bluuebunny/crossref_metadata_2025 huggingface dataset:
Formalism of Euler’s Graph Theory is Suitable for Natural Etiopathogenetic Pathways and Clusters Mapping ‐ Integrative Study of Pathophysiology
Authors: Kovac Zdenko
Published: April 2020

Leonard Euler (1707–1783) introduced the graph theory, a special part of geometry, as a tool to “… deal with relation dependent on position alone, and investigates properties of the position; it does not take magnitude into consideration, nor it does involve calculation …”. Graph theory is useful concept for nonlinear complex systems. As non‐numerical and non‐metric mathematical reasoning, it describes a complex system as a net of multiple events, seen as a whole. Similarly, graphic representation of physiological networks has an advantage in comparison with traditional narrative descriptions.

We invented matrix guided analysis and re‐synthesis method to outline and visualize etiopathogenetic pathways and their natural interconnectivity. It is four steps method (consisting of a patient problem exposition, repetition of relevant knowledge, a design of algorithm and systematization) that enforces the horizontal, vertical and longitudinal integration of knowledge. The algorithmic pathways and networking of processes are crystallized out of plethora of clinical data (signs, symptoms, dysfunctions, outcomes, etc) and knowledge of sub‐systems (nano‐molecular, thermal, macromolecular, cellular phenomena, etc.), which are largely acquired via reductionistic methodologies.

Algorithmic pathophysiology elaboration has revealed a natural tendency to form common crossing points of reactivity of heterogeneous pathways. The pathways spontaneously converge and form etiopathogenetic clusters (EPCs). EPCs integrate multiple inputs and multiple exits. They look like natural integrators, the common “hubs” of human body response in various diseases. 91 EPCs have been identified at multiple levels of hierarchy of human body disease reactivity. EPCs are shared in natural development of heterogeneous conditions and they show tendency to form a network. Among the EPCs connecting pathways serve as “the EPC‐driving force”. Thus, the EPCs may be seen as “a new emerging feature” of pathophysiology foundation of disease.

Following Euler’s strategy directed links in‐between elements of algorithm indicate a “physiological flux”, mutual oriented influence, and/or cause‐consequence. Physiological relations among acting elements have directionality of action, feedback and feed‐forward regulatory loops. Graphic maps outline the parallel pathways and easily put together relevant events of nano‐scale analyses with the clinical macro‐scale level. The (EPCs) are crossing and integrating points of complexity networks. The network visualization of physiology of disease and health underlying processes places individual mechanisms within the complex interconnectivity and may contribute to explanation of nonlinearity phenomena.
Amino Acid Substitution at Phe80 of Mammalian Phenylalanine Hydroxylase Destabilizes Both Resting‐State and Activated Conformations Increasing the Population of Intermediates.
Authors: Arturo Emilia C. , Merkel George W. , Borne Elias , Hansen Michael Riis , Lisowski Sophia , Gupta Kushol , Jaffe Eileen K.
Published: April 2020

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria (PKU). PAH equilibrates among long‐lived conformations, including resting‐state (RS‐PAH) and activated (A‐PAH), whose equilibrium position depends upon allosteric Phe binding to the A‐PAH conformation. The RS‐PAH conformation contains a stabilizing cation‐pi sandwich between Phe80, Arg123, and Arg240 (PDB entry 5DEN), which cannot exist in the A‐PAH conformation. Intrinsic protein fluorescence, enzyme kinetic analysis, native PAGE, size exclusion chromatography, limited proteolysis, and behavior on ion exchange resin are reported for F80A, F80D, F80L, and F80R, many as a function of [Phe]. These data indicate that amino acid substitutions at Phe80 destabilizes both the RS‐PAH and A‐PAH conformations so that intermediate, on‐pathway conformations are longer lived. The addition of Phe allows stabilization of the A‐PAH conformation. Kinetic characterization of F80A and F80D reflects allosteric activation while F80L and F80R are constitutively active. The reaction rates of all Phe80 variants suggest relief of a rate determining conformational change present in the wild type protein. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a central C‐terminal 4‐helix bundle, reflecting dynamic dissociation of the PAH tetramer. Under these conditions, the Phe80 variants show proteolytic hypersensitity in a linker region that repositions in the RS‐PAH to A‐PAH conformational interchange; this is protected by addition of Phe. We conclude that manipulation of Phe80 dramatically affects the conformational space sampled by PAH, increasing the population of intermediates between RS‐PAH and A‐PAH.

Support or Funding Information

NIH 5R01‐NS100081; NIH P30 CA006927
Advanced Glycation End Products Downregulate Placental Growth Factor Expression in Endothelial Cells Via a Macrophage‐Mediated Mechanism
Authors: Semola Yemi , Lovern Pamela
Published: April 2020

In coronary artery disease, atherosclerosis chronically reduces blood flow. Placental growth factor (PLGF), a protein produced by endothelial cells (EC), can promote collateral artery remodeling to increase blood flow. However, collateral growth is impaired in people with type II diabetes. Our group has previously demonstrated that PLGF levels are reduced in a mouse model of diet‐induced type II diabetes. However, the mechanism for this reduction remains to be defined. Inflammatory cytokines and advanced glycation end products (AGE) are implicated in diabetic vascular dysfunction. We therefore hypothesized that AGE and TNFα signaling would inhibit PLGF expression in EC. Human coronary artery EC were treated with AGE, bovine serum albumin (BSA, as a control for AGE), or TNFα. AGE had no direct effect on PLGF in EC; however, TNFα reduced PLGF in EC (P<0.001). Next, murine macrophages (RAW cell line) were treated with AGE or BSA. RAW media was then transferred to murine endothelial cells (EOMA cell line). Treatment with both RAW‐BSA and RAW‐AGE media significantly reduced PLGF levels in EOMA cells compared with control RAW media (BSA, 481.75 ±21.00 pg/mL; AGE, 631.29 ±41.88 pg/mL; control, 1022.48 ±115 pg/mL; P<0.001, n=4). However, TNFα levels were only increased in RAW‐AGE media compared to RAW‐BSA media and RAW‐control media (AGE, 811.11 ±48.37 pg/mL; BSA, 305.68 ±13.95 pg/mL; control, 276.07 ± 10.40 pg/mL; P< 0.001, n=3). This finding suggests that cytokines other than TNFα may also be inhibitory for PLGF. Analysis of RAW media using a Multiplex Pro mouse 6‐panel cytokine assay revealed that TNFα was increased in RAW‐AGE media compared to RAW‐BSA and RAW‐control media (P<0.007). There was also a trend towards increased IL‐10 and IFN‐γ with AGE treatment, which approached significance (P<0.06), suggesting that these cytokines may also be important targets for future investigation. Identifying signaling pathways by which AGE and inflammatory mediators affect PLGF will give new insights into the pathology of diabetic cardiovascular disease.

Support or Funding Information

NIH R56 HL084494 (PL), OSU CVM
Substrate Binding to the Catalytic Domains of Lysine Deacetylase 6
Authors: Eyassu Daniel G. , Williams Ayanna J. , Toro Tasha B. , Watt Terry J.
Published: April 2020

Thousands of acetylated protein sequences have been identified in humans. Lysine deacetylases (KDACs or HDACs) are enzymes known to reverse acetylation by catalyzing hydrolysis of acetyllysine residues. Proper regulation of acetylation levels by KDACs and the opposing lysine acetyltransferases plays a role in many biological processes. Irregular activities of KDACs have been associated with a variety of diseases including various cancers. KDAC6 is the only known human KDAC to have two catalytic domains (CDs). The individual contributions of the catalytic domains to the overall activity of the enzyme and interaction with substrates are not well understood. Additionally, there are not many known substrates for the enzyme. We hypothesized that each CD has independent deacetylase activity and different substrate selectivity. To test this hypothesis, the activity of each domain was determined with a variety of peptides, many derived from acetylated proteins that are putative substrates of KDAC6. The second catalytic domain appears to be responsible for the majority of catalysis. To determine why peptides were active with one domain and not the other, we used molecular dynamics simulations to model the interaction between the CDs and the peptides. We identified residues relevant in interactions between each catalytic domain and peptide pair. Activity data and apparent substrate affinity were used to validate molecular dynamics results as explanatory for differences in substrate affinity for each CD.

Support or Funding Information

NIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.
Effects of prebiotic inulin pre‐treatment on susceptibility to DSS‐induced colitis in mice
Authors: Nadolski Nolan L.W. , Prawitz Rachael , Winker Olivia , Walton Kristen L.W.
Published: April 2020

Inflammatory bowel disease (IBD) is a chronic intestinal disease that includes Crohn’s disease and ulcerative colitis. Previous studies have found alterations in the microbiome in IBD and in animal models of IBD, underscoring the role of the intestinal microbiota in IBD pathogenesis. Prebiotics are molecules in foods that support the growth or activity of beneficial microbes. Inulin, a soluble fiber molecule, is a commonly available prebiotic, and previous studies suggest that it can reduce symptoms in dextran sodium sulfate (DSS)‐induced colitis in mice. This study aimed to determine whether pretreatment with 1% inulin for 1 week, followed by 7 days of 3% DSS administration with continued inulin treatment, altered the severity of disease in C57Bl/6 mice. Clinical signs of colitis, including weight loss, fecal occult blood, and fecal consistency, were monitored daily during the DSS treatment period. At the end of the experiment, samples were collected for histological scoring, and for RNA isolation and analysis of colonic pro‐inflammatory cytokine expression by quantitative PCR. Unexpectedly, inulin pre‐treated, DSS‐exposed mice were positive for blood in the stool a day sooner than mice given DSS only. However, by the end of DSS treatment, the disease activity index was not different between the two groups. In addition, colon length and colon wet weight were similar in inulin‐treated mice and non‐inulin treated mice. Histological scoring of colitis showed no difference between inulin‐treated and non‐inulin‐treated colitic mice. In conclusion, 7 days of pre‐treatment with inulin followed by induction of acute DSS‐induced colitis did not significantly alter susceptibility to colitis.

Support or Funding Information

Supported by the Missouri Western State University PORTAL program
Expression and Protective Functions of the sst4 Somatostatin Receptor in the Indomethacin‐Induced Gastrointestinal Mucosal Injury Model of the Mouse
Authors: Helyes Zsuzsanna , Csekoe Kata , Kormos Viktoria , Zelena Dora , Iarushkina Natalia , Sudalina Maria , Horvath Adam , Kecskes Angela , Gaszner Balazs , Filaretova Ludmila
Published: April 2020

Introduction

Gastrointestinal ulceration is associated with high mortality despite advanced management, therefore, understanding of the mucosal protective mechanisms is important. Somatostatin is present in the endocrine cells and sensory nerves, and its analogues were shown to reduce mucosal blood flow, pepsin and gastric acid secretion. Our group discovered that its anti‐inflammatory and analgesic actions are exerted via the somatostatin receptor subtype 4 (sst4). Therefore, we investigated its expression and role in gastrointestinal injury.

Methods

Sst4 gene‐deleted (sst4−/−) and wildtype mice were treated s.c. with indomethacine (IDM; 35 mg/kg) or vehicle. RNA scope in situ hybridization was performed to localize, qPCR to quantify sst4 and somatostatin mRNA. Macroscopic and microscopic lesions in the stomach and small intestine were evaluated after 4 and 48 h. Plasma protein extravasation was measured by fluorescent, neutrophil myeloperoxidase (MPO) activity bioluminescent ex vivo imaging, TNFα, IL‐1 and COX2 mRNA by qPCR.

Results

Somatostatin mRNA is present in the neuroendocrine D‐cells and significantly upregulated 48 h after IDM. Sst4 mRNA in located in the myenteric plexus neurons, it’s expression did not change in response to mucosal injury.

IDM‐induced gastric and small intestinal macroscopic lesions (4, 24 h), gastric MPO activity (24, 48 h) and plasma extravasation (30 min) were significantly greater in sst4−/− mice. TNFα, IL‐1, COX2 expressions significantly increased after 4 h in the stomach of sst4−/− mice, but in wild‐types the same pattern was observed only later, at the 48 h time‐point. IDM‐evoked histopathological alterations and small intestinal length reduction were not altered by the sst4 deletion.

Conclusion

This is the first evidence for sst4 expression in myenteric plexus neurons and its protective roles against chemically‐induced gastrointestinal mucosal damage and inflammation. This suggests the safety of sst4 agonist drug candidates in the gastrointestinal tract, and even their potential use for gastroprotective indication.

Support or Funding Information

Supported by: GINOP‐2.3.2‐15‐2016‐00050 – PEPSYS, GINOP‐2.3.2 STAY ALIVE, EFOP‐3.6.2‐16‐2017‐00008, ÚNKP‐19‐3‐III‐PTE‐211
Pilot Study: The role of chronic stress on brain microvessel density in male and female mice
Authors: Jent Wendy , Burrage Emily N. , Price Christian , Colbentz Tyler , Childers Ryan , Bryner Randy , Chantler Paul D.
Published: April 2020

The cerebrovasculature, an intricate configuration of blood vessels, is responsible for the exchange of blood to and from the brain. Reduction of cerebrovascular vessels can reduce or prevent the flow of nutrients & oxygen that is necessary for proper brain function and increase risk of cerebrovascular diseases. Chronic stress is also a risk factor to various cerebrovascular diseases.

In this pilot study, brain microvessel density in both female and male chronically stressed mice were analyzed. Beginning at the age of 18 weeks, mice were subjected to the unpredictable chronic mild stress (UCMS) paradigm for 5 days a week/seven hours a day for 8 weeks. Upon euthanization brain samples were flash frozen and sectioned at 8um using a cryostat. Immunofluorescence was utilized to observe endothelial cells via a CD31 antibody. Images were taken using a Nikon Eclipse 800 at a 40X objective and analyzed and quantified for density using ImageJ.

Preliminary data (n=3/group) suggest that the male mice have a 26% higher microvessel density compared to females (p<0.05). When stressed, males exhibit a 17% lower microvessel density whereas stressed females did not display a loss in microvessel density (p<0.05).

Here we showed that the brain of male and female mice can be physiologically diverse in microvessel density and that males and females display a different response when exposed to chronic stress.

Support or Funding Information

NIH Cobra Grant 5P20GM109098; NIH INBRE P20GM103434
Probing the Role of Chloride in Facilitating System xc− Function
Authors: Catalfano Kevin , Chase Leah
Published: April 2020

System xc− is a heterodimeric transporter that functions as a Na+‐independent antiporter exchanging extracellular cystine for intracellular glutamate. The transporter functions as an obligate heterodimer and is comprised of two proteins, xCT, which functions specifically in amino acid exchange, and 4F2HC, which appears to play a role in transporter stability. System xc− belongs to the SLC7 family of transporters, which is further subdivided into the cationic amino acid transporters (CAT) and the L‐type amino acid transporters (LAT), the latter of which is the subfamily which includes System xc−. In recent years, there have been advances in our understanding of the structure of the SLC7 transporters as crystal structures of bacterial homologs of CAT and LAT transporters have been published. As a result, we can now begin to ask important questions about the mechanism by which System xc− exchanges cystine and glutamate across the membrane. Previous studies in the Chase lab have demonstrated that System xc− is Cl−‐dependent. More recently we have shown that transport of one molecule of cystine requires more than one Cl− ion, and that Cl− binding affects the affinity of the transporter for cystine. We have also shown that chloride dependence of cystine transport is pH dependent. The K0.5 of chloride increases as the pH increases, suggesting that the Cl− binding affinity decreases at higher pH. Using this collective information, we examined the structure of xCT to identify the most likely binding site for cystine and glutamate. Specifically, we used Chimera 1.13.1 and Pyrx to dock cystine and glutamate into its most energetically favorable positions on xCT and identified several ionizable side chains within the putative permeation pathway associated with transmembrane domains 3 and 6 that appear to interact with substrate. These residues include R135, T138, Y246 and Y251. We are currently assessing this model in the presence of Cl− to identify putative residues which may be important for the docking of Cl− near the substrate binding site and using site‐directed mutagenesis to determine whether these residues are responsible for Cl−‐dependent cystine transport.

Support or Funding Information

This research is supported by the Campbell Foundation and Schaap Endowed Funds for Undergraduate Research.
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 Information

The 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 Information

K12 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 Information

HL096787‐08; 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

Background

Our 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.

Purpose

To characterize the acute cardiovascular response to a bout of IMST (comprising 5 sets of 6 breaths) in healthy young adults.

Methods

Fourteen 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.

Results

There 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.

Conclusion

IMST 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 Information

National 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 Information

NIH‐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 Information

Funded 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 Information

NHLBI/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 Information

Supported by HL057244, HL075316
Relationship between food intake and expression of messenger RNA encoding aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase (TH), and catechol‐O‐methyltransferase (COMPT) in channel catfish.
Authors: Moyers Macy Grace , Kostner Danica , Wertz Blaine , White Taylor , Kobayashi Yass
Published: April 2020

Obesity is linked to various metabolic disorders and associated complications, including major depressive disorder and anhedonia. How dopamine metabolism affects food intake and the development of obesity in fish, however, is not clear. Our objectives were to characterize the tissue expression of AADC, TH, and COMPT mRNA and if their expression is influenced by food intake in channel catfish. Expression of these mRNAs were examined in the brain, hypothalamus, liver, muscle, kidney, spleen, large intestine, and small intestine, as well as within different parts of the brain to examine their tissue expression profile. To study the relationship between hypothalamic expression of these mRNAs and food intake, samples were harvested at −1 hour prior to (−1), at feeding (0), and 1 hour after feeding (+1, n=4 per time point). Expression of all transcripts encoding these enzymes was detectable in all tissues examined, with increased expression in the liver. These mRNAs were more highly expressed in the hypothalamus compared to other parts of the brain examined. The expression of AADC mRNA and TH mRNA was not altered by timing of food exposure. Given that these mRNA transcripts had increased expression in the hypothalamus, the dopaminergic neural system may influence the regulation of food intake. Lack of changes of expression of these enzymes within the hypothalamus in response to food exposure may indicate that the changes in the dopamine system in response to food intake may be at the enzyme activity level rather than changes in transcription.

Support or Funding Information

P20 GM103418
Increased extracellular superoxide and MMP9 attenuated COMP stabilization of BMPR2 potentially participate in pulmonary hypertension development
Authors: Alruwaili Norah , Kandhi Sharath , Kelly Melissa R. , Sun Dong , Wolin Michael S.
Published: April 2020

The role of bone morphogenetic protein type 2 receptor (BMPR2) in the development of pulmonary hypertension was emphasized after identifying mutations of the gene encoding BMPR2 among patients with idiopathic pulmonary artery hypertension. Since then many studies were devoted to investigate restoring BMPR2 signaling as a therapeutic strategy. Also, roles for increased oxidant mechanisms have been identified in the lungs and right ventricle (RV) of several animal models of PH. However, it is unclear whether altered redox state plays a role in the regulation of the BMPR2 pathway in pulmonary vascular cells. Our current studies provide evidence for extracellular superoxide regulating BMPR2 expression via a Matrix Metallopeptidase 9 (MMP9)/cartilage oligomeric matrix protein (COMP) dependent pathway. Mice deficient of extracellular SOD (SOD3) show evidence of BMPR2 depletion both in the absence and presence of chronic hypoxia. Incubation of bovine pulmonary arteries under hypoxic conditions (1% O2 for 48h) increased MMP9 activity and expression which was associated with increased superoxide production and BMPR2 depletion. Moreover, adding exogenous superoxide dismutase (SOD) to the media inhibited MMP9 activation in response to acute hypoxia. In cultured human PASMC, 24 hours of hypoxia (1% O2) induced significant increase of MMP9 activity and reduced COMP expression. MMP9 contributes to COMP/BMPR2 depletion in chronic hypoxia based on MMP9 deficient mice showing a preserved expression of both COMP & BMPR2 in lung tissues. Moreover, MMP9 deficient mice exposed to chronic hypoxia or Sugen/hypoxia showed a marked reduction of the elevated RV systolic pressures and Fulton index measurements of right ventricular hypertrophy compared to wild type mice under the same experimental conditions. ECHO measurements of PAAT/ET ratio, which correlate inversely and linearly with mean pulmonary artery pressure, in hypoxia‐exposed MMP9 KO mice showed increased ratios compared to animals exposed to chronic hypoxia or Sugen/hypoxia. These data are consistent with depletion of MMP9 reducing the development of pulmonary hypertension. Thus, increased extracellular superoxide appears to promote pathological loop of MMP9 mediated depletion of COMP leading to destabilization of BMPR2 and loss of its pulmonary hypertensive protective actions.

Support or Funding Information

Supported by NIH Grants HL115124 & HL129797
Identification, Characterization, and Treatment for a Taurine Transporter (SLC6A6) Variant Resulting in Taurine Deficiency and Pathologies in a Consanguineous Family.
Authors: Henry L. Keith , Ansar Muhammad , Ranza Emmanuella , Shetty Madhur , Paracha Sohail A. , Azam Maleeha , Kern Ilse , Iwaszkiewicz Justyna , Farooq Omer , Pournaras Constantin J. , Malcles Ariane , Kecik Mateusz , Muzaffar Waqar , Qurban Aziz , Ali Liaqat , Aggoun Yacine , Santoni Federico A. , Makrythanasis Periklis , Ahmed Jawad , Qamar Raheel , Rivolta Carlo , Sarwar Muhammad T. , Antonarakis Stylianos E.
Published: April 2020

More than 200 Pakistani consanguineous families with multiple individuals having recessive visual impairments were evaluated by exome sequencing and genotyping. One of the families, F315, from the Kohat region of Pakistan contained 2 affected individuals with a homozygous deleterious variant Gly399Val (NM_003043.5:c.1196G>T) in the taurine transporter SLC6A6 (MIM:186854) which segregated with the phenotype of progressive retinal degeneration. Gly399 is highly conserved in vertebrates and molecular modeling suggests that the increased bulk of the valine substitution displaces Tyr138 which participates in substrate coordination and transport and is consistent with the hypomorphic variant exhibiting only ~15% of the transport capacity of the native protein as determined by taurine uptake analyses in HEK‐293 cells transiently or stably expressing SLC6A6 or the Gly399Val variant. The reduced function was attributed to a catalytic effect rather than plasma membrane levels and analysis of patient‐derived fibroblasts of affected individuals mirrored these findings. Affected individuals exhibited cone‐rod retinopathy and cardiomyopathy and remarkably taurine supplementation for 24 months resulted in resolution of the cardiomyopathy and clinical stabilization of the retinopathy.

Support or Funding Information

ProVisu foundation and ERC grant 219968 to S.E.A., and NIH grant DA027845 to L.K.H.