bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lung Immune Regulation by Vectorial Transport of Endothelial Mitochondria
Authors: Islam Mohammad Naimul , Rowlands David John , Gusarova Galina , Bhattacharya Jahar 
Published: June 2021

The mitochondrial regulation of lung immunity and repair remains unclear. Here, we consider the role of vectorial mitochondrial transport in endothelial cells (EC) of alveolar capillaries. In axons, which have high length‐diameter ratios, the mitochondrial Rho‐GTPase, Miro determines microtubule‐guided, vectorial mitochondrial transport. Cytosolic Ca2+ elevation increases Ca2+ binding to Miro, causing mitochondrial transport arrest. To determine whether similar mechanisms occur in lung ECs in situ, which are linearized cells with 30–50 μm long cytoplasmic extensions, we viewed live capillaries by confocal microscopy of isolated, blood‐perfused mouse lungs. To visualize EC mitochondria, we microinfused the capillaries with the fluorescent dye, mitotracker green (MTG) that specifically localizes to mitochondria. By vascular transfection, we induced EC expression of a Miro mutant, Mirokk, which does not cause Ca2+‐dependent mitochondrial transport arrest. EC mitochondria were evident as distinct, fluorescent spots of diameter 2–3 um, each representing a cluster of 1–3 mitochondria, that distributed linearly along the cell length. Time‐dependent imaging revealed that the clusters were in continuous motion at 1 um/min, such that a single cluster traversed the distance from the nucleus to the cell periphery in ~40 min. Vascular microinfusion of tumor necrosis factor‐alpha (TNFα) elevated EC cytosolic Ca2+, arrested mitochondrial transport and induced peri‐nuclear mitochondrial clustering. By contrast, Mirokk expression inhibited the transport arrest, while decreasing lung inflammation, as reflected in leukocyte counts in the broncho‐alveolar lavage, and mouse mortality. We show for the first time, that vectorial mitochondrial transport occurs along the cytoplasmic length of capillary EC. We report a novel, Miro‐dependent mechanism of lung immune regulation in which agonist‐induced cytosolic Ca2+ elevation blocks the mitochondrial transport, promoting lung inflammation and injury. Miro may be a therapeutic target for lung repair (support HL122730).Support or Funding Informationsupport HL122730 to JB and AHA Grand‐in‐Aid to MNIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coexpression of proximal tubule claudins‐2 and ‐10a increases conductance and decreases permselectivity in a renal epithelial cell line
Authors: Curry Joshua N. , Li Jiahua , Zhuo Min , Yu Alan 
Published: June 2021

The mammalian nephron is composed of a series of tubule segments with distinct patterns of tight junction morphology, ion selectivity, and protein expression. Paracellular permeability of epithelial tissues is determined in part by claudins, a family of tight junction membrane proteins. The most abundantly expressed claudins in the proximal tubule (PT) are claudin‐2 and ‐10a. In general, claudin‐2 is expressed in leaky epithelia and functions as a cation‐permeable paracellular channel, whereas claudin‐10a has been shown to form anion‐selective pores. We hypothesized that coexpression of these PT claudins leads to distinct paracellular permeability properties, and contribute to ion transport in this segment.We established a double‐inducible, titratable gene expression system using Madin‐Darby canine kidney cells (MDCK I). Utilizing tetracycline‐controlled transcriptional activation (Tet‐Off), we developed stable cell lines which expressed claudin‐2 in the absence of tetracycline antibiotics. From this parent line, we generated stable transfectants expressing claudin‐10a under the control of a cumate‐inducible switch (SBI). Induction of claudin‐2, ‐10a, or both isoforms, all reduced TER compared to cells with no claudin overexpression (p<0.0001 using 2‐way ANOVA). We found a marked increase in PCl in cells induced to express claudin‐10a alone (17.4 ± 1.7 * 106 cm/s vs. 0.55 ± 0.06 * 106 cm/s with no induction), and a marked increase in PNa in cells induced to express claudin‐2 alone (12.2 ± 0.6 * 106 cm/s vs. 0.59 ± 0.07 * 106 cm/s with no induction). When both claudin‐2 and ‐10a were expressed, PCl and PNa were both increased (to 9.5 ± 1.5 * 106 cm/s and 6.2 ± 0.4 * 106 cm/s, respectively) and PNa/PCl was unchanged (0.76 ± 0.10 vs. 1.07 ± 0.05 with no induction). Calcium transport is believed to occur via a paracellular route in the PT, and so we measured the effect of PT claudin overexpression on unidirectional 45calcium radiotracer flux. Claudin‐2 expression increased calcium flux by over 7‐fold (p<0.0001), while claudin‐10a had no effect compared with no expression (p=0.99). No difference was detected in calcium flux between claudin‐2 expression and claudin‐2 and ‐10a in combination (p=0.89). These results further support claudin‐2 as the mediator of calcium reabsorption in the PT, and suggest that an interaction between PT claudin isoforms reduces the permselectivity of the PT to sodium and chloride.Support or Funding InformationSupported in part by NIH grant F30DK109605This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Scleraxis Regulation of Snail1 and Twist1 Gene Expression in Epithelial – Mesenchymal Transition
Authors: Czubryt Michael Paul , Al‐Hattab Danah Sufian , Safi Hamza Ayoub , Nagalingam Raghu Sundaresan 
Published: June 2021

Epithelial‐Mesenchymal Transition (EMT) is a process by which epithelial cells lose their connectivity with each other and convert to a migratory, mesenchymal phenotype, and plays an important role in development as well as pathogenesis of diseases such as cancer and fibrosis. EMT is induced in part by the up‐regulation of the transcription factors Twist1 as well as Snai1, which directly represses transcription of E‐cadherin, an integral component of adherens junctions: tight cell‐cell connections prominent in epithelial cell sheets. Our previous data in mice demonstrated that gene knockout of the transcription factor scleraxis, which is involved in cell phenotype conversion, resulted in fewer cardiac fibroblasts, which arise developmentally from proepicardial cells undergoing EMT. We observed an increase in epithelial markers concomitant with a loss of mesenchymal markers in scleraxis null hearts, suggesting that EMT was impaired by scleraxis deletion. Our data indicated that scleraxis directly transactivates the Twist1 and Snail1 gene promoters, and that scleraxis was sufficient to induce EMT in A549 epithelial cells, but the mechanistic details of this regulation were unclear.Here we examined the Twist1 and Snail1 gene promoters for E‐boxes – CANNTG sequences to which scleraxis binds. By in silico analysis, we identified one and four putative E‐boxes in the Twist1 and Snail1 proximal promoters, respectively. Using luciferase reporter assays, we found that scleraxis directly transactivated both promoters. The single E‐box in the Twist1 promoter was required for this effect, as mutation of the E‐box abolished scleraxis‐mediated reporter induction. Conversely, three out of four E‐boxes within the Snail1 promoter were similarly required. Results for both studies were confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, which demonstrated that scleraxis directly interacted with the E‐boxes in question. Scleraxis over‐expression resulted in up‐regulation of both Snail1 and Twist1, and increased migration of A549 epithelial cells as measured via scratch assay, indicating progression of EMT. TGFβ1, a known inducer of both EMT and scleraxis expression, increased Snail1 and Twist1 expression, but scleraxis knockdown via shRNA attenuated this induction, demonstrating that TGFβ1‐mediated EMT requires scleraxis. These findings suggest that scleraxis is an important factor in driving EMT, and may be a novel therapeutic target to arrest cell conversion in EMT‐dependent pathological processes.Support or Funding InformationDSA was supported by funding from the GETS program of the University of Manitoba. MPC was supported by an Open Operating Grant from the Canadian Institutes of Health Research (MOP136862).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IRBIT expands signaling repertoire: Regulation of the Ca2+‐activated Cl− channel Bestrophin 2 by synergy between Ca2+ and cAMP at membrane contact sites
Authors: Chung Woo Young , Park Seonghee , Muallem Shmuel 
Published: June 2021

The scaffolding protein IRBIT (Inositol 1,4,5‐Trisphosphate (IP3) Receptor Released with IP3) regulates the surface expression and activates several transporters, including NBCe1‐B, NHE3, CFTR, and SLC26A6, to coordinate secretory cells fluid and HCO3− secretion. IRBIT also mediates the synergy between cAMP and Ca2+ signaling. One mechanism involves PKAmediated phosphorylation of the IP3Rs that dissociate IRBIT from the IP3Rs and makes it available to activate the transporters. However, whether IRBIT affect Ca2+ and cAMP activation of the transporters and the molecular mechanism of such a regulation is not known. Here, we discovered that IRBIT is a potent activator of the Ca2+‐activated Cl− channel Bestrophin 2 (Best2), but not of Best1. IRBIT achieves this by two mechanisms. IRBIT recruits Best2 to the ER/PM junctions to increase surface expression of Best2 and increase the Ca2+‐activated Clcurrent density. At the ER/PM junctions IRBIT assembles a complex of Best2‐Adenylys cyclase 8‐PKA to confer Best2 regulation by the cAMP/PKA pathway. PKA‐mediated phosphorylation prominently increases Best2 apparent affinity for Ca2+. Best2 regulation by PKA requires intact ER/PM junctions with dissociation of the junction by knockdown of ER/PM junctions tether proteins eliminates activation of Best2 by IRBIT and regulation by PKA. Notably, activation of Best2 by Ca2+‐mobilizing receptors is markedly reduced in the salivary gland ducts of IRBIT−/− mice. These findings show the importance of localization in the ER/PM junctions for synergy in signaling and how IRBIT expands signaling repertoire by converting purely Ca2+‐activated protein to a Ca2+‐activated and cAMP/PKA regulated protein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IRBIT stimulates zebrafish NBCe1 (Slc4a4) activity and stimulates functionally impaired human NBCe1‐B cSNP activity
Authors: Chang Minhwang , Wang Yi‐Fang , Romero Michael F. 
Published: June 2021

We previously cloned and analyzed the electrogenic Na+‐Bicarbonate Cotransporter from zebrafish (zNBCe1; 80% identity to mammalian NBCe1; Sussman 2009, PMC2770747). As other fish NBCe1 clones, zNBCe1 is the only NBCe1 isoform, verified by functional data, and is most similar to the pancreatic form of NBCe1 (Slc4a4‐B). IRBIT, an inositol 1,4,5‐trisphosphate (IP3) receptor‐binding protein, specifically binds to and activates this N‐terminus (B & C isoforms) but not the shorter kidney‐type/A‐isoform. This binding depends on IRBIT phosphorylation at multiple serine residues. We tested the hypothesis that zNBCe1, as a NBCe1‐B, could functionally interact with zIRBIT rather than no regulatory interaction observed in NBCe1‐A isoforms. The zIRBIT cRNA was co‐expressed with zNBCe1 or an N‐terminal truncation (Δ91aa) of zNBCe1 in Xenopus oocytes and functionally tested using two‐electrode voltage clamp. We also investigated whether IRBIT binding would activate NBCe1‐B specific human cSNPs (identified NHLBI 6500 genome) in the autoinhibitory domain (AID). Our data demonstrate that zNBCe1 is a functional ortholog of mammalian NBCe1‐B, which both binds IRBIT via the N‐terminus and stimulates transport activity. Nevertheless, zNBCe1 transport activity was not augmented by AID removal at SPAAER motif (Δ91aa) as observed in mammalian NBCe1‐B or NBCe1‐C. Moreover, while human NBCe1‐B specific coding SNPs (G50A, N63S) show diminished transport function (vs. WT NBCe1‐B), IRBIT can still stimulate transport to NBCe1‐A levels. Together these data implicate that IRBIT regulation of NBCe1‐function is evolutionary preserved in teleost precedes NBCe1‐A appearance and that this regulation fine‐tunes NBCe1‐B transporting capacity in the presence of certain AID‐cSNPS. This IRBIT‐control might also play a physiological role when NBCe1‐A is deleted from particular cells or tissues.Support or Funding InformationMayo FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA Profile in Swimming Trained Female Rats and its Therapeutic Potential in Spontaneously Hypertensive Rats
Authors: Soci Ursula Paula Renó , Nobrega Clara Paula Renó , Fernandes Tiago Menezes , Barauna Valério Garrone , Irigoyen Maria Cláudia Menezes , Oliveira Edilamar Menezes 
Published: June 2021

IntroductionBoth aerobic exercise training (ET) and Hypertension (HY) induce different cardiac hypertrophy (CH) phenotypes which molecular differences and may lead to new targets for therapies in cardiovascular disease, as microRNAs (miRNAs).MethodsWe selected 15 miRNAs that were differentially expressed by ET from miRNAarray data and compared them with other from SHR (Spontaneously Hypertensive Rats) miRNAarray data. Four miRNAs were selected for qRT‐PCR confirmation: miRNA‐27a, 27b, 126 e 29c. Among then, miRNA 29c was chosen to be modulated by lentiviral vector due its role in fibrosis regulation. Intramuscular cardiac injection of the lentiviral vector particles 0.6×109 vp/was performed for two different times (7 and 14 days) (SHR7 and SHR14). The transduction efficiency was assessed by GFP expression by Western Blot. Blood pressure (BP) was measured by caudal plethysmography, CH was analyzed by ratio LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) and by cardiomyocytes diameter (dCMO). qRT‐PCR was used to assess miRNA‐29c expression and its targets COLIAI and COLIIIAI gene expression. The LV collagen content was assessed by histology (Picrossirius red), by collagen volume fraction, and by hydroxiproline concentration.ResultsBoth groups that received the low doses were GFP positive in the heart and liver tissue. BP was increased to SHR7, but it was prevented by miR‐29c lentivirus treatment to SHR14. The miRNA‐29c expression increased in all treated groups versus their control (CSI). COLIAI expression decreased in treated groups, while COLIIIAI did not change. Collagen volume fraction decreased in all treated groups, which shows that the treatment was efficient to decrease the cardiac collagen. HW/BW ratio decreased 7–11% in SHR14, while the dCMO was increased in all treated groups.ConclusionsThese results suggest that miR‐29c lentivirus treatment induces beneficial cardiac remodeling in SHR, increasing dCMO and decreasing cardiac fibrosis. These results open perspective for further investigation of antifibrotic therapies for cardiovascular diseases.Support or Funding InformationFAPESP, CNPq, CAPES and NAPmirThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tpcn2 Knock‐Out Mice Have Improved Insulin Sensitivity and Are Protected Against High‐Fat Diet Induced Weight Gain.
Authors: Woods Leah Solberg , Holl Katie , He Hong , DeBehnke Sarah , Yeo Chay Teng , Hansen Polly , Gebre Abraham , Leone‐Kabler Sandra , Ruas Margarida , Parks John , Parrington John 
Published: June 2021

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two‐pore channel 2 (Tpcn2) knock‐out mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared to wild type mice. We tested the hypothesis that improved insulin sensitivity inTpcn2 knock‐out mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knock‐out (KO), heterozygous (Het) and wild‐type (WT) mice were fed a low fat (LF) or high fat (HF) diet for 24 weeks. HF diet significantly increases body weight in WT mice relative to those on the LF diet, whereas HF diet consumption does not significantly affect body weight in Het or KO mice. We confirm that male KO mice have improved insulin sensitivity relative to WT mice. Unfortunately, because HF diet does not lead to insulin resistance in any of the mice, we are unable to claim a direct relationship between diet‐induced protection from insulin resistance and weight gain in the KO mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet‐fed mice where there is increased cholesterol and triglycerides across all genotypes. Fasting glucose, insulin and glucose tolerance levels do not differ by genotype or diet in male mice. In contrast, females consuming the HF diet exhibit glucose intolerance and hyperinsulinemia relative to females on the LF diet, but these effects are independent of genotype. These data demonstrate that male KO mice have improved insulin sensitivity and both male and female KO mice are protected against diet‐induced weight gain independent from changes in glucose tolerance or lipid levels.Support or Funding Informationby R01 DK088975, R01 DK106386, Department of Pediatrics at the Medical College of WisconsinThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALDH2 rs671 polymorphism is associated with athletic status and muscle phenotypes in the Japanese population
Authors: Kikuchi Naoki , Tajima Takafumi , Yamanaka Yoshiaki , Menuki Kunitaka , Okamoto Takanobu , Sakamaki‐Sunaga Mikako , Sakai Akinori , Hiranuma Kenji , Nakazato Koichi 
Published: June 2021

Mitochondrial aldehyde dehydrogenase‐2 (ALDH2) scavenges reactive aldehyde products, such as 4‐hydroxy‐2‐nonenal. Previous studies confirmed that the ALDH2 rs671 polymorphism is associated with hypertension and cancer, probably because of increased oxidative stress. Undesirable effects of ALDH2 rs671 might also be expected in skeletal muscles. The purpose of the present study was to investigate the association of ALDH2 re671 polymorphism with athletic status and muscle phenotypes in the Japanese population.In the present study, 1,464 athletes (672 sprint/Power athletes, 631 mixed athletes, and 161 endurance) and 1,341 non‐athletes (538 men and 803 women) were included in the analysis. Saliva samples were collected with the Oragene DNA self‐collection kit. The ALDH2 rs671 polymorphism was genotyped using a TaqMan SNP genotyping assay. The non‐athletes completed a questionnaire about exercise habits and were subjected to a battery of tests to assess their muscular functions (including grip strength and the 30‐second chair stand test)The ALDH2 genotype frequencies among athletes and non‐athletes were in Hardy‐Weinberg equilibrium. The frequencies of the ALDH2 genotypes GG, GA, and AA were 56%, 37%, and 7% in the athletes, and 54%, 37%, and 9% in the non‐athletes, respectively. There was a significant difference of genotype frequencies between the athletes and the non‐athletes (p=0.008). Among the non‐athletes, those with GG and GA genotypes showed higher fitness score of grip strength (p=0.001) and the 30‐second chair stand test (p=0.013) than those with the AA genotype after adjusting for age, sex, and exercise habits.Our study shows that ALDH2 rs671 polymorphism is associated with athletic status and muscle phenotypes in the Japanese population, suggesting that impaired ALDH2 activity might attenuate muscular function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐protein‐Coupled Bile Acid Receptor Attenuates Liver Injury in a Murine Model of Acute Parenteral Nutrition
Authors: Gomes Charles Klazer , Rao Prahlad , Stephenson Erin , Puchowicz Michelle , Makowski Liza , Han Joan K. , Yin Dengping , Chang Eugene B. , Pierre Joseph F. 
Published: June 2021

Parenteral Nutrition (PN) provides essential caloric support in patients unable to feed enterally. Unfortunately, PN without enteral feeding is associated with hepatic injury, especially in neonates where PN associated liver disease (PNALD) affects 60–70% of recipients. PN alters intestinal physiology and host‐microbial interactions, including bile acid enterohepatic circulation, which regulates metabolism through nuclear and cell membrane bile acid receptors. Here, we examined the role of G‐protein coupled bile acid receptor deficiency (TGR5−/−) upon hepatic phenotype in a murine model of acute PN. TGR5−/− and wild type (WT) received PN for 96 hours. PN significantly elevated serum triglycerides, resistin, PAI‐1 and hepatic fatty acid synthesis gene expression in both genotypes. However, PN‐treated TGR5−/− animals had significantly larger liver weights compared with WT, but displayed no changes in triglycerides. Histological analysis instead suggested edema. Accordingly, TGR5−/− animals on PN displayed elevated hepatic IL‐6 expression and significantly higher serum AST and ALT levels compared with WT. Furthermore, TGR5−/− animals on PN also displayed significantly decreased bile acid synthesis genes, including Cyp7A1, Cyp8B1, Cyp7B1, and BAT, compared with WT. Therefore, we examined serum bile acid composition. PN elevated total conjugated primary bile acid levels, specifically TCA, TCDCA, TbMCA, and TaMCA, in both genotypes compared with chow. However, TGR5−/− on PN displayed markedly higher levels of the unconjugated primary bile acids, CDCA, aMCA, and the secondary bile acids, oMCA and DCA, compared with WT on PN. Predictive metagenomics of 16S sequencing suggested altered microbial enzymes in TGR5−/−, including BSH and 7alpha hydroxylase, consistent with serum bile acid changes. These results suggest a protective role for TGR5 in liver function during PN that potentially includes direct attenuation of hepatic inflammation or indirect manipulation through microbiota‐bile acid interactions. Greater mechanistic understanding of TGR5 signaling during PN might improve interventions to treat or prevent PNALD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.