bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intestinal TMEM16A function as a luminal chloride channel
Authors: Kazi Mirajul Hoque , Saha Tultul , Aoun Joydeep , Hayashi Mikio , Sheikh Irshad Ali , Leblanc Normand , Sarkar Paramita , Ameen Nadia , Woodward Owen M. 
Published: April 2020

There has been a high level of uncertainty whether TMEM16A channels are truly expressed on the apical membranes of enterocytes and contribute to luminal Cl− secretion. Here, we describe a series of independent experimental approaches including qPCR, western blot, immuno‐staining and electrophysiology using Tmem 16a+/− mouse intestinal tissue, human colonic tissue and T84 cells to characterize the expression, localization and physiological function of TMEM16A and its regulation by the multi‐PDZ domain‐containing protein NHERF1.qPCR analysis revealed that Tmem16a transcripts are differentially expressed across the intestinal epithelial tissue with highest expression in the colon. Tmem16a mRNA expression did not correlate with TMEM16A protein expression. Western blot analysis of TMEM16A in mouse intestinal tissue revealed that one (Boster Bio PA2290) out of five commercially available polyclonal antibodies appreciably recognized a band at the expected size of below 100 kDa monomeric protein in jejunum, ileum, proximal and distal colon, which was absent in Tmem16a+/− mice. Ussing chamber experiments confirmed that TMEM16A protein abundance correlated with Cl− secretion in different regions of native intestine activated by the Ca2+‐dependent agonist carbachol (CCH). In Tmem16a+/− colon, basal and CCH stimulated Isc was largely reduced. Immunostaining of intestinal tissue with PA2290 antibody confirmed luminal expression of TMEM16A proteins compared to Tmem16a+/− mice. Luminal but not serosal application of MONNA, a TMEM16A specific inhibitor, resulted in significant reduction of CCH stimulated Isc, indicating that TMEM16A is primarily expressed and functions as a luminal Cl− channel. Luminal expression of TMEM16A in human colon was confirmed by confocal microscopy. Localization of TMEM16A to the luminal membrane may be facilitated through interactions with the scaffold protein NHERF1 which we found immunoprecipitated TMEM16A. Our results indicate that TMEM16A is prominently expressed in different segments of native mouse intestine as a luminal membrane protein and in human colonic tissue. TMEM16A also interacts with NHERF1. These data are consistent with TMEM16A localizing to the luminal membrane via the actin cytoskeleton and NHERF1 to influence CCH stimulated luminal Clsecretion.Support or Funding InformationUniversity Grant Commission, and Indian Council of Medical Research, Govt. of India
Resting Muscle Sympathetic Nerve Activity Is Not Different in Symptomatic Concussed College Athletes versus Healthy Controls
Authors: Worley Morgan L. , Sackett James R. , Schlader Zachary J. , Leddy John J. , Johnson Blair D. 
Published: April 2020

Heart rate variability, primarily an indicator of cardiac parasympathetic function, has been shown to be reduced at rest and during physiological stressors (e.g., aerobic exercise, handgrip exercise, and face cooling) following a concussion, which suggests altered autonomic function. Previous work in our lab has demonstrated attenuated increases in sympathetically‐mediated increases in blood pressure due to unaltered total peripheral resistance during face cooling and the cold pressor test in symptomatic concussed college athletes (CA) compared to healthy controls (HC). These responses suggest that CA have blunted sympathetic activation. It is unknown if a direct measure of sympathetic nerve activity (e.g., muscle sympathetic nerve activity (MSNA)) is altered at rest in CA compared to HC.PURPOSEWe tested the hypothesis that MSNA at rest would be lower in CA compared to HC.METHODSSeven CA (age: 19±2 y, 4 females, 4±2 days post‐injury) and seven HC (age: 21±3 y, 3 females) completed one study visit. Heart rate (3‐lead electrocardiogram), mean arterial pressure (photoplethosmography), and MSNA (microneurography) from the left radial nerve were continuously recorded at 1 kHz for 30 s following at least 20 minutes of supine rest. MSNA burst frequency, burst incidence, and total activity were assessed using commercially available software. Cardiac output was calculated as the product of heart rate and stroke volume. Total peripheral resistance was calculated as mean arterial pressure divided by cardiac output. We used unpaired t‐tests to determine if the main outcome variables were different between groups. Values are reported as mean ± SD.RESULTSResting heart rate (CA: 58±10 vs. HC: 61±12 bpm; P = 0.31), mean arterial pressure (CA: 95±9 vs. HC: 88±10 mmHg; P = 0.11), cardiac output (CA: 6±1 vs. HC: 7±3 L/min; P = 0.31), and total peripheral resistance (CA: 16±3 vs. HC: 16±11 mmHg/L/min; P = 0.45) were not different between CA and HC. Resting MSNA was not different between CA and HC for burst frequency (CA: 27±6 vs. HC: 29±9 beats/min; P = 0.33), burst incidence (CA: 47±10 vs. HC: 46±11 bursts/100 heartbeats; P = 0.44), or total activity (CA: 4883±1520 vs. HC: 6321±1689 AU/min; P = 0.07).CONCLUSIONThese preliminary data indicate that muscle sympathetic nerve burst frequency, burst incidence, and cardiovascular variables during resting conditions are not different in symptomatic concussed college athletes compared to healthy controls. Research is warranted to determine if muscle sympathetic nerve activity is different between symptomatic concussed colleges athletes and healthy controls during a sympathoexcitatory maneuver.Support or Funding InformationSupported by the National Center for Advancing Translational Sciences of the National Institutes of Health Pilot Grants Award Program under award Number UL1TR001412.
Variations of the Premaxillary Suture in Fetal and Infant Development: a Study of Position, Completeness, and Branching
Authors: Cusick Abbey Marie , Zdilla Matthew J. 
Published: April 2020

The premaxillary suture joins the pre‐maxillary (i.e., incisive) and proper maxillary regions of the maxilla. This suture is important in maxillary development at early stages in life, including intrauterine and infantile growth. By adolescence, the suture becomes fused and is usually unapparent. This study aims to classify the premaxillary suture in maxillae from individuals aged five‐months intrauterine to five‐months post‐natal. Sutures were categorized based on completeness, positioning with regard to the dental arch, and branching pattern. The development of the premaxillary‐maxillary junction likely plays an important role in the pathogenesis of cleft lip. Therefore, a thorough understanding of the variations in the premaxillary sutures may elucidate the pathogenesis of cleft lip.Support or Funding InformationFunding: WV Research Challenge Fund [HEPC.dsr.17.06]
Standard and High Fat Food Intake is Suppressed by PF5190457, the Ghrelin Growth Hormone Secretagogue 1α Receptor Inverse Agonist/Antagonist
Authors: Brehm Victoria Dobbie , Anastasio Noelle C. , Merritt Christina R. , Moeller F. Gerard , Cunningham Kathryn A. 
Published: April 2020

Ghrelin is a 28‐amino acid peptide produced by epithelial cells in the stomach mucosa and is well‐characterized to modulate gastrointestinal function, growth hormone secretion, glucose regulation, and appetite, and is increasingly implicated in substance use disorder (SUD) phenotypes. Ghrelin actions are transduced through the growth hormone secretagogue receptor 1α (GHS1αR) which is noted to exhibit constitutive activity in in vitro recombinant cell lines, and in dispersed pancreas islet cells, studies which support the concept of endogenous ghrelin tone. The small molecule PF5190457 exhibits relatively equipotent actions as a neutral GHS1αR antagonist and inverse agonist without apparent partial agonist actions (Kong et al. Br J Pharmacol 173:1452, 2016). To expand our knowledge of the physiological impact of GHS1αR function in vivo and determine an effective dose of PF5190457 for future studies of SUD phenotypes, we tested the hypotheses that PF5190457 would antagonize GHS1αR agonist‐induced intake of standard chow (SD) or high fat food (HFF) and act as an inverse agonist to inhibit basal HFF intake and weight gain.MethodsMale Sprague‐Dawley rats (n=16) maintained on ad libitum SD were pretreated with intraperitoneal injection of PF5190457 (20 mg/kg) or vehicle 60 min prior to subcutaneous (SC) injection of the GHS1αR agonist hexarelin (80 μg/kg) and assay of SD intake over a 6‐hr period. In a second study, rats (n=32) were administered acute or repeated PF5190457 (20 mg/kg) prior to maintenance on ad libitum HFF for three days. On day 3, all rats were treated with PF5190457 or vehicle 60 min prior to hexarelin (80 μg/kg; SC). Food intake and body weight were recorded throughout the study.ResultsPF5190457 significantly suppressed hexarelin‐induced SD intake vs. vehicle (p < 0.05). Both acute and repeated PF5190457 administration suppressed hexarelin‐induced HFF intake vs. control (p < 0.05). Repeated PF5190457 suppressed basal HFF intake and body weight gain (p < 0.05).ConclusionsThe novel GHS1αR inverse agonist/antagonist PF5190457 suppressed measures of both hexarelin‐induced SD and HFF intake, demonstrating antagonist activity in vivo. Similar efficacy of acute and repeated PF5190457 to suppress hexarelin‐induced HFF intake suggests that tolerance did not develop. Further, PF5190457 suppressed basal HFF intake and HFF‐associated weight gain, indicating inverse agonist activity in vivo. Together, these data suggest that PF5190457 will be a useful pharmacological tool to study the involvement of GHS1αR in SUD phenotypes.Support or Funding InformationSupported by NIDA UG3 DA050317 and F30 DA049501
The Inflammatory Lipid, 2‐Chlorohexadecanal: Cell Toxicity and Potential Scavenger Protection
Authors: Shakya Shubha , Ford David A. 
Published: April 2020

Hypochlorous acid is produced by either leukocyte myeloperoxidase activity or from chlorine gas reactions with water. 2‐Chlorofatty aldehydes (2‐ClFALD) are: 1) formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and 2) produced following leukocyte activation as well in the lung of mice exposed to chlorine gas. The biological role of 2‐ClFALD is largely unknown. Recently, our lab showed the electrophilic α‐chlorinated carbon of the 2‐ClFALD molecular species, 2‐chlorohexadecanal (2‐ClHDA) is targeted by nucleophilic attack of the cysteinyl‐sulfhydryl group of glutathione. Here, we synthesized an alkyne analog of 2‐ClHDA (2‐ClHDyA) to demonstrate 2‐ClHDyA protein modification in RAW 264.7 cells using click chemistry strategies. Furthermore, confocal microscopy showed that 2‐ClHDyA co‐localizes to the endoplasmic reticulum, Golgi, and mitochondria. Additionally, diffuse distribution of 2‐ClHDyA suggested cytoplasmic protein modification. The intensity of the fluorescence signal of 2‐ClHDyA increased in a dose‐dependent manner suggesting increased protein modification. Cell viability/metabolic activity assessed by 3‐(4,5‐Dimethyl‐2‐Thiazolyl)‐2,5‐Diphenyl‐2H‐Tetrazolium Bromide (MTT) test revealed that 2‐ClHDA reduced RAW 264.7 cell viability in a dose‐dependent manner. Cell viability was further reduced in RAW 264.7 cells treated with 2‐ClHDA in DMEM with Fetal Bovine Serum (FBS) supplementation. Click chemistry showed 2‐ClHDyA modified FBS proteins. These observations suggested 2‐ClFALD modified proteins might have accounted for cell death via cell signaling outside or inside the cell. Next, we investigated traditional aldehyde sequesterers including N‐acetyl cysteine (NAC), carnosine, and aminoguanidine to identify potential protectors that would prevent cellular 2‐ClFALD‐protein interaction resulting in reduced toxicity caused by 2‐ClFALD. Among tested sequesterers, NAC proved to be an effective sequesterer of 2‐ClHDA. NAC prevented 2‐ClHDyA interaction with FBS proteins and rescued cell death significantly. Next, we characterized the conjugate of 2‐ClHDA and NAC using direct infusion electrospray mass spectrometry. The conjugate appeared to be a result of a nucleophilic substitution reaction where nucleophilic sulfur of NAC attacked the alpha carbon of 2‐ClHDA leading to the release of chloride ion. These studies demonstrate that the interaction of 2‐ClFALD with cellular proteins with free sulfhydryl groups cause toxicity and the interaction of 2‐ClFALD with N‐acetyl cysteine may protect against toxicity caused by 2‐ClFALD.Support or Funding InformationNIH grants: R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697 and U01 ES028182
RhoA induces mitophagy through PINK1 stabilization to confer cardioprotection
Authors: Tu Michelle , Yu Justin D. , Kono Maiko , Tan Valerie P. , Smith Jeffrey M. , Miyamoto Shigeki 
Published: April 2020

An established feature of cardiac disease involves an increasing presence of dysfunctional mitochondria in the heart. Mitophagy is a key player in maintaining mitochondrial quality and thereby regulates cellular homeostasis. For instance, inhibition of mitophagy has been shown to exacerbate cardiac injury induced by oxidative stress. RhoA is a small G‐protein that serves as a proximal downstream effector of numerous GPCRs and is also responsive to oxidative stress. Activation of RhoA has been previously shown to protect the heart against stress; however, its specific role in mitophagy has not been determined. Here, we explored the possibility that RhoA signaling regulates mitophagy and in turn, provides cardioprotection. Adenoviral overexpression of RhoA in neonatal rat ventricular myocytes increased both endogenous and exogenous PINK1 levels as well as mitochondrial Parkin levels, leading to mitophagy assessed by EM analysis. Although RhoA expression did not affect PINK1 mRNA levels, it did inhibit PINK1 protein degradation, thus stabilizing PINK1 protein. Depolarization of the mitochondrial membrane potential is well established to stabilize PINK1 at the mitochondria. Interestingly, however, RhoA expression did not change the mitochondrial membrane potential. Thus the basis for PINK1 accumulation at mitochondria in response to RhoA appears to be unique. siRNA‐mediated knockdown and pharmacological inhibition of PKD, an established downstream effector of RhoA, reversed RhoA‐induced stabilization of PINK1. However, overexpression of PKD alone was not sufficient to stabilize PINK1. Active RhoA accumulates at mitochondria and this response is completely inhibited by PKD inhibition, suggesting a potential role of PKD dependent mitochondrial RhoA distribution in regulating PINK1 stability. While extrapolating the role of RhoA signaling in cardiac disease, myocardial infarction induced by ligation of the left anterior descending artery was reduced in adult mice expressing constitutively active RhoA through cardiac‐specific MLC2v promoter driven adeno‐associated virus serotype 9 when compared to that of wild‐type (WT) mice. Furthermore, although both WT and cardiac specific RhoA knockout (KO) mice around 18 weeks old revealed no overt signs of cardiac dysfunction, at around 45 weeks old, KO mice not only showed hypertrophy and fibrosis, but also revealed contractile dysfunction. Currently, we are investigating the specific contribution of RhoA‐induced mitophagy in these cardiac disease models. Taken together, our results suggest that RhoA stabilizes PINK1 through its PKD‐mediated translocation to the mitochondria, subsequently inducing mitophagy in a manner uniquely independent of the conventional pathway and thus, could confer cardioprotection against stresses.Support or Funding InformationThis work was supported by American Heart Association grant 19TPA34910011 to S. Miyamoto.
Docking of inhibitors to the structure of the human K‐Cl cotransporter
Authors: Delpire Eric 
Published: April 2020

Following the release of the cryo‐electron microscopy structure of the human K‐Cl cotransporter, hKCC1, we used a ligand‐protein docking program (Autodock Vina from the Scripps Research Institute) to assess the binding of KCC inhibitors to the K‐Cl cotransporter. We hypothesized that the inhibitors were likely to utilize the ion permeation pathway and overlap with some of the ion binding sites. We first modeled the binding of ML077 starting from the position of the 3 ions at SCl1, SK, and SCl2. ML077 is a potent inhibitor of KCC function, as it binds rat KCC2 with an EC50 of 560 nM. The top binding energies for the best solutions from these three positions were: −7.6, −7.6, and −8.5 kcal/mol, indicating that the compound clearly fits better in the lower portion of the permeation pathway. We observed that the ring structures of the inhibitory compound nicely fitted within the hydrophobic groove or cavity which is located at the bottom of the transporter core. Furthermore, the most distal methyl group of ML077 was conveniently positioned 3.6 Å from the side chain of Tyr216. Since this residue also coordinates the placement of K+, this data is consistent with previous report showing that the binding of ML077 is competitive with regard to K+. When we docked the stilbene derivatives DIDS and H2DIDS to the structure, we observed binding modalities with binding energy around −7.5 kcal/mol. The inhibitors were located in the groove with the nitrogen atom or sulfur atom of an isothiocyanate group placed 3.3 – 3.5 Å from Tyr216 and one the sulfonic acid group oriented either towards the Cl− ion at SCl2 or TM8. The second sulfonic acid group orients towards the outside of the cavity, while the second isothiocyanate group nicely fits within a lower portion of the groove. As these conformations do not involve Tyr216 and do not seem to overlap with the bound K+, this docking is consistent with non‐competitive DIDS inhibition with regards to K+. Finally, docking of furosemide to the hKCC1 structure also preferentially placed the loop diuretic within the cavity with a possible hydrogen bond between the amine group of the ligand and the hydroxyl group of Tyr216 (distance 1.8 Å). Involvement of the tyrosine residue in coordinating furosemide binding is also consistent with the competitive inhibition of furosemide with respect to K+. Thus, it seems that the binding of ML077, DIDS and H2DIDS, and furosemide are consistent with some older functional studies. Single site mutagenesis and functional experiments should confirm these predictions.Support or Funding InformationThis work was supported by NIH grant DK93501 and by grant 17CVD05 from the Leducq foundation.
Pattern of distribution of lipocalin 2 in the pancreas of diabetic rat
Authors: Adeghate Ernest A. , D’Souza Crystal , Al-Jaberi Saeeda 
Published: April 2020

Lipocalin 2 (LCN2), discovered in 1989, is an innate, inducible glycoprotein and a cytokine implicated in the regulation of inflammation and iron homeostasis. Although, LCN2 was thought to be present only in neutrophils, it has now been reported to be secreted by many other cells including adipocytes, hepatocytes and more recently, osteoblasts. LCN2 induces insulin secretion and suppress appetite by activating melanocortin‐4 receptors in the hypothalamus. Even though LCN2 has been associated with glucose homeostasis, the pattern of distribution and function of LCN2 in the pancreas of an animal model of diabetes mellitus has not been elucidated. The aim of this study was to determine whether LCN2 is present in the pancreas of diabetic and non‐diabetic Wistar rats. Immunofluorescence method was used to identify the pattern of distribution of LCN2 in pancreatic islet cells of Wistar rats. Co‐localization staining was also performed to determine whether LCN2 is located with either, insulin, glucagon, somatostatin, or pancreatic polypeptide (PP) in the islets of Langerhans. Immunofluorescence study shows that a large number of cells in the central region of rat pancreatic islet contains LCN2, where it co‐localizes mainly with insulin. LCN2 does not co‐localize with any other islet peptide. The number of LCN2 and insulin‐containing cells in pancreatic islets decreased significantly (p < 0.05) after the onset of diabetes. In conclusion, LCN2, a resident of pancreatic beta cells, may play a role in the regulation of insulin and glucose metabolism.Support or Funding InformationThis work was supported by UAE University Research Grant # G00002701
Proximal tubular mitochondrial substrate overload is an independent factor in developing kidney disease
Authors: Stadler Krisztian , Noland Robert , Mynatt Randall L. , Kruger Claudia 
Published: April 2020

Alterations in mitochondrial function are linked to the development of chronic and diabetic kidney diseases (CKD/DKD). Proximal tubular epithelial cells (PTCs) are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. It is suggested, but not entirely clear whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage. Here we modeled mitochondrial substrate overload ‐ an important aspect of metabolic disease and diabetes ‐ by creating mice lacking the enzyme carnitine acetyl‐transferase (CrAT) in the PTC. CrAT is crucial in serving as a “relief valve”, linking excess acetyl‐CoA to carnitine and allowing transport from the mitochondria. Deletion of CrAT therefore models a scenario of substrate excess where the export of carbons is limited. Mice developed tubular disease and interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis and fibrosis markers, increased oxidative stress and abnormal profiles of acyl carnitines/organic acids including those derived from branch chain amino acid and glucose metabolism. These results indicate that during mitochondrial overload in the kidney cortex, all three major forms of metabolic pathways are impacted. When mice with CrAT deletion were placed on a high fat diet, kidney disease was more severe and developed faster. Primary PTCs isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology. Next generation sequencing analysis from cortices at different time points revealed that dysregulation of genes related to mitochondrial function precede development of pathology. Mitochondrial overload also led to the enrichment of genes that are deregulated in CKD/DKD. Importantly, mice with heterozygous CrAT deletion in the PTC also developed kidney disease, suggesting that even moderate impairments in substrate efflux impede PTC function. Our findings support the hypothesis that derailments of mitochondrial substrate metabolism in PTC alone may be causative to CKD. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell metabolism could be an important preventative strategy.Support or Funding InformationThis research is supported by NIH grants R01‐DK115749‐01A1 (to KS) and R01‐DK103860 (to RN)