bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal Structure of CYP2C9*8 Genetic Variant Bound to an Anti‐Hypertensive Drug Losartan.
Authors: Kamat Sumit S. , Parikh Sonia , Shah Manish B. 
Published: April 2020

Cytochrome P450 (CYP) enzymes are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics, and more recently to pharmacogenomics. The human CYP2C9 is involved in the metabolism of over 15% of clinical drugs that include warfarin, losartan, tolbutamide, etc. More than eighty genetic variations have been identified in CYP2C9, and many of these have demonstrated significantly reduced activity compared with the wild‐type (WT) enzyme. The CYP2C9*8 allele, prevalent among African‐American population with a frequency of around 0.06, is associated with altered clearance of several drug substrates of CYP2C9. The *8 represents an amino acid variation from arginine to histidine at position 150 (R150H). The R150H variant was generated using CYP2C9 WT construct by site‐directed mutagenesis, and the enzyme was expressed in E. coli followed by protein purification and crystallization. The CYP2C9*8 was crystallized in the presence of the drug substrate losartan and the structure was determined using X‐ray crystallography at 2.3 Å resolution. The R150H, found on the surface of the protein on D‐helix that is distal from the active site, illustrates minimal effect on the overall conformation of the protein compared to the WT. Despite subtle changes in the structure itself, there were clear differences in the binding of losartan compared to the previously solved CYP2C9 WT complex. One molecule of losartan was bound in the active site and one on the surface, consistent to that observed in the WT complex. However, unlike the WT complex, the losartan in the access channel was not observed in the *8 complex. The region near the access channel was more compact than the WT enzyme. Furthermore, the losartan turn‐over rates measured using enzymatic assays differed significantly, with the variant demonstrating marked reduction in activity than the WT enzyme. Together, our findings from multiple techniques suggest an alternate mechanism may be involved in reduced activity of this variant located on the surface that leads to differences in binding of losartan near the active site. The results yield insights into the role of simultaneous binding of multiple substrate molecules, orientation of important amino acid side chains and altered hydroxylation profile of losartan with this variant.Support or Funding InformationThe X‐ray data collection at the Stanford Synchrotron Radiation Lightsource (SSRL) was done remotely. Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE‐AC02076SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (P41GM103393). The research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.
Identifying Regulators of O‐GlcNAcylation in the Broken Heart
Authors: Narayanan Bhargavi , Sinha Prithvi , Henry Roger , Reeves Russell , Kohr Mark , Zachara Natasha 
Published: April 2020

The post‐translational modification of intracellular proteins by monosaccharides of O‐linked b‐N‐acetylglucosamine (O‐GlcNAc) has emerged as a critical regulator of cellular function. Previous research has demonstrated that enhanced O‐GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury. The cycling of O‐GlcNAc is regulated by the collective efforts of two enzymes: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), which catalyze the addition and hydrolysis of O‐GlcNAc respectively. The goal of these studies is to understand the mechanisms by which the heart regulates O‐GlcNAcylation in response to injury. Sex and gender have been linked to the pathophysiology of cardiovascular disease; however, the impact of sex on O‐GlcNAc cycling has not been studied. Thus, to address our goal, we harvested male and female hearts from mice and extracted the total heart proteome and assessed the levels of O‐GlcNAc, OGT, and OGA as well as key enzymes within the hexosamine biosynthetic pathway (HBP). We observed a modest elevation in O‐GlcNAc levels in female hearts. Counter‐intuitively, OGA levels appeared to be elevated in the female hearts. To assess the impact of sex on O‐GlcNAc cycling in response to injury, we used the Langendorff perfused mouse heart model to collect male and female hearts exposed to either control, ischemic or I/R conditions. Hearts were partitioned into a soluble fraction enriched in cytosolic and mitochondrial proteins, as well as a myofibrillar fraction enriched in contractile proteins. Our data demonstrate that the majority of the O‐GlcNAc‐modified proteins are detected in the soluble fraction. Ischemia results in a suppression of O‐GlcNAcylation; however, O‐GlcNAc levels were modestly elevated in hearts subjected to I/R injury. Future work will assess the impact of sex on the activity of OGT and OGA, as well as the nucleotide sugar UDP‐GlcNAc. Collectively, these findings will provide a foundation for understanding whether sex is an important factor to consider when assessing molecular mechanisms regulating O‐GlcNAc cycling in the heart.Support or Funding InformationK12 HL141952, U01 CA2300978, R01 HL129640, P01 HL107153
Sex‐ and Time‐Dependent Differences in Glutamate Receptor Subunit (GLUN1) in the Rat Rostral Ventrolateral Medulla
Authors: Mueller Patrick J. , Azar Toni A. , Fyk-Kolodziej Bozena E. 
Published: April 2020

The cardioprotective effects of ovarian hormones are evident by the fact that women have a lower risk of cardiovascular disease compared to men, at least until menopause, at which time the relative risks reverse. Several studies have also demonstrated the importance of ovarian hormones in the regulation of sympathetic outflow, potentially via actions in the rostral ventrolateral medulla (RVLM), a brain region critical to sympathetic outflow and blood pressure. However, the time course and mechanisms of sex‐dependent changes in excitation of RVLM neurons have not been fully characterized. Therefore, the purpose of this study was to compare glutamate receptor (GluN1) subunit expression in the RVLM of male and female rats, focusing specifically on ages of development in female rats which precede the onset of the estrous cycle (4 wks old) versus when the estrous cycle is fully established (8 wks old). We hypothesized that eight week‐old female rats would exhibit reduced GluN1 compared to four week‐old females. In contrast, we also predicted that eight week‐old male rats would have higher GluN1 compared to four week‐old males. To test our hypothesis, we obtained brain punches from RVLM subregions of male and female Sprague‐Dawley rats (n=3 ea; 4 and 8 wks old) based on their anatomical location relative to the facial nucleus (FN). Punches were processed for western blotting of GluN1 subunit in four subregions: FN‐480; FN‐240; FN+240 and FN+480. At FN‐480, GluN1 was lower in eight week‐compared to four week‐old female rats (0.67 ± 0.11 vs. 1.11 ± 0.08 protein/GAPDH, respectively). In contrast, at FN‐480, eight week‐old male rats exhibited increased NR1 expression compared to four week old males (1.05 ± 0.06 vs. 0.65 ± 0.10; protein/GAPDH, respectively p<0.05). These results suggest that lower levels of ovarian hormones in prepubescent females may increase the excitability of RVLM neurons and lead to greater sympathoexcitation, which then decrease with the onset of the reproductive cycle. The absence of ovarian hormones in males (perhaps coupled with a lack of regular exercise) may exacerbate RVLM excitability, leading to greater sympathoexcitation in sedentary males. Collectively, these data suggest that ovarian hormones exert cardioprotective effects via actions in the RVLM, reducing sympathetic outflow and contributing to a lower incidence of cardiovascular disease in women of reproductive age.Support or Funding InformationHL096787‐08; AHA25810010
Inspiratory muscle strength training suppression of sympathetic nervous outflow in young adults: Time course and differential recovery.
Authors: DeLucia Claire M. , Debonis Dean R. , Schwyhart Sarah M. , Bailey E. Fiona 
Published: April 2020

BackgroundOur laboratory has shown that an abbreviated form of inspiratory muscle strength training (IMST) performed 5 minutes/day, 5 days/week for 6 weeks lowers casual systolic blood pressure and systemic vascular resistance in healthy young adults. While the effects of IMST have been characterized in the short term, the acute effects of the training protocol on cardiovascular function have not been documented.PurposeTo characterize the acute cardiovascular response to a bout of IMST (comprising 5 sets of 6 breaths) in healthy young adults.MethodsFourteen healthy women and men (n=14, 7F/7M, age: 22±2 years) performed five sets of six inspiratory efforts against a target resistance set at 75% of maximum inspiratory pressure (PImax). We recorded inspiratory pressure, postganglionic, multiunit muscle sympathetic nerve activity (MSNA), beat‐to‐beat changes in blood pressure (SBP and DBP), and heart rate (HR) at baseline, during IMST and in recovery.ResultsThere were no differences between women and men in regard to PImax (F: −79.9±17.7 and M: −78.3±21.7 mmHg, p=0.8794), or baseline measures of MSNA (F: 7.3±1.8 bursts/15s bin and M:6.2±1.4 bursts/15s bin, p=0.6377), SBP (F:114.0±9.1 and M:114.0±4.7 mmHg, p=0.9855), and HR (F: 75±8 bpm and M:74±15 bpm, p=0.6994). During IMST, women and men attained the same target pressure (75% PImax) (F: −58.6±13.0 mmHg and M: −58.7 ±16.3 mmHg, p=0.8794) and performed the same amount of inspiratory work per breath (F: 84.1±22.2 and M: 85.0±25.9 mmHg.s, p=9176). Relative to baseline, HR increased 25±10%, (p<0.0001) and MSNA decreased 43±20% (p<0.0001) in all subjects. In recovery, MSNA suppression persisted in women but not in men (p=0.015). There were no acute effects of IMST on systolic (p=0.895) or diastolic (p=0.761) blood pressures.ConclusionIMST exerts an acute inhibitory effect on MSNA in young men and women. Our data highlight a disparity in the time course of the recovery of MSNA post IMST for women vs. men.Support or Funding InformationNational Institutes of Health Interdisciplinary Training Grant in Cardiovascular Sciences T32 HL007249
Analysis of DNA Methylation Changes in Mice Mediated by Early‐life Exposure to the Antidepressant S‐citalopram
Authors: Rodriquez Meghan , Shetty Madhur , Black Elizabeth , Kretschmar Matthew , Perley Danielle , De Kumar Bony , Henry L. Keith 
Published: April 2020

Multiple studies in rodents and humans have identified increased anxiety and depression in adults who are transiently exposed to selective serotonin reuptake inhibitors (SSRIs) during early development. This persistent reprogramming suggests a significant risk to human health and in efforts to understand the molecular mechanism leading to this long‐lasting effect, we initiated DNA methylation analysis to determine if there are SSRI‐mediated marks that could encode expression changes leading to these behavioral outcomes. To do this, we are using nanopore sequencing and comparing recently developed post‐analysis methods to identify methylation changes on a genome‐wide scale in the mid‐brain of post‐natal day 35 male mice. The methylation changes were then correlated to methylation patters from treated transgenic mice harboring a single amino acid substitution in the serotonin transporter that spares serotonin transport while conferring virtual insensitivity to many SSRIs. This comparison allows for the identification of specific methylation changes associated with serotonin transporter blockade.Support or Funding InformationNIH‐NIGMS‐P20 GM104360
Divergent Muscle Sympathetic Nerve Activity during Cold Pressor Test and Perceived Pain
Authors: Cunningham Hannah A. , Carter Jason R. 
Published: April 2020

The cold pressor test (CPT) is often used as a noxious stimulus to induce pain, and results in robust and reproducible increases in blood pressure, heart rate, and muscle sympathetic nerve activity (MSNA) in humans. In contrast to the relative consistency of CPT, some laboratory techniques (e.g. mental stress) result in divergent MSNA responses, where some individuals exhibit an early‐onset increase in MSNA (i.e. positive responders) and others an early‐onset decrease (i.e. negative responders). The purpose of the present study was to compare perceived pain in subjects based upon their early‐onset MSNA responsiveness (i.e., positive vs. negative) during the CPT. Forty‐three young, healthy adults (21±1yrs; 20 men and 23 women) participated in an experimental visit where continuous BP (finger plethysmography) and MSNA (microneurography) were measured during a 3–5 minute baseline and 2 minute CPT. Perceived pain was recorded in 15 second intervals during CPT using a standard pain scale. The initial 15 seconds of CPT were used to categorize subjects for analysis based on MSNA responsiveness (burst/min), which resulted in the following two groups: 1) positive responders (n=17; 8 women; mean, Δ8 ± 8 burst/min; range Δ0 – 31 burst/min), 2) negative responders (n=26; 15 women; mean, Δ‐4±4 burst/min; range Δ‐14 – 0 burst/min). Reactivity and blood pressure data were analyzed using a mixed ANOVA with time as a within subjects factor and group as a between subjects factor. Independent‐samples t‐tests were used to analyze changes in perceived pain (p<0.05). While there was a significant time×group interaction for MSNA reactivity patterns when expressed as burst frequency (p=0.031) and bursts per 100 heart beats (p=0.043), all groups demonstrated similar systolic (time×group, p=0.888) and diastolic (time×group, p=0.753) blood pressure responsiveness. Heart rate reactivity was also similar between groups (time×group, p=0.777). In contrast to the hemodynamic responses, change of pain during CPT was augmented in responders (8.5±0.4 a.u.) compared to non‐responders (7.4±0.6 a.u.; p=0.007). This was also observed when pain was expressed as a peak change (responders, 10.6 ± 0.4 a.u., non‐responders, 9.2 ± 0.7 a.u.; p=0.011). In summary, our findings suggest a potential relationship between the early‐onset divergence of MSNA reactivity and the changes in perceived pain during the CPT.Support or Funding InformationFunded by the National Institutes of Health (R01 AA‐024892‐01‐A1, R15 HL098676, R15 HL‐122919) and the Portage Health Foundation.
Fibroblast Growth Factor‐2‐Induced Endothelial Cell Migration Is Regulated by Junctional Adhesion Molecule‐A Through Its Tyrosine Phosphorylation and Interaction With Afadin
Authors: Naik Meghna U. , Chatterjee Sharmila , Naik Ulhas P. 
Published: April 2020

Neovascularization, or angiogenesis, plays an important role in tumor growth and ischemic diseases. Fibroblast growth factor‐2 (FGF‐2) is a major angiogenic growth factor. We have previously shown that FGF‐2‐induced angiogenesis requires junctional adhesion molecule‐A (JAM‐A). However, the signaling mechanism through which JAM‐A regulates the FGF‐2‐induced signaling pathway is currently unknown. To assess the role of JAM‐A in FGF‐2 signaling, we generated various JAM‐A mutants as well as Jam‐A null ECs and performed cell adhesion and migration assays, western blot analysis, co‐immunoprecipitation assays, and proximity ligation assays. Here we show that FGF‐2‐induced endothelial cell (EC) adhesion and migration on vitronectin is dependent on JAM‐A. FGF‐2‐induced ERK1/2 activation and Rap1 activation in EC also requires the presence of JAM‐A. We show JAM‐A to be phosphorylated on serine 284 residue in its cytoplasmic tail and that it associates with EC integrin avβ3 and CD9 in unstimulated conditions. However, upon FGF‐2 stimulation, JAM‐A gets phosphorylated on tyrosine 280, which is accompanied by dephosphorylation of phosphoserine 284 and dissociation from the integrin avβ3/CD9 complex. We also found that JAM‐A associates with PDZ domain‐containing protein zona occludens‐1 (ZO‐1) in resting conditions but dissociates from ZO‐1 and associates with afadin (AF‐6) through its PDZ‐domain binding motif present in its cytoplasmic tail upon FGF‐2 treatment. To test the role of specific residues of JAM‐A involved in regulating FGF‐2‐induced cell migration, we ectopically expressed various mutants of JAM‐A in human umbilical vein endothelial cells (HUVECs). Expression of PDZ domain‐binding motif‐deleted mutant JAM‐A showed significantly attenuated FGF‐2‐induced Rap1 activation, ERK1/2 activation, as well as cell migration. We found that tyrosine 280 phosphorylated JAM‐A associates with C‐terminal Src kinase (CSK) upon FGF‐2 stimulation and this association is found to be through the SH2 domain of CSK. Ectopic expression of JAM‐A tyrosine 280 to phenylalanine substitution mutant resulted in abrogation of FGF‐2 induced cell migration. Our results suggest that FGF‐2‐induced signaling in ECs is dependent on the tyrosine 280 residue and the PDZ domain‐binding motif of JAM‐A. This regulates its interaction with signaling proteins such as AF6 to regulate Rap1 and ERK1/2 activation induced by FGF‐2 which subsequently leads to EC migration and angiogenesis.Support or Funding InformationNHLBI/NIH 2R01 HL119374‐06 to UPN.
Citation Format Matters: Undergraduate Student Comprehension of Peer‐Reviewed Scientific Manuscripts
Authors: Cowan Mackenzie , Merritt Edward K. , Mikan Vanessa 
Published: April 2020

Primary research articles from peer‐reviewed journals are often used as teaching tools in undergraduate science courses. With their unique writing style, field specific terminology, and complex topics, students often struggle to comprehend the articles. Educators use a variety of methods to teach students how to improve their comprehension including annotating the articles, asking targeted questions, or providing specific strategies for reading them. However, one aspect often overlooked in student understanding of primary research articles is how the in‐text citation format affects reading comprehension. The purpose of this study was to determine if in‐text citation format in scientific journals affects reading comprehension in undergraduate students enrolled in science courses. We hypothesize that more complex in text citations (e.g. parenthetical “Author, et. al, Year”) will decrease reading comprehension compared to simpler in text citations (e.g. numeric “[1]”).Undergraduate students (n = 30) enrolled in an upper‐division science course were tasked with reading the introductions of two primary research articles with differing in‐text citations in a randomized, cross‐over design. Each article was modified to have numeric or parenthetical, Author‐Year, in‐text citations. Other than style of in‐text citation, the articles were formatted as published. Each student had 6 minutes to read an article with one in‐text citation format, and then 6 minutes to take a quiz to determine his or her understanding of the article. Students then had 6 minutes to read the second article with the other in‐text citation format followed by 6 minutes to take its quiz. Following the readings and quizzes, students completed a questionnaire that asked about basic demographic information, perceived understanding of the article, and difficulty of reading the article.Student reading comprehension as assessed by post‐reading quizzes was 34% higher when reading articles with numeric in‐text citations compared to articles with parenthetical, Author‐Year, in‐text citations (p = 0.03). The difference in reading comprehension was most pronounced in articles that students perceived as more difficult to read and understand regardless of in‐text citation format. Students performed 70% worse when reading difficult articles with Author‐Year in‐text citations compared to numeric citations (p = 0.01). These results highlight the importance of seemingly minor formatting decisions by editors and journals on reader understanding. Educators should consider providing undergraduate students with primary research articles with modified, simpler, in‐text citations to improve their reading comprehension.
Aggravated Endothelial Inflammasome Activation and Neointima Proliferation by Hepatic Steotosis‐intensified Exosome Release in Liver‐Specific Asah1 Gene Knockout Mice
Authors: Yuan Xinxu , Bhat Owais M. , Comus Sarah , Zhang Yang , Li Pin-Lan 
Published: April 2020

Nonalcoholic fatty liver disease (NAFLD) has been reported to be independently associated with increased traditional risk for cardiovascular diseases (CVDs). However, the mechanism underpinning the association between NAFLD and CVD remains poorly understood. The present study hypothesized that circulatory exosomes released from liver during NAFLD contribute to vascular endothelial inflammasome activation and neointima proliferation. In mice with liver‐specific deletion of Asah1 gene, a gene coding mouse acid ceramidase (Asah1fl/fl/Albcre), we found significantly enhanced liver lipid deposition when they were fed the high fat diet (HFD), as shown by HE staining and Oil Red Staining. These mice also had augmented expression of exosome marker, CD63 in the liver, and exosomes in their plasma was significantly increased as measured by Nanoparticle Tracking Analysis (NTA) compared to WT/WT mice. In partial ligated carotid artery, we observed that Asah1fl/fl/Albcre mice with HFD treatment markedly increased Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome formation as shown by the colacolization of NLRP3 with apoptosis‐associated speck‐like protein gene (ASC) or caspase‐1. Enhanced colocaziation of fluorescent labeled inhibitor of caspases (FLICA) with endothelial cell marker, vWF and increased IL‐1β level in the carotid intima demonstrate NLRP3 inflammasome activation. Correspondingly, more severe vascular endothelial injury was found in Asah1fl/fl/Albcre mice as shown by reduction of vWF and endothelial junction protein, ZO‐1, which was accompaned by increased neointima proliferation. These results suggest that acid ceramidase importantly controls exosome release from the liver and its decifiency intensifies NAFLD and thereby releasees more exosomes to promote endothelial NLRP3 inflammasome activation and consequent neointiam proliferation.Support or Funding InformationSupported by HL057244, HL075316