bluuebunny/crossref_metadata_2025 huggingface dataset:
hATTR : neurotrophic factors expression in Schwann cell line after Let7 transfection
Authors: M'Hammed Aguennouz , Francesca Polito , Rosaria Oteri , Ragusa Marco , Maria Di Giorgio Rosa , Giuseppe Vita , Anna Mazzeo
Published: April 2020

Neurotrophic factors (NF) promote the survival of specific neuronal populations. BDNF and GNF have physiological effects on neurons such as inducing morphological differentiation, enhancing nerve regeneration, stimulating neurotransmitter expression, and otherwise altering the physiological characteristics of neurons. These properties suggest that NF are highly involved in pathologies of the peripheral nervous system. Hereditary transthyretin amyloidosis (hATTR) is a life‐threatening disease transmitted as an autosomal dominant trait with phenotypic variability expression among patients. Recently, we characterize a differential miRNA cluster circulating in plasma of hATTR patients. In order to investigate how these microRNAs can modify the epigenetic of nerve tissues, we transfected a Schwann cell line with let7 to show the functional proteomic manifestations. Changes of BDNF and NGF expression patterns were studied using qPCR, Western blot analysis, ELISA and immunohistochemistry. Our preliminary data showed a down expression of BDNF and NGF in this cells after transfection. This suggest that Let7 could be involved in the pathogenesis and progression of hATTR.
Acute eCig Vapor or SHS Exposure Induces Inflammatory Signaling in the Adult Murine Lung
Authors: Hirschi-Budge K. , Tsai K. Y.F. , McCusker H. Graviet , Homer K. , Rock S. , Davis T. , Llavina S. , Fowers R. , Long M. , Jensen T. , Arroyo J. , Graff T. , Reynolds Paul R.
Published: April 2020

The detrimental effects of exposure to tobacco smoke have been confirmed repeatedly over the last several decades. Primary smoking produces the greatest damage; however, secondhand smoke (SHS) is also notably detrimental. More recently the development of electronic cigarettes (eCigs) has introduced a new aspect to the study of smoke exposure. A heating element is used by eCigs to vaporize an oil‐based carrier that contains nicotine and various flavorings. eCig liquid and aerosols have been linked to increased cytotoxicity, inflammation, and production of reactive oxygen species. Negative effects on respiratory health have also been confirmed. Both tobacco smoke and eCig vapor have been found to alter cellular signaling and activate inflammatory responses. Of note, inflammatory pathways include signaling through receptors for advanced glycation end‐products (RAGE). Our objective in this study was to examine effects of acute exposure to eCig vapor compared to SHS in the murine lung. Wild type mice were exposed to vapor containing Apple or Cinnamon flavoring, or to SHS for a period of four days using a nose‐only delivery system (Scireq). Exposure consisted of 30 minutes of eCig vapor or SHS generated by three cigarettes over a period of 30 minutes. Control mice were exposed to room air. Animal use was in accordance with approved IACUC protocols at Brigham Young University. After exposure, lung tissue was collected: the left lung lobe was inflated for histological analysis, and the right lung lobes were snap frozen for protein and RNA analysis. H&E stained lung sections did not show changes in morphology and staining for pulmonary cell types using cell‐specific antibodies (SPC, CCSP, T1a and Fox J1) revealed evidence of cell population alterations potentially explaining increases in pro‐apoptotic cleaved caspase‐3 following exposure. Immunoblotting for RAGE and signaling intermediates activated by RAGE such as NF‐kB and pERK revealed differential expression following eCig or SHS exposure. These results indicate that activation of inflammatory signaling pathways occurs following an acute four‐day exposure. Such signaling in eCig vapor exposed mice has potential to lead to chronic inflammatory disease characteristics with longer exposures potentially leading to chronic obstructive disorders resulting from tobacco smoke exposure.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).
CRISPR in the Classroom – Engaging with Ethics
Authors: Smith Emmett M
Published: April 2020

CRISPR‐Cas9‐mediated human genome editing has rocketed into the scientific world, creating a frenzy of speculation about the future of humanity. Education surrounding this methodology is critical for an enlightened discussion of the technology and its potential application to humankind. I designed an undergraduate course “The Perfect Human: Promises and Perils of Genetic Engineering” to address this concern. This course is dedicated to discussing the science of CRISPR, then extending that to consideration of ethics, socio‐economic factors, politics and religion. It is open to first‐year students of any major or background on campus. We cover 5 modules: 1) The Science – An Introduction to Genetics 2) The Revolution: Editing the Human Genome 3) The Pathway: Can We Create Designer Embryos? 4) The History: Eugenics and 5) The Implications: Where Do We Go From Here? Students engage with primary literature, review articles and position papers from top scientists around the globe. We closely follow current events surrounding CRISPR technology. Most classroom sessions are centered around discussion. Students are asked to write two 5–6 page opinion papers as the semester progresses. Their final project is to create a policy document that can be handed to the Indiana State Government addressing whether or not human germline editing should be allowed in Indiana reproductive clinics. Students enjoy the challenge of this course. One stated “The most positive features about this course were learning about prevalent scientific discovery and the pioneering of genetic modification, which is and will be very important in the lives of our generation. After learning about different processes, we were able to have discussions on ethics and what the future may hold. With the knowledge I have now, I am able to go online and have educated conversations as well as understand news about CRISPR‐Cas9, PGD and the history of eugenics.” Another student shared “The most positive features of this course were the delivery process of informing all of us into the basics, history, perils, and promises of genetic engineering. Well informed and educated to know at least the general overview of current and past genetic engineering articles. Also, this class made me fully aware of current events. Finally, I loved learning how genetic engineering works, had worked, and its potential. An exciting future awaits!” Here I share strategies to incorporate ethics discussion into genetics classrooms and provide activities and prompts that can be used in many classroom settings.
Total and Afferent Renal Denervation Blunts Hypertension and Central and Renal Inflammation in the Developmental Phase of 2‐Kidney, 1‐Clip Hypertension
Authors: Lauar Mariana Ruiz , Van Helden Dusty , Banek Christopher T. , Evans Louise C. , Menani José Vanderlei , Osborn John W.
Published: April 2020

Hypertension (HTN) is a multifactorial disease that affects one third of the population of western countries. Although 95% of cases are of unknown origin (i.e., primary HTN), it is well established that renal ischemia results in secondary renovascular HTN. Experimental renovascular HTN is induced in rats by placing a silver clip on the left renal artery creating a stenosis, which decreases renal perfusion pressure and increases renin release from the clipped kidney (2K1C‐HTN). Previous studies indicate a role of renal nerves in 2K1C‐HTN since renal denervation (RDN) attenuates HTN in 2K1C‐rats. Moreover, activation of the sympathetic nervous system in this model is associated with inflammation in the hypothalamus, a key site for regulation of sympathetic activity and an area that also receives input from afferent renal nerves. In the present study, we tested the hypothesis that renal nerves modulate the levels of hypothalamic and renal proinflammatory cytokines and mean arterial pressure (MAP) during the development of 2K1C hypertension in rats. Male Sprague‐Dawley rats (150–180 g, n=5–8/group) were instrumented with telemeters to measure MAP. One week later a clip was placed on the left renal artery of the rats to generate 2K1C HTN. In the same surgery rats also underwent either total or afferent RDN (2K1C‐TRDN or 2K1C‐ARDN) of the clipped kidney. In sham rats (2K1C‐sham) the left renal artery was exposed but not denervated. MAP was continuously measured for the following 6 weeks after which rats were euthanized and brains and kidneys removed for measurement of hypothalamic and renal cytokine content. MAP at the end of the study was 160±6 mmHg in 2K1C‐sham rats. In contrast, MAP was significantly lower in both 2K1C‐TRDN (135±7 mmHg) and 2K1C‐ARDN (133±7 mmHg) rats. TRDN and ARDN statistically reduced hypothalamic protein levels of TNF‐α (0.24±0.01 and 0.2±0.01 pg/mg, respectively) compared to 2K1C‐sham rats (0.3±0.02 pg/mg). Similarly, ARDN statistically reduced hypothalamic IL1β, IL‐2 in 2K1C rats. TRDN trended to the same effect but this was not statistically significant. Renal inflammatory cytokines/chemokines (MCP‐1, IL‐6, TNF‐α) were increased in clipped compared with non‐clipped kidney in 2K1C‐rats. Moreover, ARDN and TRDN statistically reduced this inflammatory response. These results suggest that RDN decreases hypothalamic and renal inflammation in 2K1C hypertensive rats and this may be one mechanism by which RDN decreases MAP in the developmental phase of the 2K‐1C hypertension model.

Support or Funding Information

NIH R01 HL116476, FAPESP (2018/11221‐1) and PROPE/FUNDUNESP
Evidence of the Intrinsic Coagulation Pathway and Contact System Activation in Sickle Cell Patients During Steady State
Authors: Pawlinski Rafal , Henderson Michael W. , Ilich Anton , Sparkenbaugh Erica , Key Nigel S.
Published: April 2020

A chronic hypercoagulable state and increased risk for venous thromboembolism are prominent features of sickle cell disease (SCD). This hypercoagulable state is mediated by activation of both the intrinsic and extrinsic coagulation pathways. There is growing interest in targeting components of the intrinsic coagulation pathway (factor XII and XI) to prevent thrombotic disorders, primarily because this goal may be attainable without incurring any significant bleeding risk. In addition, FXIIa can also activate plasma prekallikrein to kallikrein (PK), which initiates the contact pathway. We have previously reported that FXIIa contributes to the prothrombotic state and vascular inflammation in sickle mice. In this study we investigated if the intrinsic and contact pathways are activated in SCD patients.

Fifty four African American outpatients with SCD and twenty three healthy African‐Americans were recruited. The study was approved by the University of North Carolina’s Institutional Review Board for human subjects. Platelet‐poor plasma was prepared from blood drawn into 3.2% sodium citrate (9:1). To detect activation of FXII, FXI, FIX and PK we have developed sandwich ELISAs to quantitate plasma levels of complexes formed between active forms of these proteases and their natural inhibitor C1 esterase inhibitor (C1). We used mouse monoclonal antibodies specific to the proteases to capture the complexes and a sheep polyclonal antibody to recognize C1. The polyclonal antibody is biotinylated, and detected with streptavidin‐horseradish peroxidase. Protein standards are generated by combining ~6μM purified proteases with ~12μM recombinant C1 for 18 hours at 37C, and quenched with 25μM PPACK. These standards are diluted to achieve the working linear range of the assay.

Compared to healthy controls, plasma levels of FXIIa‐C1 (1.07±0.19 vs 2.33±0.29nM; mean ±SEM), FXIa‐C1 (0.47±0.09 vs 0.98±0.10nM) and FIXa‐C1 (0.84±0.13 vs1.43±0.16nM) were all significantly elevated in SCD patients (p<0.05 for all groups). Similar to that plasma levels of PK‐C1 complexes were also elevated in SCD patients (12.50±1.06 vs 16.74±1.05; p<0.05). In the SCD patient group, highly significant correlations were observed between activation of FIX and activation of both upstream components of the intrinsic pathway (r=0.91; p<0.0001 for FXIIa and r=0.91; p<0.0001 for FXIa). Interestingly, a significant correction was also observed between FIX‐C1 and PK‐C1 complexes (r=0.64; p<0.0001).

Our data strongly suggests that both the intrinsic coagulation pathway and contact system are activated and contribute to the chronic hypercoagulable state observed during steady state in SCD patients. The contribution of these two pathways to the pathology of SCD should be further investigated.

Support or Funding Information

The authors wish to acknowledge support from NIH grants 1UO1HL117659 and R01HL142604
Molecular Pathogenesis of Bone Degenerative Disease and Associated Inflammatory Processes
Authors: Joshi Priya , Amour Maryanne , Siddiqui Fakiha , Liles Jeffrey , Hoppensteadt Debra , Fareed Jawed , Hopkinson William
Published: April 2020

Introduction

Osteoarthritis is one of the most common forms of arthritis, which is a result of the degeneration of protective cartilage at the end of bones, resulting in friction in the joints. This is due to a complex pathophysiologic process where inflammation and physical alterations of the bone contribute to the overall pathology. During this process, several mediators of inflammation and matrix degradation are formed. Profiling of some of these mediators and related biomarkers provides a molecular approach to understand the processes involved in the progression of this disease. The aim of this study is to profile various biomarkers of inflammation and hemostatic activation in osteoarthritis patients undergoing total joint replacement.

Materials and Method

Seventy‐four EDTA plasma samples were collected from osteoarthritis patients prior to their surgery. The control comprised of a pool prepared from normal male and female EDTA plasma. Such coagulation markers as D‐Dimer, vWF, MP‐TF, PAI‐1, TAFI, and fibronectin were performed using ELISA techniques. Matrix metallopeptidase 9 (MMP‐9) and C‐reactive protein were also measured using ELISA methods. All results were compiled as group means and compared with EDTA controls. Appropriate statistical methods were used to analyze the data.

Results

The circulating level of D‐Dimer was much higher on average in the patient samples (9178 ± 3.56 ng/ml) in comparison to the control samples (115 ± 85 ng/ml) p<0.05. The concentration of vWF was higher (125 ± 28 %) than in the control sample (104 ± 7.4%) p<0.05. There was not a significant difference between the MP‐TF or TAFI concentrations in the total joint samples vs the controls p<0.05. There was no correlation among any of the tested biomarkers.

Conclusion

This data shows that osteoarthritis patients exhibit marked elevation of inflammatory and hemostatic activation biomarkers. Wide variations are noted in the circulating level of these biomarkers in patients. This data suggests that multifactorial processes are involved in the pathogenesis of osteoarthritis. Profiling of inflammatory and thrombotic biomarkers may be helpful in the risk stratification and risk management in these patients.
Numerical method analysis of the activation and time‐dependent inhibition of midazolam metabolism by ticlopidine
Authors: Paragas Erickson Mesina , Nagar Swati , Korzekwa Ken
Published: April 2020

Drug metabolism plays an important role in the pharmacokinetic and pharmacodynamic properties of drug candidates. In most cases, drug metabolizing enzymes (DMEs) display non‐Michalis‐Menten or atypical kinetics. Atypical kinetics includes activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. These kinetics make it difficult to obtain in vitro kinetic constants to be used for in vivo predictions of drug‐drug interactions. Numerical methods offer a unique tool to estimate improved kinetic constants. In this study, we use ordinary differential equations to model the activation and time‐dependent inactivation of midazolam (MDZ) hydroxylation by ticlopidine. Ticlopidine (TCP) is the first thioenopyridine‐based prodrug developed to inhibit platelet aggregation. Time‐dependent inactivation (TDI) assays showed both activation and partial inactivation of midazolam hydroxylation. Co‐incubation of MDZ with different TCP concentrations (0–250 μM) activated the formation of 1‐hydroxymidazolam, increasing kcat by 30%. Additional experiments are currently underway to model the observed activation and time‐dependent inactivation of CYP‐mediated metabolism of MDZ by TCP.

Support or Funding Information

Funding: NIGMS grants R01GM114369 and R01GM104178.
Integrative Analysis of Long Non‐Coding RNAs and mRNAs in Lungs of Sickle Cell Mice
Authors: Kang Bum-Yong , Chang Sarah S. , Park Changwon , Archer David R. , Sutliff Roy L. , Hart C. Michael
Published: April 2020

Rationale

Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) and causes significant morbidity and mortality. Emerging evidence indicates that long non‐coding RNAs (lncRNAs) play a pivotal role in biological processes such as, cellular proliferation, differentiation, and apoptosis. However, the role of lncRNA and mRNA interaction in SCD‐PH pathogenesis has not been defined. We sought to evaluate the expression profiles of lncRNAs and mRNAs in lungs of sickle cell (SS) mice that spontaneously develop PH.

Methods and Results

Total RNAs were isolated from lungs of SS mice and littermate control (AA) mice at age 15–17 weeks and subjected to lncRNA and mRNA expression profiling using the Arrystar™ lncRNA array (Rockville, MD). Raw signal intensities were normalized in quantile method by GeneSpring GX software (Agilent Technologies, Santa Clara, CA). Volcano plot filtering was used to screen for differentially expressed (DE) lncRNAs and mRNAs with statistical significance (Fold Change >2.0, p< 0.05). To validate DE lncRNAs and mRNAs, 5 lncRNAs and 5 mRNAs were selected for quantitative real‐time PCR (qRT‐PCR). 2302 lncRNAs and 1753 mRNAs were upregulated (Up) and 2546 lncRNAs and 1455 mRNAs were downregulated (Dn) in lungs of SS mice compared to AA mice. Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics analysis was used for Gene Ontology (GO) enrichment including biological process (BP), molecular function (MF), and cellular component (CC), and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO analysis showed that the altered mRNAs were mainly distinct to negative (Up) or positive (Dn) regulation of biological processes in BP, ion binding (Up) or organic cyclic compound binding (Dn) in MF, and membrane‐bounded organelle and vesicle (Up) and non‐membrane‐bounded organelle and intracellular organelle (Dn) in CC. Pathway analysis revealed that malaria and MAPK signaling pathway were significantly associated with upregulated mRNAs, whereas C‐type lectin receptor signaling pathway, focal adhesion, and JAK‐STAT signaling pathway were related with downregulated mRNAs. LncRNA‐mRNA co‐expression network analysis was evaluated by Coding‐Non‐coding gene Co‐expression network (CNC network) analysis and generated by Cytoscape software (The Cytoscape Consortium, San Diego, CA).

Conclusions

These results identify potential new targets for therapeutic intervention in SCD and provide new insights into SCD pulmonary vascular dysfunction and PH pathogenesis.

Support or Funding Information

NIH HL133053 (BYK)
Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase
Authors: Huynh Katherine , Hardin Taylor , Guzman Lucero , Castillejo Nadia , Palfey Bruce , Vey Jessica
Published: April 2020

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a further decorated form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes an sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.

Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 μM, which compares well with other Class D monooxygenases. We are also investigating the effect of pH (pH 5.5 to 10.5) on C4a‐OOH formation and elimination. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination, but has no effect on the intermediate’s formation.

Support or Funding Information

California State University Program for Education & Research in BiotechnologyNIH SCORE 5SC3GM122652
Exploring the Effects of Increased Bacteria Exposure on Late Gestation Fetal Sheep
Authors: Yu Kevin , Keller-Wood Maureen , Wood Charles E.
Published: April 2020

The concept of the fetal microbiome is beginning to gain traction as new evidence of bacterial presence in fetal tissues arise. With evidence of bacteria in fetal tissues mounting, it becomes important to find out how the fetus responds to increasing presence of bacteria. In our previous studies, we reported that small numbers of bacteria (too few to cause fever or behavioral response) inoculated into the maternal sheep during late gestation can be detected in fetal tissues. In the present study, we hypothesize that transfer of these low numbers of bacteria from mother to fetus stimulate a transcriptomic response in the fetal brain, and that the response is related to inoculation dose. In this study, we used tissues from our previous study in which we inoculated Staphylococcus aureus bacteria into maternal sheep during late gestation. We studied three experimental groups: Naïve (n=6), 300 CFUs (colony forming units injected into the maternal bloodstream) (n=7), and 30,000 CFUs delivered to maternal bloodstream, mouth, and vagina (n=7). Inoculation doses were chosen to ensure low enough dose to not cause fever or anorexia in the sheep. Fetal cerebral cortex samples were collected 3–5 days post inoculation. RNA was extracted and gene expression analyzed using RNAseq. Validation of RNAseq differential expression results was performed using qPCR. Gene expression in the three groups were minimally overlapping in Principal Component Analysis. Using conservative criteria (p<0.05 after Bonferroni correction, we identified 53 differentially expressed genes (DEG) comparing Naïve to 300 CFU and 60 DEG comparing Naïve to 30000 CFU. We conclude that bacteria passing from mother to fetus stimulate transcriptomics responses in the fetal brain. We speculate that this altered gene expression might alter the course of fetal development.

Support or Funding Information

This work was supported by HD033053, AI120195, and HL083810
Differential white blood cell numbers in response to lipopolysaccharide exposure in marine and terrestrial mammals
Authors: Zenteno-Savín Tania , Symon Taryn Elizabeth , Reyes-Ramos Carlos Armando , Lugo-Lugo Orlando , Gaxiola-Robles Ramón , Vázquez-Medina José Pablo , Ramírez-Jirano Luis Javier , Hernández-Camacho Claudia Janetl
Published: April 2020

Marine mammals, such as bottlenose dolphins (Tursiops truncatus) are often seen with wounds, bruises or shark bites down to the muscle, apparently with no signs of infection. In terrestrial mammals, including humans, similar injuries would be associated with infections, inflammation and potentially death. The objective of this study was to compare the differential leukocyte numbers in marine and terrestrial mammals under control conditions and following exposure to lipopolysaccharide (LPS). Blood samples were collected following routine procedures from healthy human anonymous volunteers from the blood bank at Instituto Mexicano del Seguro Social, La Paz, Baja California Sur, Mexico, and from healthy dolphins at CaboDolphin. All samples were taken with ethylenediaminetetraacetic acid (EDTA) as anticoagulant, transported to the laboratory on ice and processed within 24 h. Leukocytes were separated by centrifugation with ficoll hypaque and subjected to cell culture at 35°C with 5% CO2 and constant humidity. Leukocytes (106 cells) from both species were exposed to 10 μg LPS μL−1 for 24 or 48 h and number of each leukocyte cell type was registered; cell type was assigned based on morphological characteristics. Under control (no LPS) conditions, number of lymphocytes from both species was higher at 24 and 48 h as compared to the initial numbers. In humans, but not in dolphins, lymphocyte numbers were lower in cells exposed to LPS for 24 or 48 h as compared to untreated cells. In both species, at all times and regardless of exposure to LPS, an inverse relationship was observed between lymphocyte and neutrophil numbers. In humans, basophil number was lower in cells exposed to LPS for 24 and 48 h as compared to untreated cells. In T. truncatus only one cell with morphology similar to basophils was observed in only one individual. These differences in leukocyte types between species in response to LPS exposure could contribute to explain the apparent lack of infection or inflammation in injured marine mammals. This research was conducted in accordance with institutional ethical standards and following the 1964 Declaration of Helsinki and later amendments and in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. The research protocol (2018‐785‐010) was approved by the Ethics Committee of Instituto Mexicano del Seguro Social (CONBIOÉTICA‐09‐CEI‐009‐2016061).

Support or Funding Information

Consejo Nacional de Ciencia y Tecnología (CONACYT CB‐2016‐01‐283669)
Discovery of First‐in‐Class Small Molecule Adhesion GPCR Modulators
Authors: Vizurraga Alexander , Yeung Jennifer , Tall Gregory
Published: April 2020

GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs). These GPCRs are unique in their possession of an autoproteolytic extracellular domain that cleaves the receptor prior to activation. GPR56 is a model AGPCR that couples to G12/13 family G‐proteins and we recently discovered that it functions as a platelet collagen receptor important for hemostasis. Based on experimental evidence thus far, we believe that GPR56 is a first‐acting collagen sensor for platelets – enabling them to first recognize blood vessel injury and to initiate the platelet program that leads to repair. Despite being biologically relevant in a number of cellular processes and diseases, there is a distinct lack of available small molecule tool compounds for AGPCRs, including GPR56. We conducted a large‐scale high throughput screen to find agonists and antagonists of GPR56. Over 160,000 compounds were screened in conjunction with UM’s Center for Chemical Genomics (CCG) using a cell‐based serum response element (SRE) luciferase gene reporter assay in HEK293T cells. Primary screening yielded ~30 promising candidate full agonists and 8 candidate antagonists that robustly activated or inhibited GPR56 in cell‐based and biochemical assays. These compounds are currently being used in orthogonal platelet activation assays. Knowledge of these receptors is still sparse; the discovery of high affinity agonists and antagonists will be useful probes to explore AGPCR activities in endogenous contexts. These compounds may also serve as leads for the development of GPR56‐targeted pro‐ or anticoagulants.

Support or Funding Information

GM120110, NS103946, T32‐GM007315
Assessing the ABCG2 Protein Interactome: a Potential Strategy to Disrupt ABCG2 Function
Authors: McHarg Ailsa Gavan , Gose Tomoka , Schuetz John D.
Published: April 2020

The ATP‐binding cassette sub‐family G member 2, ABCG2, is an important plasma membrane transporter that removes potentially harmful endogenous toxins and xenobiotics from cells. ABCG2 was identified as an important contributor to the multidrug resistance phenotype in cancer cells because it exported a wide variety of structurally diverse chemotherapeutic agents, including mitoxantrone, topotecan and the tyrosine kinase inhibitors. High ABCG2 expression in a variety of tumours is associated with poor clinical outcomes. In our present study, we hypothesized that proteins that interact with ABCG2 modulate its function. This knowledge would allow us to discover novel therapeutic targets that could be exploited to alter ABCG2 expression and/or function.

To identify interacting proteins for ABCG2, we employed a site‐specific biotinylation system utilizing a 15 amino acid peptide tag known as AviTag, fused to ABCG2. In the presence of the E. coli biotin ligase (BirA), biotin can be covalently attached to the AviTag peptide and the complex can be purified using the non‐covalent streptavidin‐biotin interaction. A plasmid harboring both AviTag‐ABCG2 and an IRES directed BirA was constructed. We stably expressed it in HEK293 cells and confirmed that AviTag ABCG2 was expressed at the plasma membrane and retained typical ABCG2 transport activity. Importantly, the cells expressing the AviTag‐ABCG2 and BirA readily labelled AviTag‐ABCG2 with biotin. The AviTag‐ABCG2 protein complexes captured by streptavidin agarose beads were purified and eluted according to an empirically developed protocol.

Using a proteomic approach with mass spectrometry, we identified 39 proteins (both known and novel ABCG2 interacting proteins) at a p‐value threshold of 0.05 in the AviTag‐ABCG2 protein complex compared to the negative control. As expected, ABCG2 was one of the top 10 proteins in abundance (high spectral counts). Among the 39 interacting proteins, we confirmed by immunoblotting that several important cancer‐related proteins interact with ABCG2. These proteins appear capable of regulating ABCG2 function in cancer cells.

Future studies will elucidate how these interactions affect ABCG2 function. These findings might aid in the development of new strategies for blocking ABCG2 function in multidrug resistant cancers, which could have important implications in improving patient’s response to chemotherapy.

Support or Funding Information

This work was supported by NIH and ALSAC
Evaluation of the mitragynine content, levels of toxic metals and the presence of microbes in kratom samples purchased in the Western Suburbs of Chicago
Authors: Prozialeck Walter C. , Edwards Joshua R. , Plotkin Balbina J. , Sigar Ira , Grundmann Oliver , Veltri Charles A.
Published: April 2020

Kratom (Mitragyna speciosa) is an herbal product, indigenous to Southeast Asia, that has both stimulant and opioid‐like effects. Kratom leaf products are being increasingly used in the West for their reputed benefits in the treatment of pain and opioid use disorder. Recently, the US FDA and CDC have raised concerns regarding the contamination of some kratom products with toxic metals (Pb and Ni) and microbes such as Salmonella. To further explore this issue, 8 different kratom products were legally purchased from various “head”/”smoke” shops in the Western Suburbs of Chicago and then tested for microbial burden, a panel of metals (Ni, Pb, Cr, As, Hg, Cd), and levels of the main psychoactive alkaloid mitragynine. All of the samples contained significant, but variable, levels of mitragynine (3.9–62.1 mg/g) indicating that the products were, in fact, kratom. All but 2 of the samples tested positive for the presence of various microbes including bacteria and fungi. However, none of the samples tested positive for Salmonella. The same 6 samples that contained microbes also showed significant levels of Ni (1.7–7.9 μg/g), Pb (0.32–1.7 μg/g) and Cr (0.23–4.1 μg/g) while the other 2 samples were negative for metals. These data indicate that many kratom products contain variable levels of mitragynine, and can contain significant levels of toxic metals and microbes. These findings highlight the need for more stringent standards for the production and sale of kratom products.

Support or Funding Information

This work was supported by personal funds from Dr. Walter Prozialeck and by restricted funds from Midwestern University.
East Coast Versus West coast: Medical Student’s Anatomy Lab Experience
Authors: Nair Rakesh Ravikumaran , Mohan Gisha , Nausheen Fauzia
Published: April 2020

Objectives

Anatomy labs are traditionally taken as first operation room of medical students where future doctors make their first incision. Although, some new medical school are not dissection based but they implement many proactive pedagogies to make anatomy learning an interesting, interactive, engaging and exciting experience. We looked into similar teaching methodologies of two US medical schools : Florida International university‐COM and California University of Science and Medicine‐SOM.

The objective of this study is to compare the student’s perception of anatomy lab teaching strategies of two medical schools based on student’s end of the course evaluations and final grade

Methods

we developed a collaboration between FIU‐COM and CUSM‐SOM anatomy faculty to share the curriculum, evaluation comments and performance of medical students after completion of Anatomy course. We are in the processes of getting exempt from IRB. The study data will include students feedback comments on teaching strategies and their final grade average for the course.

Results

As a result of our collaboration, we reviewed the Course curriculum, evaluation comments and performance of medical students after completion of anatomy course between 2 universities. FIU curriculum is discipline based in year 1 and system based in year 2. The lectures are mostly didactic, and labs are led by the instructors. The teaching methodologies in lab include: a checklist of objectives, the identifications on plastic models, plastinated specimens and prosected cadavers. Students learn the structures and confirm their understanding of the anatomy with facilitators. During the discussion, facilitator review the high yield structures to the whole group using the DOC‐cam projector.

CUSM‐SOM has a system‐based curriculum that is built on the active learning, student centered teaching strategies. Classes are mostly teams‐based and flipped classrooms. The labs are mostly student‐centered with multiple stations that student rotate every 20–30 min. One lab of 4 hours typically have 6–8 stations. Various aspects of anatomy of the same parts are learned at each station. These include: the gross anatomy specimens, embedded quizzes on specimens, videos, radiology images, hands‐on ultrasound experience and application questions. Final data analysis of the variables will be done after obtaining IRB exempt.

Conclusion

The student evaluations from two different school’s anatomy lab teaching methodologies will hopefully identify the most effective teaching strategies that is most received and accepted by the students of this era. Collaborative efforts can improve anatomy lab teaching and student learning.
Regulator of G protein Signaling 10 (RGS10) functionally interacts with store‐operated calcium entry machinery to regulate COX‐2 and TNF‐α in Microglia
Authors: Wendimu Menbere Yisfashewa , Alqinyah Mohammed , Vella Stephen , Moreno Silvia N.J. , Hooks Shelley
Published: April 2020

Activation of microglia is a driving factor of neuroinflammation and a hallmark of neurodegenerative diseases. The central inflammatory enzyme cyclooxygenase 2 (COX‐2) and the pro‐inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α) mediate critical features of microglial activation responses, and their upregulation in the brain is a characteristic feature of many neurodegenerative diseases and brain injury. The Regulator of G protein Signaling 10 (RGS10), a small RGS protein enriched in microglia, has G protein‐independent functions in suppressing lipopolysaccharide (LPS)‐induced expression of COX‐2 and TNF‐α. Herein, we identify a biochemical and functional interaction between RGS10 and the endoplasmic reticulum (ER)‐localized calcium sensor STIM2, which is an essential component of the store‐operated calcium entry (SOCE) machinery. We determined that STIM2 and its downstream calcium pathway, particularly Orai channels and the Ca2+‐dependent phosphatase calcineurin, are essential for LPS‐induced COX‐2 and TNF‐α production in microglia, and likewise this pathway mediates the inhibitory effect of RGS10 on LPS‐signaling. We further demonstrated that ER calcium depletion amplifies LPS‐induced COX‐2 and TNF‐α expression and that RGS10 functions to suppress SOCE triggered by ER depletion. Collectively, this physical and functional interaction between RGS10 and STIM2 in microglia suggest a complex regulatory network connecting RGS10, LPS‐signaling, and SOCE, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.

Support or Funding Information

NIH‐NINDS
Gut to Eye Axis: Probiotic Mitigates Dysbiosis and Improves Ocular Functions in Mice
Authors: Singh Mahavir , Homme Rubens P. , Sandhu Harpal , George Akash K. , Stanisic Dragana , Malonee Carissa J. , Molnar Jack , Tyagi Suresh C.
Published: April 2020

It is known that a dysbiotic gut together with bacterial infection can disrupt ocular functions. As demonstrated by our laboratory, and others that obese phenotype in mice is metabolically prone to ocular hypertension phenotype, including retino‐vascular occlusion. These physiological perturbations arising presumably from alterations in the microbial population(s) of the host lead to loss of ocular homeostasis. We thus hypothesized that restoration of the gut‐microbiome via long‐term probiotic administration may help improve ocular functioning that has been derailed by the dysregulated “1‐carbon metabolism” as a result of high fat diet (HFD) consumption. To test this hypothesis, we used 4 different experimental groups of mice, designated as: Group‐I: WT mice (C57BL/6J); Group‐II: WT + HFD, Group‐III: WT + HFD + PB (Lactobacillus rhamnosus), and Group‐IV: WT + PB (Lactobacillus rhamnosus). The PB (Lactobacillus rhamnosus) intervention was done orally employing 2.5x105 CFU/day for a continuous period of 16 weeks. Visual function analysis, and detailed retinal angiography of the WT + HFD mice revealed somewhat abnormal vascular patterns, and impaired ocular functions. These conditions were subsequently improved by PB (Lactobacillus rhamnosus) supplementation. Further experimentations using electro‐retinography (ERG), ocular coherence tomography (OCT), and the fluorescein angiography (FA) analyses on WT+ HFD + PB (Lactobacillus rhamnosus) treated mice and other experimental groups supported the observed beneficial effects. Taken together, our findings indicate that PB (Lactobacillus rhamnosus) treatment could restore ocular metabolism, in part, via mitigating the adverse retino‐vascular remodeling phenotype as induced by the dysbiotic environment, most likely, via modulation of the “gut‐to‐eye axis” through reconstitution of the gut‐microbiota.

Support or Funding Information

HL139047 and AR071789
SERO‐PREVALENCE OF HIVANTIBODIES AMONG STUDENTS OF IMO STATE POLYTECHNIC UMUAGWO
Authors: Uchegbulam Henry Ugochukwu , Emeka-Nwabunnia (Nee Kalu) Ijeoma , Etteh Casmir Chinedu , Azuonwu Obioma , Nwankpa Promise , Ezekwe Ahamefula Sunday
Published: April 2020

Human Immunodeficiency Virus (HIV) is the name given to the virus that causes HIV infection and it’s continuous attack on the body defensive mechanisms leads to Acquired Immune Deficiency Syndrome (AIDS). Since its discovery in 1981, the virus has been widespread and has shown to be of global public health concern, thus, empirical evidence strongly suggest that HIV attacks the CD4+T‐lymphocyte‐ a special type of T‐lymphocyte that fights off disease in our immune system and render them ineffective to perform it’s task as required

The aim of the study was to investigate the HIV Sero‐prevalence among students of Imo State Polytechnic Umuagwo extraction.

A total of 1301 apparent healthy students aged between 19–24 years, were recruited through convenient random sampling technique, between June to November, 2009 respectively. However, out of these number, 536(41.20%) were males and 765(58.8%) were females and a rapid screening kit (Unigold kit‐Trinity Biotech, Ireland) which is based on antibody and antigen reaction was explored according to manufacturer’s instruction for the assay. The Microsoft excel software was used to represent the results in bar and pie chart respectively.

The result from the assay showed that 11 (0.85%) of the males tested positive whereas 10(0.77%) were discordant while 18(1.38%) females tested positive whereas 8(0.62%) were discordant. These probably suggest that among the youths of these ages, females were more infected than the males. Thus, there is need for continues health education among youths as abstinence from premature sex and avoidance of risky habits could be a strong veritable tool of prevention among active youths in our global communities.
Cross Interaction Between OGT and mTOR in T Cells and its Consequences in Autoimmunity
Authors: Castrillo Savannah Danielle , Gorelik Gabriela
Published: April 2020

Background

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that has been studied extensively, yet an understanding of the mechanisms of this disease remains unclear. It is known that epigenetic mechanisms are involved in the disease. We found O‐GlcNAc Transferase (OGT), an X‐linked gene, overexpressed due to hypomethylation in T cells from lupus patients (J. Autoimmunity, 2013). Furthermore, it was reported that the mechanistic target of rapamycin (mTOR) modifies T cell cytokine patterns and contributes to the TReg defect in patients with lupus. (J. Immunology, 2014).

Purpose

To investigate the role OGT has in T cell signaling in order to understand the contribution of its overexpression to lupus. In particular, we investigated the relationship between OGT and the mTOR pathway in T cells to understand their participation in the disease.

Methods

Healthy female donors were recruited by advertising. The research was reviewed and approved by the University of South Alabama Review Board for Human Subject Research. Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation, then T cells were isolated by magnetic beads. One fraction was transiently transfected for 48 h with 2μg of OGT siRNA using a nucleofector device (Amaxa). Other fractions were subjected to different treatments. Cells were stimulated after treatments with PMA/Ionomycin for 4h and the whole cell extracts were separated by SDS‐PAGE electrophoresis. Protein expression was analyzed by immunoblotting using specific antibodies. ß actin was used as loading control.

Results

Increasing the levels of glycosylated proteins with 100 mM Pugnac, a competitive inhibitor of O‐GlcNAc‐β‐N‐acetylglucosaminidase (OGA), or with 5mM Glucosamine, increased the phosphorylation of p70S6K, substrate of mTORC1, which indicates an increase in mTOR activity. On the other side, when OGT is knocked down by siRNA, as expected, the protein expression of OGT levels as well as glycosylated protein levels decreased. Interestingly, the activity of mTOR also decreased. Similar results were observed with 10 nM MHY1485, potent activator of mTOR, or with 50 nM rapamycin and 10 nM Torin‐1, both inhibitors of mTOR, which increased or decreased the phosphorylation of its substrate respectively. However, they were unable to modify the levels of glycosylation.

Conclusions

These results strongly suggest that mTOR activation is OGT‐dependent and is located downstream of OGT in T cells. Overexpression of OGT could be responsible for the increase in mTOR activity in T cells from lupus patients.

Support or Funding Information

NIH 5R03 AR067518‐02Summer undergraduate research fellowship (SURF) University of South AlabamaDepartment of Biomedical Science, College of Allied Health, University of South Alabama
Loss‐ And Gain of Function Mutations in the Type 1 IP3 Receptor (IP3R1) Modify Agonist‐Evoked Vasoactive Responses in Cerebral and Skeletal Muscle Resistance Arteries From Adult Mice
Authors: Mishra Ramesh C. , Zhu Hai-Lei , Chen S.R. Wayne , Cole William C. , Braun Andrew P.
Published: April 2020

Calcium mobilization within vascular smooth muscle and endothelial cells of small resistance arteries plays a critical role in agonist‐evoked vasoconstriction and dilation, respectively. Despite the recognized contribution of IP3‐mediated intracellular Ca2+ release for agonist‐evoked vasoactive responses, it has been difficult to study this process acutely in native arteries, due to the lack of selective pharmacological agents or experimental strategies to target the predominant IP3R1. In humans, heterozygous mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function phenotypes for Ca2+ release that impact cerebellar activity (e.g. ataxia), yet it remains unclear how these global mutations affect the cardiovascular system. To facilitate such investigation, we have engineered novel genetic knock‐in mice harbouring a single amino acid mutation at position Asp2594 in the transmembrane S6 helix of the endogenous IP3R1 Ca2+ release channel that confers either a LOF (Asp2594Ala) or GOF (Asp2594Lys) phenotype, respectively. In permeabilized HEK293 cells expressing these mutant receptors, the LOF mutation shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right, whereas the GOF mutation produced a 2.3‐fold leftward shift compared with wild‐type (WT) IP3R1. In endothelium‐denuded cerebral arteries pressurized to 10 mmHg, the sensitivity of serotonin‐evoked vasoconstriction was reduced in vessels from heterozygous LOF mice compared with WT, whereas agonist responsiveness was enhanced in arteries from heterozygous GOF mice. Vasoconstriction in response to the thromboxane receptor agonist U46619 was modestly enhanced by the GOF mutation, but largely unaffected in arteries from LOF mice. In cannulated cremaster skeletal muscle arteries from LOF and GOF mice pressurized to 70 mmHg, the sensitivity of endothelium‐dependent, acetylcholine‐mediated inhibition of myogenic tone was right shifted (3.7‐fold) and left shifted (2‐fold), respectively, compared with vessels from WT. Comparable changes in functional sensitivity were observed in response to the endothelium‐dependent vasodilator bradykinin. In contrast, dilation evoked by the NO donor sodium nitroprusside was not different among the three genotypes. Collectively, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization in small resistance arteries functionally impacts agonist‐evoked vasoactive responses. These observations highlight the importance of IP3R1 activity in the vascular wall, and further implicate vascular impairment as a contributing factor to the neurological dysfunction observed in patients with LOF and GOF IP3R1 mutations.

Support or Funding Information

This work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC), MOP‐142467 (APB) and PJT‐155963 (WC, WCC and APB).