bluuebunny/crossref_metadata_2025_split huggingface dataset:
Blunted Autophagy and Heat Shock Responses in Peripheral Blood Mononuclear Cells of Elderly Adults During Prolonged, Extreme‐Heat Exposure
Authors: McCormick James J. , Meade Robert D. , McGarr Gregory W. , Notley Sean R. , Fujii Naoto , Kenny Glen P. 
Published: April 2020

With rising global temperatures and accompanying increases in the incidence and severity of extreme heat events, there are growing concerns about the impact of heat stress on human health. Increases in the activation of autophagy and the heat shock response are essential to cellular survival under stressful conditions, such as extreme heat. Accumulating evidence from various animal models suggests an age‐related decline in the autophagy and heat shock response pathways during heat stress; however, this has not yet been assessed in humans. We therefore sought to evaluate the hypothesis that the autophagy and heat shock response would be blunted in elderly compared to young adults following a prolonged heat exposure. To assess this hypothesis, peripheral blood mononuclear cells (PBMCs) were harvested from 10 young (23 [SD 3] years; 3 women) and 9 elderly adults (71 [SD 3] years; 2 women) prior to and following 9‐hours of resting exposure to extreme heat (40°C, ~15% relative humidity). Changes in microtubule associated protein 1 light chain 3 beta (LC3)‐II (indicative of autophagy) and heat shock protein 70 were analysed via Western blot, while their associated genes (LC3B and HSPA1A, respectively) were measured via quantitative reverse transcription polymerase chain reaction. All values were normalized to β‐actin and reported as fold changes relative to baseline. Core temperature (indexed by rectal temperature) was measured throughout the exposure. Data are presented as means and standard deviations (SD) and were compared between‐groups using unpaired (2 tailed) t‐tests (α=0.05). After 9‐h of heat exposure, the elevation in core temperature from baseline values were greater in the older (1.0°C [SD 0.3]) vs. young adults (0.6°C [SD 0.2]; p<0.01). Despite this, LC3‐II protein was lower in elderly (2.02 [SD 2.00]) when compared to young adults (4.45 [SD 5.93]; p=0.04). This was further revealed by a reduction in LC3B gene expression in elderly (1.39 [SD 0.39]) compared to young (2.57 [SD 3.78]; p=0.04) adults suggesting a blunting in the autophagic response during heat stress. In elderly, HSP70 protein and HSPA1A gene expression were both lower when compared to young (1.21 [SD 0.43] vs. 1.80 [SD 0.65]; p=0.02 and 1.58 [SD 1.64] vs. 2.88 [SD 4.29]; p=0.04, respectively) suggesting an age‐associated decline in the heat shock response. These preliminary findings demonstrate that despite greater elevations in core temperature, elderly adults experience blunting in both the autophagy and heat shock response during a prolonged extreme‐heat exposure.Support or Funding InformationCanadian Institutes of Health Research
Microglial Targeting as a Therapeutic Strategy for Social Stress Susceptibility
Authors: Pope Brittany S. , Harrington Evelynn N. , Bouknight Samantha J. , Martinez- Muniz Gustavo A. , Augenblick Lee M. , Morgan C. Grant , Wood Susan K. 
Published: April 2020

Social stress is a common risk factor for anxiety and cardiovascular disease (CVD). Women are more likely than men to suffer from comorbid anxiety and CVD. While the underlying neural mechanisms linking these pathological conditions remain unclear, the locus coeruleus (LC) is a stress‐sensitive region known to facilitate both behavioral and cardiovascular responses to social stress. Importantly, neuroinflammation has been shown to play a causative role in generating anxiety‐like behavior, yet whether pharmacological manipulation of microglia represent a strategy to regulate stress susceptibility in a female population remains to be determined. Here, we utilize mannosylated liposomal clodronate, a small hydrophilic molecule toxic to microglia. Both the liposomes and the drug clodronate (CLD) are selective for macrophages/microglia, making this intra‐LC administration a targeted depletion of microglial cells. Two separate studies were conducted to test the hypothesis that microglial targeting is effective in regulating susceptibility to social stress. In study #1, following recovery from surgical implantation of cardiovascular transmitters (HD‐S11, DSI) and bilateral LC cannulae, rats were treated with liposomal CLD injections (5 or 25 μg/side) or empty‐liposomal vehicle (0 μg/side). Female rats were then subjected to witnessing an aggressive social defeat encounter between a male intruder and a novel male resident for 15 min on 5 consecutive days. This study identified that witness stress exposure increased anxiety‐like burying compared with controls and that partial microglial depletion in the LC attenuated this burying response. Interestingly, intra‐LC CLD had no cardiovascular effect upon the first exposure to stress, yet promoted the pressor response following repeated stress exposure (day 5). To further examine whether microglia are controlling the behavioral and cardiovascular responses to stress, female rats were treated with intra‐LC LPS (0, 1, 3 ug/rat) with or without a TLR‐4 selective microglial inhibitor (LPS‐RS and (+)‐ naloxone) prior to witness stress exposure. While these treatments had no effect under non‐stress conditions, witnesses treated with LPS alone exhibited greater burying duration compared with those treated with vehicle, and this was blocked by the microglial inhibitor. Taken together, these studies reveal a novel role for microglia in regulating behavioral and cardiovascular responses to social stress and demonstrate that selective targeting of microglia may be a therapeutic strategy to alter stress susceptibility.Support or Funding InformationSupport provided by 1R01MH113892‐01A1, NARSAD Young Investigator Grant 26809, I21 BX002085.
Regulation of Neutrophil Function by Selective Targeting of Glycan Epitopes Expressed on the Integrin CD11b/CD18
Authors: Brazil Jennifer Catherine , Kelm Matthias , Lehoux Sylvain , Azcutia Veronica , Cummings Richard D. , Nusrat Asma , Parkos Charles A. 
Published: April 2020

Polymorphonuclear neutrophils (PMN) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMN and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease and chronic obstructive pulmonary disease. The glycoprotein CD11b/CD18 plays a well described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMN. MS analyses revealed a surprising lack of sialylation on CD11b glycans. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis (GNA) lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin (PHA‐E) lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN associated tissue damage in chronic inflammatory diseases.Support or Funding Information
This work was supported by the Crohn’s and colitis foundation (SRA # 544596)
The balance of life and death may hinge on the intracellular localization of the small GTPase, Rap1b in the megakaryocyte cell line (DAMI)
Authors: Wisinski Jaclyn , McElfresh Regan , Muntifering Grace , Nguyen Tam , Veleke Carter 
Published: April 2020

Platelets are specialized cell fragments that circulate in the bloodstream and clot to prevent blood from leaving damaged vessels. The number of circulating platelets is governed by the balance of platelet production by mature megakaryocytes and clearance of platelets from the bloodstream. Normal platelet function requires the signaling protein, Rap1b, whose abundance also increases as megakaryocytes mature. The intracellular location of Rap1b is determine by its phosphorylation state. When Rap1b is not phosphorylated, it associates with membranes; conversely when Rap1b is phosphorylated, it detaches from membranes and reside in the cytosol. We hypothesize that Rap1b, specifically in its phosphorylated, cytosolic state, promotes megakaryocytes survival and proliferation. To address this hypothesis, the human megakaryocyte cell line (DAMI cells) have been modified 1) by disrupt the rap1b gene creating a Rap1b‐knockout cell line, 2) stable overexpression of a Rap1b phospho‐mimetic mutant, 3) stable overexpression of a Rap1b phospho‐deficient mutant or 4) stable overexpression of wild‐type Rap1b (to be used as a control). Using the modified DAMI cell lines we are beginning to understand the impact of Rap1b phosphorylation on proliferation on in MTS survival assays. Changes in cell survival can be attributed to changes in cell cycle progression and/or susceptibility to apoptosis. To address possible changes in cell cycle progression, we have evaluated each cell line based on the percentage of cell undergoing DNA synthesis at a given time by BrdU incorporation assays. To evaluate apoptosis susceptibility, mitochondrial membrane integrity and activation/cleavage of Caspase 9 was determined in the presence or absence of the apoptotic inducer ABT‐737. The analysis of the role of Rap1b in megakaryocytes has the potential to increase our collective understanding of regulation of platelet count and could be a novel target to combat thrombocytopenia (low platelet count) or thrombocythemia (high platelet count).Support or Funding InformationUniversity of Wisconsin ‐LaCrosse Faculty Research Grant
The Early Progression of Renal Injury in Obese Dahl Salt‐Sensitive Rats is Associated with Increased M2 Macrophage Infiltration
Authors: Poudel Bibek , Shields Corbin A. , Cornelius Denise C. , Williams Jan M. 
Published: April 2020

Previously, we reported that, prior to puberty, the obese Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) strain displays proteinuria and renal injury independent of hyperglycemia and elevations in arterial pressure. Recently, we observed that the early development of renal injury in the obese SSLepRmutant strain was associated with a 5‐fold increase in renal infiltration of macrophages compared to SSWT rats. In this study, we examined whether depletion of macrophages with clodronate would reduce renal injury in prepubescent obese SSLepRmutant rats. Four week‐old SSWT and SSLepRmutant rats were separated into four groups (n=8/group): (1) SSWT and (2) SSLepRmutant rats treated with vehicle and (3) SSWT and (4) SSLepRmutant rats treated with clodronate (50 mg/kg, i.p., twice/week) for 4 weeks. While administration of clodronate did not alter the macrophage population in SSWT rats, clodronate reduced macrophages in the kidneys from SSLepRmutant rats compared to vehicle‐treated rats (8±4 vs. 33±10 % gated, respectively; p<0.05 vs. vehicle) without altering the monocyte population. Interestingly, we did not observe any effects on MAP in animals chronically treated with clodronate. At baseline, protein excretion was significantly higher in SSLepRmutant rats compared to SSWT rats (70±14 vs. 10±3 mg/day, respectively; p<0.05 vs. SSWT) that further increased over the course of the study (335±82 vs. 45±14 mg/day, respectively; p<0.05 vs. SSWT). After 2 weeks of treatment with clodronate, protein excretion was markedly reduced in the SSLepRmutant strain (159±32 vs. 303±52 mg/day; p<0.05 vs. vehicle). However, after 4 weeks of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (346±195 vs. 399±50 mg/day). Chronic treatment with clodronate significantly decreased glomerular injury and renal fibrosis in the obese SSLepRmutant strain. Since clodronate depletes all macrophages: M1 (pro‐inflammatory) and M2 (anti‐inflammatory or pro‐inflammatory), we also examined macrophage polarization in the vehicle treated lean SSWT and obese SSLepRmutant strain prior to puberty at 8 weeks of age. We found that the M1/M2 ratio was significantly higher in the lean SSWT versus obese SSLepRmutant rats (1.60±0.05 vs. 0.54±10, respectively; p<0.05 vs. SSWT), demonstrating more M2 macrophages in the kidneys of the obese SSLepRmutant strain. Overall, these data show that the early development of renal injury in the obese SSLepRmutant strain is associated with increased renal M2 macrophages and suggests that M2 macrophages may be a therapeutic target in obesity‐induced renal injury.Support or Funding InformationThis study was supported by DK109133.
Effects of In Vivo Administration of an Endothelial KCa Channel Activator on Recovery of Cardiac Function Following Acute Myocardial Infarction in Mice
Authors: Kim Tae yeob , Belke Darrell D. , Mishra Ramesh C. , Braun Andrew P. 
Published: April 2020

Cessation of coronary blood flow, due to stenosis or acute blockage, produces prolonged ischemia that results in myocardial infarction (MI) and impaired cardiac function and blood pumping activity of heart. Increasing coronary blood flow to the ischemic area and/or reducing myocardial work are predicted to mitigate the damage typically associated with an acute MI injury. Recent studies from our group have demonstrated that pharmacological enhancement of endothelial KCa2.3 and KCa3.1 channels by the activator SKA‐31 improves agonist‐evoked vasodilation in small resistance arteries from multiple vascular beds and species. The goal of our present study is to investigate the potential cardio‐protective effects of SKA‐31 administration in male mice (12–15 weeks of age, C57B1/6 strain) following an acute myocardial infarct (MI) induced by permanent surgical ligation of the left‐anterior descending (LAD) coronary artery. Within 48 h post‐surgery, MI and sham surgery mice were treated orally with either vehicle or 10 mg/kg of SKA‐31 for 6–7 weeks. Myocardial damage in MI mice was confirmed by histological staining. Echocardiographic assessment of cardiac structure and function 2 weeks post‐surgery revealed that left ventricular (LV) wall thickness during systole and diastole was similar in sham animals treated with either vehicle or SKA‐31. In contrast, LV wall thickness during systole and diastole was ~30% greater in MI surgery mice receiving SKA‐31 treatment. At 4 weeks post‐surgery, sham animals exhibited LV wall thickness values during systole (1.00 mm ± 0.03, n=3) and diastole (0.82 mm ± 0.10, n=3) that were similar to those observed at 2 weeks, indicating that SKA‐31 treatment did not adversely affect these parameters. In MI surgery animals receiving SKA‐31 administration for 4 weeks, LV wall thickness during systole (0.97 mm ± 0.10, n=5) and diastole (0.80 mm ± 0.08, n =5) decreased to the values observed in sham surgery mice. At the end of the SKA‐31 treatment period, cardiac output (CO) and LV ejection fraction (EF) measured by pressure‐volume loop analysis were both higher in sham mice (CO = 6.49 mL/min, EF = 50.9%) compared with MI surgery animals (CO = 5.83 mL/min, EF = 40.7%). Following completion of ongoing measurements and analyses, we will present additional data describing changes in cardiac function in MI surgery mice treated with drug vehicle, and histological assessment of myocardial integrity in sham and MI surgery mice. Our preliminary findings thus far suggest that enhancement of endothelial KCa channel activity by SKA‐31 administration improves recovery of cardiac function following ischemic damage associated with blockade of the LAD coronary artery.Support or Funding InformationThis work was supported by research funding to APB from the Canadian Institutes of Health Research (MOP 142467) and the Natural Sciences and Engineering Research Council of Canada (RGPIN‐2017‐04116).
Exploring a 13‐nucleotide Motif Capable of Driving Cap‐independent Translation
Authors: Kharbanda Pankhuri , Flores Megan A. , Juba Amber N. , Wellensiek Brian P. 
Published: April 2020

In the classical model of eukaryotic translation, a 7‐methylguanosine cap at the 5’ end of the messenger RNA (mRNA) acts as the recognition signal to recruit the ribosome to the RNA strand. The ribosome then scans the mRNA for the translation initiation codon, AUG. However, under stressful circumstances—such as apoptosis or viral infection—the recognition signal can be lost. Despite this, there is increasing evidence that translation still seems to proceed under certain circumstances. This cap‐independent mode of translation has been seen to occur through various regulatory elements along the mRNA. In an attempt to identify sequences that drive cap‐independent translation, the human genome was surveyed using mRNA display. The results of this survey led to the identification of many translation enhancing elements (TEEs) scattered throughout the genome. Further characterization of these TEEs provided evidence that they are able to recruit the ribosome to internal mRNA sequences, therefore bypassing the cap structure. A combination of sequence function and phylogenetic analysis led to the isolation of a short 13 nucleotide motif within several TEEs that is capable of driving cap‐independent translation. Further evaluation of the 13mer suggests that the functional motif maybe larger than originally thought, and the sequence context surrounding the 13mer can influence its effectiveness. When exploring the sequence around the 13mer, protein translation appeared to be the most efficient when the upstream nucleotides, preceding the start site, followed a triplicate pattern. To further investigate this phenomenon, we have explored exact start codon usage, performed cryptic promoter analysis, and conducted mutational mapping of the sequence the 13mer was originally discovered within. The results of our study suggest that the ribosomal binding site is larger than 13 nucleotides and translation initiation likely occurs at a position other than the original ribosomal binding site.Support or Funding InformationMidwestern University Intramural funds
The Them1 START Domain Regulates Nuclear Trafficking in Brown Adipocytes
Authors: Baqai Mahnoor , Li Yue , Tillman Matthew , Ortlund Eric A. , Cohen David E. , Hagen Susan J. 
Published: April 2020

Thioesterase superfamily member 1 (Them1) is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is a fatty acyl‐CoA thioesterase with tandem hotdog fold thioesterase domains, an N‐terminal set of phosphorylation sites at S15, 18, and 25, and a C‐terminal lipid‐binding steroidogenic acute regulatory protein‐related lipid transfer (START) domain, which modulates thioesterase activity. Cell fractionation studies have demonstrated that Them1 thioesterase activity is localized in microsomes and nuclei, suggesting that it is targeted to specific intracellular compartments. Our aim was to determine the mechanism by which Them1 traffics to the nucleus and its function therein.MethodsIdentification of a putative nuclear localization signal was determined using cNLS mapper (
http://nls‐mapper.iab.keio.ac.jp/cgi‐bin/NLS_Mapper_form.cgi). Full‐length, mutant, or truncated THEM1 constructs were cloned and linked to EGFP. Plasmids or adenoviral vectors were transfected into a differentiated brown adipocyte cell line (MUCAR cells) that does not express Them1. The localization of Them1 was visualized by confocal microscopy via EFGP and mRNA expression was determined using qPCR. Confocal z‐stacks were used to produce 3‐D images with Volocity software. Them1 nuclear localization was evaluated in the presence or absence of the nuclear export inhibitor leptomycin B. Cyclohexamide was used to block new protein synthesis.ResultsPhosphomimic mutations that simulated Them1 phosphorylation at S15 resulted in a diffuse Them1 localization. Stimulation of cells with norepinephrine resulted in Them1 phosphorylation and its reorganization from discrete puncta to a diffuse localization. In the diffuse state, Them1 was transported into the nucleus. Treatment with cyclohexamide suggested that the nuclear Them1 pool was not newly synthesized. We identified a nuclear localization signal in a hinge region located between the 2nd thioesterase domain and the START domain. Deletion of either the nuclear localization signal or the START domain in full length Them 1 blocked nuclear targeting. Conversely, mutant Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus, as evidenced by exit blockade using leptomycin B. Nuclear Them1 regulated mRNA expression levels for a number of genes that regulate lipid metabolism in brown adipocytes.ConclusionsOur results demonstrate that trafficking of Them1 to the nucleus occurs via a nuclear localization signal within the START domain. We speculate that nuclear Them1 regulates the transcription of lipid metabolic enzymes by a mechanism that is distinct from its acyl‐CoA thioesterase activity.Support or Funding InformationThis work was supported by NIH DK103046 to DEC, SJH and EAO.
White Matter Hyperintensities and Carotid Arterial Stiffness in Amnestic Mild Cognitive Impairment
Authors: Curtis Bryon Matthew , Tomoto Tsubasa , Chiles Collin , Austin Courtney , Zhang Rong 
Published: April 2020

IntroductionWhite matter hyperintensities (WMH) is a risk factor of cognitive impairment and Alzheimer’s disease (AD). Arterial stiffening is associated with increased arterial pressure and cerebral blood flow (CBF) pulsatility which may lead to WMH pathogenesis. However, the relations among WMH, arterial stiffness, and pulsatile change in arterial pressure and CBF remain unclear. Therefore, this study investigated the associations among WMH volume, carotid arterial stiffness, carotid arterial pressure, and CBF pulsatility in patients with amnestic mild cognitive impairment (MCI), a prodromal stage of AD.MethodsFifty‐four amnestic MCI patients and twenty‐four cognitively normal subjects (CN) underwent measurements of WMH volume using T2‐weighted‐fluid‐attenuated inversion recovery imaging. Carotid β‐stiffness index and arterial pressure at the common carotid artery (CCA) were measured using ultrasound and applanation tonometry. CBF pulsatility index (PI) was measured from the middle cerebral artery using transcranial Doppler.ResultsWMH volume, PI, carotid β‐stiffness index, carotid systolic blood pressure (cSBP) and pulse pressure (cPP) did not differ between groups. Greater WMH volume was correlated with higher CBF PI in both groups (MCI: r=0.373, NC: r=0.463, p<0.05), whereas it was correlated positively with carotid β‐stiffness index, cSBP, and cPP only in the MCI group (MCI: r=0.499, r=0.432, r=0.468, all p<0.01). In multiple linear regression analysis, cSBP was an independent determinant of WMH (β=0.293, p=0.005) with adjustment for age, sex, body mass index, and MCI status.ConclusionThese findings collectively suggest that carotid arterial stiffness, carotid SBP, and pulse pressure may play an important role in WMH pathogenesis in MCI.Support or Funding InformationThis work was funded by the National Institutes of Health ((R01AG033106, R01HL102457, P30AG012300)