bluuebunny/crossref_metadata_2025_split huggingface dataset:
G protein‐coupled receptor 37L1 participates in epigenetic gene regulation in human renal proximal tubule cells
Authors: Konkalmatt Prasad R. , Fan Caini , Cuevas Santiago , Armando Ines , Jose Pedro A. 
Published: April 2020

G protein‐coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormality in the renal luminal sodium transport plays an important role in the pathogenesis of essential hypertension. There are a growing number of GPCRs and their interacting protein components that are localized on the nuclear membrane or within the nucleus, where they retain their signaling functions. In most cases, nuclear GPCRs are constitutively expressed on the nuclear membrane. The functions of these nuclear GPCRs are not well understood. Recently we showed that G protein‐coupled receptor 37L1 (GPR37L1), expressed on the apical membrane of renal proximal tubules (RPT), regulates luminal sodium transport and blood pressure via modulating NHE3 function. However, the GPR37L1 signaling pathway in the RPT has not been studied. Co‐immunoprecipitation (Co‐IP), combined with mass spectrometry analyses, revealed the association of GPR37L1 with nuclear import proteins: IPO7, IPO8, KPNA2, KPNB1; nuclear export proteins: XPO1 and XPO7; nuclear pore complex proteins: NUP210; kinases involved in signaling: mTOR, PRKDC, IRS4; and a DNA (Cytosine‐5‐)‐Methyltransferase 1 (DNMT1) in the GPR37L1 protein complex. Immunoblot analyses of nuclear preparations from the human RPT cells (hRPTCs) confirmed the presence of GPR37L1 in the nucleus. Confocal fluorescence imaging of hRPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1‐GFP) showed distinct nuclear membrane expression of GPR37L1. Association of GPR37L1 with mTOR, PRKDC, and DNMT1 proteins was confirmed by Co‐IP followed by immunoblot analyses. Overexpression of GPR37L1 in hRPTCs increased the phosphorylation of PI3, and AKT and decreased the expression of DNMT1, and NHE3 promoter methylation. Our data show that GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNMT1. Decreased DNMT1 function reduces CpG methylation on the NHE3 promoter that in turn increases NHE3 transcription. This is the first report showing the direct role of a nuclear GPCR in the regulation of renal sodium transport through epigenetic gene regulation.Support or Funding InformationR56DK116828: Prasad Konkalmatt
Selective Peripheral Chemoreceptor Hyperreflexia Evoked after Sympathetic Stimulation
Authors: Felippe Igor Simoes Assuncao , Paton Julian F. R. 
Published: April 2020

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P<0.05; differences were tested using paired student‐t test.The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.Support or Funding InformationHealth Research Council of New Zealand
Understanding PM2.5‐Induced Oxidative Stress In Alveolar Macrophages
Authors: Kaur Mandeep , Raval Kosha , Flores David , Olivas Micah , Waterston Anthony , Hasson Alam , Dejean Laurent 
Published: April 2020

In situations of oxidative stress, the antioxidant processes of any eukaryotic organism can eventually become overwhelmed. Exposure to ambient particulate matter 2.5 (PM2.5), composed of transition metals and organic compounds 2.5μm or less in diameter, has been shown to cause oxidative stress in alveolar macrophages (AMs). This can lead to various adverse health effects, including cardiovascular and pulmonary diseases. In this study, we examine the capacity of ambient PM2.5 samples collected during periods of peak air pollution in Fresno and Claremont, CA to trigger ROS production in AM cells. These PM2.5 samples were characterized for their chemical content using chromatography and mass spectrometry. A DCF‐fluorescence based microplate assay was used to estimate the potential of the PM2.5 samples to trigger ROS production in NR8383, rat AM cells. Two types of quinones (1,2‐Naphtaquinone and 1,4‐Naphtaquinone) and two transition metals (Cu2+ and Fe2+) were associated with samples that had a high capacity to induce ROS generation in NR8383. While establishing a cytotoxic link between PM2.5 constituents and ROS, we also seek to understand the underlying biological mechanisms through which PM2.5 interacts with AM cells to produce excessive ROS. Previous observations suggest that the upregulation of NADPH Oxidase (NOX1), an enzyme that enhances ROS production, may contribute to cellular oxidative stress in macrophages. In this study, NOX1’s potential role in a PM2.5‐ induced oxidative stress pathway is investigated in NR8383 cells incubated with concentrations of iron and copper relevant with those found in the collected PM2.5 samples. The change in NOX1 expression was quantified using fluorescence‐based western blotting, with zymosan, a well‐known inducer of oxidative stress in macrophages, used as positive control. Our preliminary results indicate that 4‐hour treatments with increasing relevant iron concentrations triggered both an increase of ROS and NOX1 protein levels in NR8383. Our current experiments consist of testing the potential of relevant concentrations of other chemical constituents of PM2.5 suspected of triggering increased ROS production and NOX1 expression (these include naphtaquinones, phenanthraquinones, and copper). Understanding which chemical components of PM2.5 are able to induce significant amounts of ROS will allow us to understand how pollution is pernicious and advocate for the global reduction of PM2.5 emission.Support or Funding InformationThis work is supported by Fresno State ASI, the Division of Research and Graduate Studies, and Fresno State’s CSU‐LSAMP program funded by NSF under grant #HRD‐1826490.
Serum Extracellular Vesicles Derived From Obese Mice Control Energy Expenditure and Promote Liver Steatosis in Lean Mice
Authors: Cunha e Rocha Karina , de Mendonça Mariana , Rodrigues Alice Cristina 
Published: April 2020

Cells can communicate with neighboring or distant cells trough extracellular vesicles (EVs). EVs have been shown to participate in many diseases, however, little is known about their role in metabolic diseases. Obesity is a major risk factor for noncommunicable diseases, and represents a threat to global public health. Indeed, obesity is rising in incidence, prevalence, and economic burden. Once the number of released EVs and its cargo are altered during obesity, we hypothesize that EVs intercellular communication may play a role in obesity pathophysiology. In order to investigate the metabolic changes caused by EVs in obesity, C57BL/6 male mice fed a control balanced diet were injected (i.p.) with serum EVs derived from diet‐induced obese mice (400ug/mouse) every 3 days for 8 weeks. Body weight was assessed every week. Visceral and subcutaneous fat depots, liver and gastrocnemius and soleus muscles were dissected and weighed. Serum biochemical analysis were conducted, and the content of hepatic triglyceride and total cholesterol was measured. Glucose, insulin and pyruvate tolerance tests were performed, as well as body composition analysis and indirect calorimetry. Differences at p<0.05 were considered significant. Compared to the control group, EVs injection attenuated weight gain, reduced the weight of subcutaneous fat depot, gastrocnemius muscle and liver, decreased the respiratory exchange ratio and increased hepatic triglyceride content, oxygen consumption and insulin sensitivity. Our results suggest that EVs released in obesity can modulate lean mice metabolism. Altogether, these data provide initial insights on EVs function and signaling during obesity.Support or Funding InformationThis study was financed in part by grants #2018/05426‐0 and #2019/14999‐6, São Paulo Research Foundation (FAPESP) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001.
24‐Hour Total Sleep Deprivation Alters Cardiovagal Baroreflex Sensitivity and Blood Pressure Reactivity in Mid‐Life Adults
Authors: Colling Morgan E. , Greenlund Ian M. , Gonzalez Joshua E. , Carter Jason R. 
Published: April 2020

Nearly 1 in 3 American adults fail to get recommended levels hours of sleep, and epidemiological studies report a significant relationship between chronic sleep deprivation and hypertension. A number of studies from our laboratory and others have focused on neural and cardiovascular responses to a 24‐hour total sleep deprivation (TSD) model. However, these prior studies have primarily been conducted in young or older populations, with a gap in mid‐life adults. This is relevant because mid‐life can often be when chronic sleep levels reach a lifetime nadir. The purpose of this study was to determine the cardiovascular effects of 24‐h TSD on mid‐life adults. Nine participants (6 men and 3 women, BMI: 28 ± 5 vs. 22 ± 3 kg/m2) between the ages of 40 and 55 yr were tested twice, once after 24‐h TSD and once after normal sleep (randomized, crossover design). In both tests, participants were equipped with an automated sphygmomanometer (Omron HEM‐907XL, Omron Health Care) to assess mean arterial pressure (MAP), finger plethysmography to measure beat‐to‐beat arterial pressure (NOVA, Finapres), and a three‐lead electrocardiogram to record heart rate. Following a 10‐minute supine baseline, participants performed three 15‐s Valsalva maneuvers (VM) with 1‐minute recovery periods. Following another 3‐minute post‐VM baseline, a 2‐minute cold pressor test (CPT) was performed by submersing each participant’s hand in ice water. Data were analyzed using WinCPRS software (Absolute Aliens, Turku, Finland). R‐waves were detected and marked in the time series. R‐R interval (ms) vs. systolic arterial pressure (SAP, mmHg) were plotted for Phase IV of each VM, which was defined as the interval between the first SAP that exceeded the mean supine resting baseline SAP to peak SAP during Phase IV “overshoot.” Cardiovagal baroreflex sensitivity (cBRS) was determined from the slope of the regression line. The cardiovascular responses to each VM were included if the correlation coefficient exceeded 0.7 and the SAP increase was at least 15 mmHg (n=7). Irregular “square waves” were excluded from analysis. Statistical analysis included repeated measures ANOVA (p<0.05). TSD did not alter baseline MAP (84±3 vs. 85±4 mmHg) or heart rate (62±4 vs. 58±3 beats/min, p>0.05 for both). Cardiovagal baroreflex sensitivity (cBRS) was significantly reduced after 24‐h TSD (21.9 ± 10.3 vs. 14.7 ± 6.47 ms/mmHg, p<0.05). SAP reactivity was augmented following TSD during CPT (Δ16±7 vs. Δ25±6 mmHg, p=0.05), and DAP reactivity trended higher following TSD during CPT (Δ14±3 vs. Δ19±4 mmHg, p=0.09). In summary, TSD impacts baroreflex sensitivity and blood pressure reactivity in mid‐life adults.Support or Funding InformationSupport was provided by the American Physiological Society’s (APS) Summer Undergraduate Research Fellowship, Michigan Space Grant Consortium, and the Portage Health Foundation.
Smooth Muscle Cell – Extracellular Matrix Alterations in Genetic Vasculopathies
Authors: Buja Louis Maximilian , Zhao Bihong , Lelenwa Laura , Cecchi Alana , Safi Hazim , Estrera Anthony , Milewicz Dianna 
Published: April 2020

Pathogenic variants in 11 genes predispose individuals to aneurysms and dissections of the thoracic aorta and in some cases, vascular complications throughout the arterial tree, termed heritable thoracic aortic disease (HTAD) (Renard M et al. JACC, 2018;72:605–615). These genes are involved in vascular smooth muscle cell (SMC) contraction and metabolism, extracellular matrix (ECM) integrity and transforming growth factor β (TGF β) signaling. A unifying hypothesis is that the mutated gene products contribute to altered mechano‐transduction leading to vasculopathy (Humphrey JD et al. Circ Res 2015;116:1448–1461; Pinard A, Jones GT, Milewicz DM. Circ Res 2019;124:588–606). The objective of this study was to employ light (LM) and electron (EM) microscopy to evaluate the thoracic aortic and other arterial tissue from individuals harboring pathogenic variants in the 11 established predisposition genes who underwent surgical aneurysm or dissection repair. These individuals (n) harbored pathogenic variants in the following genes: FBN1 (13), ACTA2 (6), TGFBR1 (7), TGFBR2 (3), TGFβ2 (3), SMAD3 (3), LOX (1), MYLK (1), PRKG1 (1), and COL3A1 (4); total 42. Arterial specimens were evaluated using LM consensus criteria (Halushka MK et al. Cardiovasc Pathol 2016;25:247–257). EM was performed on selected cases (Davis EC. Lab Invest 1993;68:89–99). By LM, specimens were graded (0 – 3+) for mucoid extracellular matrix accumulation (MEMA), elastic fiber fragmentation and/or loss (EFFL), SMC nuclear loss/laminar medial collapse (SMCNL/LMC), and overall medial degeneration (A 1–2, B 3–4, C 5–6, D 7–9); intimal lesions (0–3+) and medial dissection (+/−) also were noted. The 42 specimens included 17 with medial dissection and 25 non‐dissected aneurysms. By LM, cases showed a spectrum of extent and severity of medial degenerative changes: A mild 8 cases, B moderate 18 cases, C moderately severe 14 cases, and D severe 2 cases. By EM, COL3A1 specimens showed reduction of collagen fibrils and shape variations of residual collagen fibrils; elastin fiber connections to SMCs were intact. In contrast, specimens with mutations in the TGF β pathway exhibited multiple foci with deficiency of elastic fibers and loss of elastic fiber connections to SMC but had normal collagen ultrastructure. These results support the hypothesis that the pathogenesis of genetic vasculopathies involves structural defects in the interaction of ECM components and SMCs, including impairment of the elastin‐contractile unit directly or through loss of structural support provided by collagen.Support or Funding InformationLocal funds
Pharmacokinetic Analysis of Ionizable Zwitterionic Oximes: Antidotes to Organophosphate Exposure.
Authors: Samskey Nathan , Ho Kwok-Yiu , Momper Jeremiah , Radic Zoran , Sit Rakesh , Sharpless K. Barry , Taylor Palmer 
Published: April 2020

Currently approved antidotes to organophosphate (OP) exposure rely on discoveries of Irwin Wilson in the 1950’s, employing site‐directed pyridinium aldoximes to reactivate OP‐conjugated acetyl‐cholinesterase (AChE). This led to approval of 2‐PAM in the US and other pyridinium aldoximes elsewhere as antidotes to exposures of OP pesticides and terrorism nerve agents. However, principal limitations of these quaternary agents reside in their inability to cross the blood‐brain barrier and reactivate CNS AChE rapidly and their inherently rapid overall body clearance. Moreover, certain pyridinium aldoximes carry risks of channel blocking side effects. Alternative compounds that are ionizable zwitterions (hydroxyiminoacetamido alkyl amines‐RS194B) show facile transfer across the blood‐brain barrier through its neutral species, limited reversible binding to OP conjugated AChE, oral bioavailability and minimal toxicity. However, high doses may be needed for acute treatment, since these agents are extruded by brain capillary transporters and are rapidly cleared as the parent compound by renal transporters. To understand and counter these actions, we employed two approaches: (a) examination of tissue disposition and pharmacokinetics in a P‐glycoprotein knock‐out strain of mice, and (b) employing small molecules to inhibit extrusion of RS194B by brain capillaries and limit rapid clearance by renal tubules. While gene knock‐out enhances brain levels in mice, this is not practical in humans, so antidote retention in the CNS and diminishing renal clearance require competing small molecules. To avoid an imposed requirement of approval of two new agents, the antidote and a clearance inhibitor, we examined current FDA‐approved drugs for repurposing in a formulation. We have achieved favorable outcomes with transport inhibitors already approved by the FDA for other therapeutic purposes. Our approach employs agents that are zwitterionic, block P‐glycoprotein transport in the CNS and kidney, and show limited liability or toxicity for short term use. We have compared various fluoroquinolones, biguanides, ion channel blockers and have achieved favorable ratios of enhanced CNS levels and diminished renal clearance of RS194B in rodents at doses not eliciting toxicity. These comparisons enable assessing efficacy of the combination in other animal species.Support or Funding Information(Supported in part by RO1 GM18360 and UO1 NS058046)
Mapping Key Active Site Residues of the PA3944 Gcn5‐related N‐acetyltransferase
Authors: Variot Cillian , Baumgartner Jackson , Houseman Charles , Zhang Brian , Joe Layton , Kuhn Misty L. 
Published: April 2020

PA3944 is an acetyltransferase from Pseudomonas aeruginosa that acetylates polymyxin antibiotics in vitro. It belongs to the large Gcn5‐related N‐acetyltransferase (GNAT) superfamily. We previously determined the 3D structure of this enzyme, but were unable to obtain a complex in the presence of polymyxin. Since it is not know which residues in the active of this protein are critical for function and recognition of the polymyxin substrate, we created point mutations of residues in the active site and assayed their kinetic activity. We found two key residues that were critical for activity of the enyzme: one was important for the catalytic mechanism and the other played a structural role. Additionally, we found two mutants that improved the enzymatic activity, which could be used to further explore the possibility of using this enzyme for biotechnological applications to modify polymyxin antibiotics with reduced toxicity. Moreover, these results provide insight into GNAT evolution and how key residues can alter substrate specificity and structure.Support or Funding InformationResearch reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).
DETERMINATION OF STRUCTURAL STABILITY AND CATALYTIC ACTIVITY OF G6PD VARIANTS USING MOLECULAR DYNAMIC SIMULATION (MDS) APPROACH
Authors: Hamza Muaawia Ahmed 
Published: April 2020

BackgroundGlucose‐6‐phosphate dehydrogenase (G6PD) is a known housekeeping enzyme that is important in the pentose phosphate pathway (PPP) and essential for basic cellular function. G6PD also aids in producing compounds to prevent build‐up of reactive oxygen species (ROS) within the red blood cells. Glucose‐6‐phosphate dehydrogenase deficiency (G6PDD) is a known hereditary mutation and X‐linked recessive genetic disorder that have affected over 400 million people worldwide. More than 186 G6PD variants have been characterized that affect enzyme catalytic activities and biochemical reactions leading to various clinical phenotypes.ObjectivesThe intention of this study was to investigate the genotype‐phenotype correlations of selected G6PD mutations via structural analysis and molecular dynamic simulation analysis (MDSA) tools.MethodESPript 3.0 software was used for multiple sequence alignments of G6PD from human and from other organisms. PyMOL molecular visualization tool was also used to shown and evaluate structural changes in ten selected variants of the protein.ResultsMultiple sequence alignment of G6PD from human and three other organisms, namely; Trypanasoma cruzi, Mycobacterium avium and Leuconostoc mesenteroides revealed three conserved regions. The findings of this study suggested that mutation to residues that are unable to adopt a cis conformation would lead to an extremely high activation energy barrier for the enzyme to adopt the correct transition state during the enzymatic reaction. The protein was subjected to several computational approaches as described in previous part. The simulation data from the molecular dynamic simulation helps to determine the protein dynamics through stability and flexibility of G6PD native and mutation.The native G6PD have high RMSD average value than Class I, class II and class III mutation, thus, have low structure stability. The G163D has the lowest RMSD value thus have the most stable structure through simulation. From RMSF analysis, the residue at 310 until 320 (loop 1), 374 until 388 (loop2), 408 until 414 (loop 3) and 424 until 434 (loop 4) responsible for structure flexibility and stability. The loop regions produce larger fluctuation thus, providing the flexibility to the structure.Findings from molecular dynamic analysis reported here will be more valuable if comparison to kinetic data of the mutants are made. Simulation of the enzyme dimer form could stipulate the relationship between structural changes and enzymatic activity of G6PD variants.Support or Funding InformationResearch Center, King Fahad Medical City, Riyadh, KSA