfalk Dryden Ray , Simon Mikaela Elizabeth , Schulze Kiana Marie , Bruells Christian S. , Poole David C. , Behnke Bradley Jon
Published: April 2020

Introduction

In skeletal muscle arterioles, cellular and vasoactive responses are influenced by the physiochemical milieu of the surrounding tissue. Understanding the mechanical properties of the blood vessel wall yields insight into vascular function in health and disease. In blood vessels, the endothelium mediates flow‐dependent vasodilation and contributes to the regulation of arterial mechanics. Prolonged mechanical ventilation (MV)(>6 h) results in large, time‐dependent reductions in diaphragmatic blood flow and impaired endothelium‐dependent (and ‐independent) vasodilation of first‐order (1A) medial costal diaphragm arterioles. The purpose of this investigation was to determine if the severe reductions in diaphragmatic blood flow (and shear‐stress) with MV impair the structural and material properties (i.e. increased stress/stretch relation and/or circumferential stretch) of 1A arterioles from the medial costal diaphragm.

Methods

Female Sprague‐Dawley rats (~5 mo) were randomly divided into two groups, spontaneous breathing (SB, n = 6) and prolonged MV (6 h MV, n =5). Following SB and 6 h MV, 1A medial costal diaphragm arterioles (~200 μm diameter, ~2 mm in length) were isolated and cannulated. After cannulation, vessels were adjusted to a fixed length (L, 1.5 mm) and pressurized. Vessels that exhibited no leaks were subjected to a step‐wise passive‐pressure diameter test (0–140 cmH2O) in calcium‐free Ringer’s solution, at a single fixed axial length (L). Inner diameter (μm) and wall thickness (μm) were measured at each pressure step and used to calculate wall: lumen ratio, incremental distensibility (%/mmHg), Cauchy‐stress (μ), and circumferential stretch (λθ).

Results

Following 6 h MV, in the undeformed condition (0 mmHg) the wall: lumen ratio was significantly smaller compared to SB (1.04 ±0.21vs. 2.60 ±0.40; p<0.05). There were no differences in incremental distensibility (%/mmHg) and the stress/stretch (μvs λθ) relation between groups (p>0.05). At every pressure step, circumferential stretch (λθ; undeformed radius (r)/current deformed radius (R)) was significantly increased with 6 h MV versus SB (p<0.05).

Conclusion

Despite no change in the distensibility and structural behavior (stress/stretch) of resistance vessels from the medial costal diaphragm, prolonged MV resulted in altered material properties (i.e. elevated circumferential stretch (λθ)) of medial costal diaphragm arterioles. Increased circumferential stretch can enhance superoxide generation in the endothelium and vascular smooth muscle cells. Therefore, elevated circumferential stretch of 1A diaphragm arterioles in combination with the severe reductions in blood flow may, in part, explain the previously demonstrated vascular dysfunction in the diaphragm following MV.

Support or Funding Information

Supported by: NIH HL137156‐01A1
Team‐Based Learning (TBL) in upper‐division biochemistry: Identifying critical components for student learning
Authors: Offerdahl Erika G. , Arneson Jessie
Published: April 2020

Team‐based Learning (TBL) is an evidence‐based instructional practice (EBIP) commonly used in medical biochemistry contexts that requires students to acquire basic content knowledge outside of class and then work in collaborative groups to apply, extend, and/or synthesize knowledge in class (Michaelsen et al. 2004). An instructional approach that can be traced back to the 1970s, the “key ingredients” of TBL are well established. TBL is comprised of a three‐step cycle beginning with an individual preparedness test, a collaborative group preparedness test, and application‐based team exercises. The instructional design is informed by several core principles that are used to prescribe how teams are formed, the readiness assurance process (i.e. individual and team readiness), provision of immediate feedback to teams, how to incentivize engagement, and the need for peer review. The efficacy of canonical TBL as compared to didactic instruction has been demonstrated in a variety of contexts (e.g. undergraduate STEM, medical school education). The fidelity with which instructors implement TBL as prescribed is variable (Haidet et al. 2014). Indeed, it is common, if not inevitable, for instructors to adapt instructional approaches in response to local contexts and conditions. Yet it is unclear from the literature how adaptations of canonical TBL affect learning outcomes. In this research, we adopt a fidelity of implementation (FOI) approach (Stains et al. 2017) to empirically determine the effects of adapting TBL on student learning in an upper‐division, two‐semester biochemistry course. Specifically, we report on the effects of adaptations of the team formation and readiness assurance processes on student performance as observed in a multi‐year study.
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 Information

This work was supported by the Arnold and Mabel Beckman Foundation.
The Role of Protease‐Activated Receptor ‐4 in Vascular Smooth Muscle Cells
Authors: Gardner-Jones Carla , Simtion Kyle , Jackson Jamill , Wright LaKeshia , Motley Evangeline
Published: April 2020

Studies have shown that thrombin stimulates the activation of extracellular regulated kinase (ERK 1/2) and Rho/Rho Kinase, which are associated with cellular proliferation and migration in vascular diseases such as hypertrophy and atherosclerosis. Thrombin activates these kinases through protease activated receptors (PARs). PARs are a subfamily of G‐protein coupled receptors activated by proteolytic cleavage. PAR‐1, the prototype receptor, is known to mediate vascular smooth muscle cell (VSMC) migration, the hallmark of atherogenesis. However, little is known in regards to PAR‐4 function in VSMCs. To elucidate a role for PAR‐4 in VSMCs, we utilized PAR‐4 activating peptide AYPGKF to probe receptor function. AYPGKF induced approximately a 4‐fold increase in migration of VSMCs. We also observed rapid phosphorylation of a Rho kinase substrate, myosin light chain phosphatase (MYPT), and sustained phosphorylation of ERK.
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 Information

Michigan 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 Information

This 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.
Structural and Functional Characterization of Glucan Phosphatases from Agriculturally Relevant Crops
Authors: Teittinen-Gordon Saana , Hong Jiayue , Cole Molly , Raththagala Madushi
Published: April 2020

Starch is a key component in many aspects of daily life. Reversible starch phosphorylation by dikinases and glucan phosphatases is an essential step in plant transitory starch metabolism. Much of the progress of glucan phosphatases has been come from studying the proteins from Arabidopsis thaliana, a model species in plant biology. However, there are several unresolved questions as to how glucan phosphatases bind and locate phosphate groups within the complex starch structure and how we can harness phosphorylation to modify starch yield and properties in plant systems. The overall goal of this project is to expand our understanding of glucan phosphatases on to agriculturally relevant crop species and utilize them to improve starch degradation in vitro and starch yield in vivo. To identify glucan phosphatases from plant and algal species, BLAST searches were performed using the A. thaliana homologs as query sequences. We cloned and overexpressed Starch Excess4 (SEX4) and Like SEX Four 2 (LSF2) from Oryza sativa (rice), Zea mays (corn), and Solanum tuberosum (potato) in E. Coli and purified them to homogeneity using nickel affinity chromatography and size exclusion chromatography. Successful purification of SEX4 and LSF2 has allowed us to obtain functional and structural mechanisms of glucan dephosphorylation in transitory starch degradation.
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 Information

March of Dimes, Stanford Microbiome Therapies Initiative
Identification of a Transcription Factor that Represses Alkane Hydroxylase Expression
Authors: Peterson Celeste , O’Connor Thomas , Abbondanza Domenic , Smolarek Brandon , Dey Arup
Published: April 2020

Some gram negative and gram positive bacteria have evolved enzymes for metabolizing alkanes. A medium‐chain alkane hydroxylase has been found in the model organism Caulobacter crescentus, belonging to the CYP153 family of cytochrome P450 enzymes. However, the molecular mechanisms regulating expression of this enzyme are still unknown. We carried out a reverse genetics screen and RNA‐seq to identify CoxR as a transcription factor regulating this alkane hydroxylase. A deletion of coxR resulted in upregulation of both the alkane hydroxylase as well as ferredoxin and ferredoxin reductase enzymes. EMSA assays showed that CoxR bound directly to a short region of the alkane hydroxylase promoter. In summary, we have identified a novel transcription factor involved in metabolism of alkanes.
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

Background

Ischemic 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.

Methods

On 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.

Results

Immunocytochemistry 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.

Conclusion

These 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.
Determining the Binding Affinities of Starch‐Protein Interactions using Concanavalin based Sedimentation Assay
Authors: Pruitt Annabel , Mak Claudia , Raththagala Madushi
Published: April 2020

Starch is ideally suited as an energy storing molecule in plant systems because of its unique structure and biophysical properties. However, these same properties limit the accessibility of hydrolyzing enzymes that break down starch at night. Phosphorylation of the glucosyl residues of the outer layers of the starch granule by dikinases makes the starch granule partially water soluble and accessible to starch degrading enzymes such as amylases. The subsequent removal of phosphate groups by glucan phosphatases is necessary for complete starch hydrolysis. Despite their important biological roles, our understanding of how dikinases, glucan phosphatases and amylases interact with the complex starch granule is not complete. In biological context, determining the affinities of starch–protein interactions are challenging due to low binding affinities and the heterogeneity and complexity of the structure of starch. Using a concanavalin based in vitro sedimentation assay, the goal of this project is to develop a novel binding assay to measure the affinities of starch ‐protein interactions. We determined the Kd app for amylopectin and glucan phosphatase Starch Excess4 and compared that to currently existing methods to determine the validity of the method. The further optimization of our method will allow us to determine binding affinities of glucan phosphatase, dikinase and amylases families of enzymes.
An Innovative Approach to Accommodate Different Learning Needs in Medical Histology course
Authors: Xu Lu , Wang Andrew , Sultan Angela , Krakosky Danielle
Published: April 2020

Background

Medical 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.

Description

Tulane 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.

Results

Student 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.

Conclusion

Using 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 Information

This 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 Information

This 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.
Using CRISPR and Saccharomyces cerevisiae As a Tool To Inspire Interest In Biotechnology at Community College
Authors: Furlong Robert James
Published: April 2020

Clustered Inter‐spaced Short Palindromic Repeats (CRISPR) is a technology used to make accurate edits to the genome of a particular organism, cheaply and quickly. Saccharomyces cerevisiae (S. cerevisiae) is a model Eukaryote organism used in both industry and research for a number of applications. 88% of publications referencing CRISPR in the PubMed data base, approximately 9000 in all, have been published since January 2015 (Wolyniak et al 2019) indicating that CRISPR has become a sensation in the media, as well as the biotechnology industry. It is more imperative than ever that undergraduate students are able to gain experience with this technology to not only better prepare them for use in industry and research, but to also generate interest in the population. This experiment will take community college students sorted into three groups; those with 9+ hours of biology course work, those with 8–4 hours of biology course work, and those with 3 or less hours of biology course work, and have them complete an experiment derived from the protocol devised by Mans et al 2018, with supplemental information from the outline developed by Wolybiak et al 2019, to perform a CRISPR knock in experiment using the Beta‐galactosidase, an enzyme conserved in many organisms that ferments lactose (Jeurs, D. et al), DNA sequences from different organisms. The student’s comfort level with the subject material, as well as technique handling the varying technologies, learned through out the experiment will be assessed at the beginning, through out, and at the end of the experiment. Their perceived interest in CRISPR as a technology will also be tracked through out the study.
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 Information

BUILD 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 Information

OIE (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.
Formolized Cadaveric Specimens Immersion‐Recovery Protocol
Authors: Obando Fabian David Camacho , Rueda-Esteban Roberto Javier
Published: April 2020

Cadaveric dissection, promoted by multiple anatomists and institutions as a fundamental area of medical education and training , requires embalming of the bodies to disinfect and preserve them. Therefore, despite being a known toxic and carcinogenic agent, formaldehyde is still widely used for its characteristics and results. Our objective is to introduce a Formolized Cadaveric Specimens Immersion‐Recovery Protocol developed at our laboratory, the chemical reagents used, performed processes and results. The protocol consists on specimen’s immersion in an aqueous solution of potassium hydroxide (KOH) for its maceration, this process causes chemical reactions like lipids saponification and proteins breakdown. The immersion time varies from one specimen to another; however, KOH concentration should never exceed 10% (w/v), otherwise tissue hydrolysis may occur. After a profuse wash, oxidative degradation is carried out: oxidizing formaldehyde into H2O and CO2 through the degradation of hydrogen peroxide (H2O2). It is extremely important to avoid the contact of this agent with the previously used KOH, which would cause the decomposition of H2O2 into H2O, O2 and hydroxyls, without obtaining a proper formaldehyde oxidation. For this purpose, sodium hypochlorite can also be used, with variable results. To ignore this chemical basis limits the achievement of ideal results. This processes allow to obtain 60% more joint movement on goniometry measures compared with formolized specimens, with a change from grey color generated by formaldehyde to ocher. Likewise, the degradation of formaldehyde allows obtaining minimum levels of it in treated specimens, thus reducing health impact on those who handle the specimens. After the recovery process, our specimens were preserved with a low formaldehyde solution. Anatomy students were invited to a peer‐guided extracurricular activity, they interacted with both formolized and recovered specimens, and a likert questionnaire was used to evaluate their experiences with both techniques. Students referred that recovered specimens looked more realistic and considered them better to work with. They also mentioned recovered corpses had less sensory hassles, like eye watering, airway irritation and coughing. Previous studies have compared Thiel solution and formaldehyde fixation. Thiel‐preserved bodies are considered to be a better choice on dissection and surgical training, since tissues offer a more realistic experience. Nonetheless, this technique has disadvantages such as higher costs, vacuum conservation, and the need of trained personal to handle the specimens. Moreover, some chemicals and infrastructure are not available worldwide. An example of the former scenario is present with Ammonium Nitrate, key component in Thiel’s, which is strictly regulated in several countries

Support or Funding Information

Anatomy Laboratory, Universidad de Los Andes, School of Medicine