bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of carbonic anhydrase IX expression impairs gastric mucosal defence against luminal acid (1109.13)
Authors: Li Taolang , Liu Xuemei , Singh Anurag , Riederer Brigitte , Gros Gerolf , Bartels Helmut , Tuo Biguang , Seidler Ursula
Published: June 2021

Background: The exoenzyme carbonic anhydrase IX is strongly expressed in tumours. In normal tissues, CA IX is expressed during embryogenesis but downregulated postnatally, except in the stomach. Aim and Methods: We studied the cellular differentiation pattern and acid secretory capacity in gastric mucosa from CAIX KO and WT mice at age 8 days to 1,3,6,9,12 months. Surface pH and mucus layer thickness, as well as the ability of the surface epithelial cells to withstand a luminal acid load was assessed in young adult CAIX KO and WT mice by in vivo two photon microscopy. Results: CAIX KO gastric mucosa developed a unique phenotype with a gradual expansion of the surface cell zone, and regression of the parietal cell zone to the lower gland area. Concomitantly, the acid secretory capacity, which was normal at young age, declined. HCO3‐ output into the lumen and mucus layer thickness were not significantly different between CAIX KO and WT. However, the ability of the surface cells to withstand a luminal acid load, as well as to maintain a more alkaline surface pH than the luminal pH, was significantly impaired in CAIX KO compared to WT stomach. Conclusion: We speculate that the CAIX at the basolateral membrane of the gastric surface cells rapidly converts extruded protons with blood‐borne HCO3‐ to CO2 and thus augments interstitial buffer capacity, surface cell pHi regulation, and maintenance of the pH microclimate in the mucus layer.

Grant Funding Source: Supported by DFG Se 13‐4 and 9‐6 to Ursula Seidler
Actin depolymerizing factor but not cofilin‐1 is necessary to have a normal intestinal brush border (1109.16)
Authors: Gao Lucy , Sarker Rafiquel , Donowitz Mark
Published: June 2021

Two highly conserved isoforms of the cofilin gene family (actin severing proteins expressed in intestinal epithelial cells) are cofilin‐1 (COF) and actin depolymerizing factor (ADF). COF and ADF take part in Arp2/3 remodeling and seem to play overlapping roles since both rescue the knockdown (KD) phenotypes of the other. Their precise role in the intestine is unknown. We hypothesized that COF and/or ADF change local actin polymerization at the base of epithelial cell microvilli by terminal web remodeling. COF and ADF were KD using lentivirus/puromycin in Caco‐2 cells, a polarized intestinal Na‐absorptive cell line. Both were normally subapical, as well as diffusely cytosolic in the cells. Structural studies were performed using confocal and electron microscopy with brush border (BB) characterization by phalloidin and wheat germ agglutinin binding. ADF KD, but not COF, significantly changed epithelial cell structure with a) shorter cell heights; b) greater cell diameter; c) shorter/sparser BB. In addition, Western blot showed increased B‐actin and villin amount in ADF KD cells, normalized to GAPDH. In contrast, COF KD had less dramatic changes in intestinal cell structure: a) slight increase in cell height; b) taller/denser microvilli; c) minimally increased B‐actin and villin. We conclude that in spite of similar sub‐apical localization, ADF but not COF is necessary to have a normal intestinal BB, including the microvilli.

Grant Funding Source: Supported by APS UGSRF, NIH NIDDK R01DK26523, R01DK61765, P01DK72084, P30DK89502
SPAK/OSR1 kinases directly phosphorylate the K+‐Cl‐ co‐transporters (1109.7)
Authors: de los Heros Paola , Zhang Jinwei , Gourlay Robert , Campbell David , Deak Maria , Macartney Thomas , Kahle Kristopher , Alessi Dario
Published: June 2021

Precise homeostasis of the intracellular chloride (Cl‐) concentration is critical for cell volume regulation, epithelial transport, and neuronal excitability, and is achieved via the coordinated activities of the Cl‐ influx N(K)CCs and efflux KCCs members of the cation‐ Cl‐ cotransporter family. Here we present biochemical, genetic, and pharmacological evidence that the WNK kinase substrates SPAK/OSR1 directly phosphorylate the KCCs at one of the two recently‐described cell swelling‐regulated C‐terminal threonines, Thr 1048 (Site 2) but not Thr 991 (Site 1) in KCC3a. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site‐2 in vitro. Second, STOCK 1S‐50699, a selective WNK pathway inhibitor, suppresses SPAK/OSR1 activation and Site 2 phosphorylation. Third, the endogenous phosphorylation of KCC2, 3 and 4 at Site‐2 is abolished in double SPAK/OSR1 knock in embryonic stem cells, where T‐loop phosphorylation by WNK isoforms is prevented. These cells exhibit an elevated basal activity of 86Rb+ uptake that was not stimulated further by KCC3a activating conditions. Fourth, we show a tight correlation between SPAK/OSR1 activity and the magnitude of KCC3a Site‐2 phosphorylation. Finally consistent with phosphorylation of KCCs promoting inhibition of function, a Site 2 alanine KCC3a mutant exhibits increased activity, while a double Site 1/2 mutant is constitutively active. We also observed phosphorylation of the KCCs by SPAK/OSR1, at a novel site: Thr5 in KCC1/3 and Thr6 in KCC2/4 (Site 3) and in the recently reported KCC3a‐specific residue Ser 96 (Site 4). These data demonstrate WNK‐regulated SPAK/OSR1 kinases directly phosphorylate the N(K)CCs and KCCs, reciprocally promoting their stimulation and inhibition, respectively.
Impaired serotonin transporter function in the small intestine of mice on a high fat diet (1110.6)
Authors: France Marion , Galligan James , Swain Greg
Published: June 2021

Background: Obesity can alter 5‐HT (serotonin) availability in the gut mucosa disrupting motility. 5‐HT availability depends on 5‐HT released from enterochromaffin (EC) cells and by 5‐HT uptake by the serotonin transporter (SERT). We determined if 5‐HT availability was altered in the jejunum of control and high fat fed (HF) obese mice.

Methods: 5‐HT mucosal levels were measured using high‐performance liquid chromatography. Amperometry measured 5‐HT availability in real time near the mucosal surface (i.e. near sites of release and uptake). We generated current approach curves in which the electrode distance in relation to the mucosa decreased in a step‐wise fashion. We obtained the slope derived from a linear fit of the ln(current) vs. electrode distance plot. A decrease in 5‐HT uptake caused by the SERT inhibitor fluoxetine (1 μmol L‐1) or cocaine (10 μmol L‐1) was indicated by a decrease in the slope.

Results: 5‐HT mucosal levels were not different in HF mice compared to controls. In HF mice, 5‐HT clearance was insensitive to fluoxetine and cocaine compared to controls suggesting SERT function was impaired and other monoamine transporters did not compensate for loss of SERT function.

Conclusions: Our data suggests SERT function is impaired in HF mice whereas mucosal 5‐HT levels are unchanged. Impaired SERT function can change GI motility by elevating extracellular 5‐HT and increasing 5‐HT signaling.

Grant Funding Source: Supported by R01DK094932
Modeling ZO‐1‐C‐terminal domain exchange at the membrane as a diffusion problem (1111.4)
Authors: Nepomuceno Helene , Nguyen Crystal , Ogawa Katherine , Hasfura‐Buenaga J. Roberto , Balreira E. Cabral , King Jonathan
Published: June 2021

Zonula occludens‐1 (ZO‐1) is a membrane‐associated tight junction protein that has been observed to participate in ATP‐dependent exchange via an intracellular pool. We hypothesized that a diffusion model can explain the ZO‐1‐C‐terminal domain exchange dynamics at the tight junction. We developed stable cell lines with GFP‐ZO‐1 carboxy‐terminus fusion constructs in Madin‐Darby canine kidney cells. Fluorescence recovery after photobleaching (FRAP) experiments were conducted using a Nikon A1R laser scanning confocal microscope to photobleach the localized construct and acquire intensity data over time. Recovery was analyzed by partitioning the bleach region of interest into one‐square micron sections or the entire bleach ROI. The local fluorescence recovery in the photobleached area yields curves that are modeled to be solutions of the diffusion process. Fitting experimental data to a one‐ or two‐dimensional diffusion model will quantitatively reveal if the exchange occurs within the membrane or via an intracellular pool, respectively. Future research will compare the behavior of different ZO‐1 constructs and relate the quantity of ZO‐1 expression to its recovery behavior.

Grant Funding Source: Supported in part by NSF‐MRI #1229702 and NSF Award #0926702.
HLA DQ/DR prevalence and microbiota disturbance in northwestern Mexican children with type 1 diabetes (1118.3)
Authors: Mejia‐Leon Maria , Petrosino Joseph , Ajami Nadim , Domínguez‐Bello Maria , Calderon de la Barca Ana
Published: June 2021

Type 1 Diabetes (T1D) is a pro‐inflammatory autoimmune disorder leading to β‐cells destruction and insulin deficiency. Its prevalence of 1:300 is increasing and cannot be explained only by HLA DR4‐DQ8 and DR3‐DQ2 predisposition. Environmental factors seem to have a crucial role in its development through microbiota modulation. The aim of this study was to determine the HLA risk prevalence and to compare the fecal microbiota structure of Mexican children with T1D at onset and after treatment for more than 2 years. A case‐control study was conducted in Mexican mestizo children, aged 7‐18 years (8 newly diagnosed, 13 controlled T1D cases after 2 years treatment and 8 healthy controls). Predisposing haplotypes were typed and the fecal DNA was extracted; the V4 region of the 16S rRNA gene was amplified and pyrosequenced. All of the T1D cases had at least one HLA risk allele, principally the DQA1*0301. The newly diagnosed T1D cases had high levels of the genus Bacteroides (p<0.004), whereas the control group had a gut microbiota dominated by Prevotella. Children with T1D treated for 蠅2 years had levels of Bacteroides and Prevotella tending to those of the control group. The gut microbiota of newly diagnosed T1D cases is altered, but involving of causes or consequences remains unclear.

Grant Funding Source: Partially Supported by CONACYT grant S0008‐2009‐01‐115212
High salt diet causes epigenetic modifications and suppresses endothelial progenitor cell function (1120.2)
Authors: Karcher Jamie , Hoffmann Brian , Liu Pengyuan , Greene Andrew
Published: June 2021

Endothelial progenitor cells (EPCs) are bone‐marrow derived cells that exhibit endothelial cell properties, promote neovascularization, and contribute to vascular homeostasis through autocrine and paracrine mechanisms. We describe for the first time how dietary salt negatively affects EPC function. EPCs were significantly decreased in the bone marrow of rats on a high salt diet (HSD, 4% NaCl) as compared to normal salt diet (NSD, 0.4% NaCl) rats. CD34, CD133, VEFGR2, and c‐Kit expression was similar between NSD and HSD EPCs. HSD EPCs had suppressed in vitro tube formation and were unable to restore angiogenesis following transplantation into a rodent model of limited angiogenesis (Sprague Dawley rats on a HSD). To identify molecular candidates that were altered by dietary salt, we preformed reduced representative bisulfite sequencing to analyze DNA methylation and we preformed mass spectrometry to identify differentially expressed protein targets. Analyses revealed increased methylation and suppressed protein expression of RET (proto‐oncogene tyrosine‐protein kinase receptor Ret) in HSD EPCs. Loss of RET expression causes thyroid tumor suppression and increased cell death providing a mechanistic explanation for HSD EPC reduced angiogenic efficacy. Overall high salt inhibits EPC angiogenic function through DNA methylation causing proteomic changes on the membrane.

Grant Funding Source: HL‐082798
The effects of age on multiscale entropy measurement of neurophysiological signal complexity (1125.6)
Authors: Tobia Michael , Meadowcroft Mark , Karunanayaka Prasanna , Yang Qing
Published: June 2021

Complex physiological signals are neither deterministic nor random, showing a mixture of long‐range dependencies and irregularities (i.e., information) over multiple time scales. In humans, aging and pathology are associated with diminished CNS neurophysiological complexity and impaired cognition. In this study, we computed multiscale entropy (MSE) to measure the time scale dependent complexity (i.e., information) of the resting state BOLD fMRI signal in humans over a large age span (N=138; ages 8‐83; 58 male). Physiological noise (heart rate, respiration, head motion and local white matter [WM] signals) was removed from fMRI time courses using linear regression. Data were then bandpass filtered (.008‐.1 Hz) to isolate low frequency fluctuations. MSE was computed with parameters m=2 and r=.2 for temporal scales 1‐18. BOLD signal in gray matter (GM) and WM gained information with increasing temporal scale and exceeded the information gain from bandpass filtered white and 1/f noise. GM sustained the information over coarse scales better than WM. Age groups stratified MSE profiles showing an age‐related loss of signal complexity in GM that was maximal at scale 18. In contrast, MSE from WM did not dissociate age groups at any scale. These results show that BOLD fMRI is a complex physiological signal characterized by a tissue‐specific age‐related reduction in complexity.
Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract prevents attenuated body weight gain and blunts neuronal activation in the hypothalamus following chronic intermittent hypoxia (1126.4)
Authors: Nedungadi T. Prashant , Bathina Chandra , Mifflin Steve
Published: June 2021

Patients with sleep apnea (SA) are at a risk of developing metabolic syndrome. In the rat, 7 days of Chronic Intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in SA patients, is associated with reduced body weight gain (p<0.005, n= 18), food intake (p<0.05, n=11), hyperinsulinemia (p<0.05, n=7), glucose intolerance and elevated fasting blood glucose levels (p<0.05, n=8). Brainstem melanocortins and other peptides has been well documented to be involved in metabolic homeostasis while tyrosine hydroxylase (TH) is known to be responsive to hypoxic stress; however the involvement of NTS TH neurons in regulating energy homeostasis following CIH has not yet been investigated. AAV‐TH shRNA virus was used to knockdown TH in the NTS of male rats, while virus with scrambled RNA was used as a control. Knockdown of TH in the NTS prevented the reduction in body weight following CIH (n=7) compared to scrambled virus injected rats and naïve rats subjected to CIH. Immunohistochemical staining for the neuronal activational marker FosB revealed elevated immunoreactivity following CIH (p<0.05, n=6) in the hypothalamic arcuate nucleus (ARH). Knockdown of TH resulted in reduced FosB staining in the ARH and dorsomedial hypothalamus while no change was observed in the lateral hypothalamic area. However, in the ventromedial hypothalamus, increased FosB staining was observed (p<0.05, n= 8). Our findings suggest that NTS TH neurons play an important role in regulating metabolic status during exposure to CIH and alter the neuronal activity in hypothalamic feeding centers, possibly via regulation of an anorexigenic signaling pathway.

Grant Funding Source: P01 HL88052
Rheumatoid arthritis and autonomic function (1132.10)
Authors: Adlan Ahmed , Shantsila Alena , Kitas George , Paton Julian , Lip Gregory , Fisher James
Published: June 2021

Rheumatoid arthritis (RA) is an inflammatory disorder associated with increased cardiovascular mortality. We sought to determine whether muscle sympathetic nerve activity (MSNA) is heightened, and cardiac baroreflex sensitivity (cBRS) is reduced in patients with RA. Whilst resting supine, blood pressure (BP; Portapres), heart rate (HR; ECG) and MSNA (microneurography) were continuously monitored in 7 chronic RA patients (5 men, 56±4 yr, 28±1 kg/m²; mean±SE), 7 healthy controls (HC: 5 men, 52±5 yr, 26±1 kg/m²) and 7 patients with hypertension as ‘disease’ controls (DC: 5 men, 54±5 yr, 28±1 kg/m²). MSNA burst frequency and spontaneous cardiac baroreflex sensitivity (sequence technique, systolic BP‐RR interval) were determined. Mean BP (p=0.001) and HR (p=0.014) were lower in HC group (88±5 mmHg, 55±2 bpm) than RA patients (106±2 mmHg, 70±3 bpm) and the DC group (116±6 mmHg, 63±4 bpm). MSNA burst frequency tended to be higher in RA patients and the DC group (34±4, 37±3 and 26±3 bursts/min in RA, DC, HC; P=0.066), whilst cBRS was reduced in RA (7.6±2.0, 10.0±3.1 and 16.7±2.1 ms/mmHg in RA, DC, HC; P<0.05 RA vs. HC and DC). These preliminary findings support the existence of an imbalance in autonomic neural control of the heart and peripheral vasculature in patients with RA.

Grant Funding Source: Supported by Arthritis Research UK
Acute leptin exposure alters renal proximal tubule function in vitro via mTOR and AMPK (1133.3)
Authors: Hryciw Deanne , Briffa Jessica , Grinfeld Esther , McAinch Andrew
Published: June 2021

Exposure to elevated leptin reduces the metabolic activity in proximal tubule cells (PTC), leading to apoptosis. Currently there is limited knowledge of the molecular pathways involved. Mammalian target of rapamycin (mTOR) and AMP‐activated protein kinase (AMPK) regulate cell growth and differentiation, and in the obese kidney, phosphorylated AMPK is decreased and phosphorylated mTOR is increased. Therefore we hypothesize that in PTCs acute elevated leptin exposure will alter tubular function via AMPK and mTOR.

We used the established Opossum kidney (OK) PTCs cell line model, that were exposed to leptin (0.05 ‐ 0.50 µg/mL) for 2 hrs. We have previously demonstrated that leptin acts via megalin and not the leptin receptor in this model. We then investigated the effect leptin has on signalling mediated expression, metabolic activity, TGF‐β1 secretion and albumin uptake and degradation.

Leptin decreased the PTC metabolic activity via mTOR activation. Leptin increased TGF‐β1 secretion, decreased megalin expression and upregulated phosphorylated AMPK‐α. Interestingly, elevated levels of leptin decreased albumin uptake and degradation. This suggests that leptin activates fibrotic pathways via mTOR, AMPK and TGF‐β1.

Therefore we have identified for the first time that leptin alters the function of PTCs and that leptin alters PTC signalling mediated expression via a megalin mediated pathway.
Dopamine D1 and D5 receptors differentially regulate paraoxonase 2 (1134.6)
Authors: Yang Sufei , Yu Peiying , Yang Yu , Jose Pedro A , Zeng Chu‐Yu
Published: June 2021

We have previously reported that dopamine D1‐like receptors (D1R and D5R) negatively regulate NADPH oxidase isoform expression and activity in renal proximal tubule (RPT) cells from rodents and humans. We tested the hypothesis that D1‐like dopamine receptors (D1R and D5R) differentially regulate the anti‐oxidant enzyme paraoxonase2 (PON2) in HEK‐293 cells heterologously expressing human D1R and D5R. Fenoldopam (1µM, 15 min), an agonist for both D1R and D5R, increased PON2 co‐immunoprecipitation with D1R (160±4% vs. control, 101±1%) and D5R (132±0.1% vs. control, 102±1%) in both cell lines. Short‐term (15, 30, and 60min) stimulation of D1R and D5R with fenoldopam (1µM) did not alter total PON2 protein. However, in D1R cells, at 15min, fenoldopam increased PON2 protein in both lipid rafts (LRs) (144±3% vs. control, 100±3%) and non‐LRs (163±25% vs. control, 100±21%) (P<0.05, n=6/group, ANOVA). In contrast, in D5R cells, fenoldopam increased PON2 protein only in non‐LRs (132±5% vs. control, 100±1.6%, n=3). Long‐term (2, 4, 8, and 24h) fenoldopam (1µM) stimulation increased PON2 protein in a time dependent manner (146% ±7/8h, and 162% ±10/24h vs. control, 100±9%) (P<0.05, n=4, ANOVA) in D5R but not D1R cells. Fenoldopam also increased PON2 mRNA (1.25‐fold) (n=3, P<0.05, t‐test) at 24h in D5R but not D1R cells. Silencing PON2 gene with PON2‐siRNA (10nM/48h) in D5R cells decreased PON2 protein (‐40±4% vs. mock‐siRNA, 100±17%, n=3) and mRNA (‐17±4% vs. mock‐siRNA, 100±2%, P<0.05, n=3, t‐test) and increased basal ROS production by 1.36‐fold, relative to mock‐siRNA. PON2 protein was decreased in D5R‐/‐ mice (‐44±13%) relative to D5R+/+ mice (100±8%, n=4‐6/group). We conclude that short‐term stimulation of either D1R or D5R leads to PON2 protein recruitment to non‐LRs and also to LRs in the case of D1R. However, long‐term stimulation of PON2 and negative ROS regulation are mainly due to D5R.

Grant Funding Source: HL023081, HL074940, DK039308
microRNA‐mediated actions of unliganded estrogen receptor beta in breast cancer (1138.1)
Authors: Nassa Giovanni , Tarallo Roberta , Bruno Giuseppina , Giurato Giorgio , Hashim Adnan , Ravo Maria , Rinaldi Antonio , Rizzo Francesca , Salvati Annamaria , Stellato Claudia , Nyman Tuula A. , Weisz Alessandro
Published: June 2021

Estrogen Receptor β (ERβ) exerts oncosuppressive functions in breast cancer (BC) also in the absence of physiological ligands. Ligand‐independent actions of ERβ were investigated by expressing it in MCF‐7 and ZR75.1 BC cells. Interaction proteomics revealed >300 nuclear interacting partners, comprising proteins involved in transcriptional gene silencing by miRNAs. miRNA profiling (smallRNA‐Seq) and quantitative proteomics (iTRAQ) of ligand‐free ERβ+ vs ERβ‐ BC cells showed >100 miRNAs and >700 proteins whose expression is modulated by ERβ. Interestingly, these results combined with transcriptome analysis revealed post‐transcriptional regulation of protein expression by unliganded ERβ mediated by miRNAs. Forced expression of a mimic of ERβ‐responsive miR‐30a‐5p led to the identification of several transcripts targeted by this small RNA in BC cells, including 11 encoding regulatory proteins whose intracellular concentration was strongly affected by unliganded ERβ. These results suggest that regulation of BC cell functions by ligand‐free ERβ via miRNAs may exert a key role in the control of the biological and clinical phenotype of BC.

Grant Funding Source: Supported by: FIRC (fellowship to GN) and AIRC (Grant IG‐13176 to AW and fellowship to MR)
Lung epithelial cells resist influenza A infection by inducing the expression of cytochrome VI C oxidase that is regulated by hsa‐miR‐4276 (1138.4)
Authors: Othumpangat Sreekumar , Noti John , Beezhold Donald
Published: June 2021

MicroRNAs regulate at least one third of all human genes and are phylogenetically well conserved. Influenza A virus infection induces several changes in host miRNA/protein profile and understanding the regulatory mechanism is essential for developing intervention approaches. In humans, mitochondrial energy metabolism involves mitochondrial respiration, electron transport and oxidative phosphorylation. Dysregulation of these processes has major pathological consequence and studies have shown that cytochrome oxidases are altered during viral infections. In these studies, influenza A infection of both human alveolar epithelial cells and human bronchial epithelial cells resulted in a significant down‐regulation of hsa‐miRNA‐4276 (miRNA‐4276). Influenza infection also triggered the overexpression of cytochrome oxidase VI C (COX6C). Transfection of epithelial cells with miRNA‐4276 mimic reduced COX6C mRNA and protein expression. Similarly, epithelial cells transfected with miRNA4276 inhibitor increased COX6C mRNA and protein expression. Finally, transfection of cells with miRNA4276 inhibitor and subsequent infection with influenza A led to reduced influenza viral copy number. Taken together the data suggests that miRNA4276 is involved in the regulation of COX6C and that COX6C plays a role in resisting influenza A survival and propagation in lung epithelial cells. Thus, miRNA‐4276 may be an important target in controlling the early stage infection of influenza A.
Expression of plasma membrane (PMCA) and sarco/endoplasmic reticulum (SERCA) Ca2+‐ATPase pumps in cellular model of Huntington disease (1138.8)
Authors: Cesca Federica , Dubsky de Wittenau Giorgia , Curcio Francesco , Lonigro Renata
Published: June 2021

Deregulation of calcium homeostasis is involved in the etiology of Huntington’s disease (HD). We investigated the expression level of calcium pumps in cellular models of HD derived from murine embryonic striatum. We compared the steady‐state level of PMCA1‐4 and SERCA2,3 pumps in wild‐type and mutant cell lines, both in undifferentiated and in glial differentiated conditions.

Comparison between mutant and wild‐type cell lines in the undifferentiated condition show that: i. the level of PMCA1‐4 RNA and protein is comparable except for a reduction of PMCA3 RNA.

ii. SERCA2 protein level is significantly reduced while SERCA3 protein level is significantly increased, suggesting a role for these pumps in calcium homeostasis deregulation in HD.

There is no correspondence between SERCA proteins and their respective RNA, probably due to post‐transcriptional regulatory mechanism/s. In a rat striatal‐derived cellular model of HD in which huntingtin expression is inducible by doxycicline, we confirmed the reduction of SERCA2 protein level as a huntingtin expression‐dependent event. These data suggest that the reduction of SERCA2 pump could be an early event occurring in HD.

Comparison between mutant and wild‐type cell lines in the glial differentiated condition show that PMCA1 and PMCA2 protein levels are reduced, while their respective RNA are increased suggesting a post‐transcriptional regulatory mechanism/s for calcium homeostasis deregulation also in glial differentiated cells.

Grant Funding Source: SNO ‐ Gobessi Fund
Dynamics of DNA methylation of human CYP3A gene promoters in pediatric liver (1141.4)
Authors: Vyhlidal Carrie , Bi Charlie , Gaedigk Roger , Leeder J. Steven
Published: June 2021

Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression in which CYP3A7 is expressed in fetal tissues followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Recently, epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. Generally, hypermethylation of cytosine in CpG dinucleotides is associated with silencing of gene expression and hypomethylation is associated with activation. The objective of this study was to investigate changes in CpG methylation of the CYP3A4 and CYP3A7 genes in pediatric human liver. The methylation status of CpG dinucleotides was determined in 16 pediatric liver samples using Agilent SureSelect Human Methyl‐Seq. Samples were separated by age into four groups (20‐96 days; 2.0‐2.6 years; 7‐8 years; and 15‐17 years). Cytosines at 182, 199, and 219 bp upstream of CYP3A7 were hypomethylated in livers from neonates vs. adolescents 15‐17 years old (p<0.001). In contrast, a cytosine 383 bp upstream of CYP3A4 was hypermethylated in liver samples from neonates vs. adolescents (p=0.00001). No differences were observed for cytosines at 667, 1452, 1521, 1547, and 1569 bp upstream of CYP3A4. In conclusion, methylation status of CpG dinucleotides in the proximal promoters of CYP3A4 and CYP3A7 correlate with changes in gene expression of the two enzymes. Current studies are underway investigating the consequences of methylation on promoter activity and transcription factor binding to differentially methylated sites.
Protein kinase C affects transport function of organic anion transporting polypeptide 1B1 (1141.7)
Authors: Hong Mei , Hong Weifang , Yu Xuan , Huang Jiujiu
Published: June 2021

Objective: Organic anion transporting polypeptides (OATPs) are a family of important uptake transporters. It was demonstrated previously that OATP uptake function is regulated by protein kinase C (PKC). However, the phosphorylation site(s) modified involved in PKC regulation remains unknown. Methods: HEK293 cells stably expressing organic anion transporting polypeptides 1B1 (OATP1B1) were treated by PKC modulators to investigate its regulatory effect on OATP1B1. In addition, site‐directed mutagenesis was used to identify the possible PKC regulation sites within OATP1B1. Results: We found out that OATP1B1 uptake function is regulated by PKC modulators. Further studies indicated that PKC may affect OATP1B1 activity through its regulation of the protein expression on the cell surface. Moreover, we found out that PKC activator PMA not only affect the internalization of OATP1B1 but its recycling as well. Mutagenesis studies revealed that several amino acids located at intracellular loops and the carboxyl terminus of OATP1B1 are involved in the phosphorylation regulation process. Conclusions: OATP1B1 transport function is regulated by PKC, the action of which leads to accelerated internalization and reduced recycling of the transporter protein.

Grant Funding Source: Supported by National Natural Science Foundation of China Grant #81373473 and #U1332124
A live‐cell reporter that differentiates between quiescent and cycling cells (1148.14)
Authors: Henderson Lindsay , Lim Curtis , Zambon Alexander
Published: June 2021

The transition of a proliferating cell into a state of cell quiescence is a key step in organogenesis and tissue homeostasis. It is currently difficult to distinguish between quiescent cells and those that have entered the cell cycle. This study’s objective was to generate a fluorescent‐based genetically encoded reporter capable of differentiating between arrested and actively cycling cells. The Ki67 promoter (Ki67p) was previously shown in our lab to selectively label cells that have entered the cell cycle. However, using Ki67p to drive GFP as a reporter gene results in fluorescent daughter cells due to GFP’s long half‐life (~26h). We hypothesized using the Ki67p to drive expression of the Fluorescent Ubiquitination‐based Cell Cycle Indicator (FUCCI) reporters would enable indirect detection of quiescent (G0) cells and cells in G1/S and S/G2/M. The existing FUCCI reporters employ a constitutively active promoter, thereby preventing discrimination between cells in G0 and cells in G1. To overcome both reporters’ limitations we generated two lentiviral vector constructs with Ki67p upstream of mcherry‐hCdt1(30‐120) and mAG‐hGem(1‐110). We then generated stable double transgenic cell lines (HT1080 and COS cells) that express mCherry‐hCdt1(30‐120) and mAG‐hGem(1‐110) under control of Ki67p. Fluorescence time‐lapse imaging of Ki67p‐FUCCI cells indicates that low‐density plating induces G0 cell cycle withdrawal and concurrent attenuation of Ki67‐FUCCI reporter activity. Low density compared to high density plating increased the number of Ki67p‐FUCCI− HT1080 cells (44% vs. 5%, P<0.05) indicating G0 entry. Moreover, serum starvation induced G1 arrest (91% Ki67p‐mCherry+ without serum vs. 47% with serum, P<0.01). These reporters will enable studies designed to segregate between quiescent and actively proliferating cells in each of the major phases of the cell cycle in living tissue and in real‐time, facilitating studies in stem cell and cancer biology.

Grant Funding Source: NIGMS 2‐R25‐GM‐083275, AHA 10SDG2630130
Answering Huxley: an exercise in the history of examinations (1149.2)
Authors: Rangachari P
Published: June 2021

Twelve students in an undergraduate health sciences program, who had completed a 2nd year (sophomore) course in Anatomy and Physiology volunteered to participate in a “history” exercise. They answered two essay questions set by Thomas Henry Huxley when he was the Examiner in Physiology and Comparative Anatomy for diverse faculties at the University of London from 1857‐1870. These were selected from a question bank kindly provided by Dr. Pauline Mazumdar (University of Toronto). In preparation for the hour‐long, hand‐written exam, they studied specific sections (liver physiology, blood, vascular system) from two 19th century texts (Foster, Broussais). They later filled out a questionnaire that asked them to contrast the experience of reading older, denser texts and answering essay‐type exams with their more contemporary experiences. Many wrote thoughtful, reflective comments on the exercise that gave them an insight into the difficulties faced by students in the past (8.0 ± 1.4) that also proved to be a valuable learning experience (8.3 ± 1.2).
Matrix metalloproteinase‐2 mediate oncostatin‐M induced cardiomyocyte dedifferentiation (1151.2)
Authors: Fan Frank , Ali Mohammad , Hughes Bryan , Launier Katherine , Schulz Richard
Published: June 2021

Dedifferentiated cardiomyocytes are an important source of cardiac progenitor cells facilitating cardiac repair. Cardiomyocyte dedifferentiation induced by oncostatin‐M (OSM) is characterized by progressive sarcomere degeneration. However, the mechanism regarding the execution of sarcomere degeneration remains unclear. Matrix metalloproteinase‐2 (MMP‐2) is a key intracellular protease abundantly expressed in cardiomyocytes. We hypothesized that MMP‐2 may be involved in cardiomyocyte dedifferentiation. We performed confocal immunofluorescence and biochemical studies to explore the role of MMP‐2 in OSM‐induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). Cells treated with OSM (10‐200 ng/ml for 1‐5 days) showed a dose and time‐dependent loss of sarcomeric α‐actinin and troponin‐I. The mature sarcomere transformed to a pattern resembling a less developed sarcomere (Z‐disks transformed into Z‐bodies usually found in immature myofibrils). Western blots and gelatin zymography showed that OSM treatment dose‐dependently increased MMP‐2 activity. Both selective MMP‐2 inhibitor (ARP100, 100 µM) and pan‐MMP inhibitors (GM6001, 5 µM) rescued OSM‐induced sarcomere degeneration. These results suggest that intracellular MMP‐2 mediates sarcomere degeneration in OSM‐induced cardiomyocyte dedifferentiation and potentially contributes to cardiomyocyte regeneration.

Grant Funding Source: The Canadian Institutes of Health Research (CIHR)
Nicotinamide adenine dinucleotide hydrate (NAD+) regulates glucose deprivation‐induced activation of protein O‐GlcNAcylation, ER stress and autophagy (1154.2)
Authors: Zou Luyun , Chatham John
Published: June 2021

Mammalian sirtuins are important regulators of metabolism and cell survival including autophagy and ER stress. The post‐translational modification of proteins by O‐linked‐N‐acetylglucosamine (O‐GlcNAc) is also metabolically regulated and regulates cell survival responses. We have previously reported that glucose deprivation (GD), which induces autophagy and ER stress is also a potent stimulus for increasing cellular O‐GlcNAc levels. Therefore, the goal of this study, therefore, was to determine whether NAD+, which activates sirtuins, alters the response of GD on protein O‐GlcNAcylation, ER stress, and autophagy. AC16 cardiomyocytes were subjected to GD for up to 24hrs with and without NAD+ (0.25mM). 24hrs after GD there was a significantly increase in O‐GlcNAc levels as well as CHOP, Bip, P‐PERK/T‐PERK and LC3‐II/LC3‐I, consistent with both increased ER stress and activation of autophagy. Treatment with NAD prevented the GD induced increase in O‐GlcNAc and also significantly attenuated both ER stress and autophagic responses. Interestingly GD resulted in a decrease in protein acetylation, which was prevented by NAD, suggesting that the effects of NAD+ may not be mediated via activation of sirtuins, which catalyze deacetylation. Therefore these data indicate that while NAD+ contributes to the regulation of protein O‐GlcNAcylation, ER stress and autophagy this appears to be sirtuin independent. These results also demonstrate for the first time potential cross‐talk between cytosolic redox state and regulation of O‐GlcNAc cycling.

Grant Funding Source: Supported by NIH grants HL101192 and HL110366
Endothelial‐dependent dilation is lower in males compared to females on a controlled sodium diet (1156.6)
Authors: Lennon‐Edwards Shannon , Matthews Evan , Ramick Meghan , Brian Michael , Edwards David , Farquhar William
Published: June 2021

Excess dietary sodium has been linked to the development of high blood pressure (BP) and other cardiovascular diseases (CVD) and recently shown to effect endothelial function independently of BP. However, it is unknown whether sodium consumption impacts endothelial function differently in males (M) and females (F). Given the varying habitual intake of sodium, we sought to fix sodium using a controlled diet study. Therefore, the aim of this study was to test the hypothesis that endothelial‐dependent dilation (EDD) would be lower in M as compared to F on a controlled sodium diet set at the recommended intake for Americans. 16 M (29±2 y; BMI 23.8±0.7 kg/m2) and 12 F (31±3 y; 24.3±0.7 kg/m2) completed the study. All subjects were apparently healthy and free of CVD. Subjects consumed a eucaloric diet providing 100 mmol/d of sodium for 7 days. On day 7 of the diet, 24‐hr mean arterial pressure (MAP) was recorded, and blood and urine were collected. EDD was assessed by brachial artery flow‐mediated dilation. Urinary sodium excretion was not different between M and F (81.4±7.7 vs. 93.9±22.8 mmol/24hr, p > 0.05) while 24‐hr MAP was higher in M (88±1 vs. 82±1.3; p<0.05). EDD was significantly lower in M than F (7.0±0.8% vs. 10.4±1.2%; p < 0.05). These data demonstrate that M have a lower EDD than F at the recommended intake of sodium. Future research should focus on exploring the mechanisms underlying this difference.

Grant Funding Source: Supported by R01 HL 104106
NFATc1 mediates Pak1 activation in the modulation of chemokine‐induced human aortic smooth muscle cell F‐actin stress fiber formation, migration, and proliferation and injury‐induced vascular wall remodeling (1158.5)
Authors: Singh Nikhlesh , Kundumani‐Sridharan Venkatesh , Kumar Sanjay , Gadepalli Ravishekhar , Rao Gadiparthi
Published: June 2021

In addition to their role in cell proliferation, cyclin‐dependent kinases (CDKs) mediate cell migration; however the underlying mechanisms are not clear. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a time‐dependent but delayed manner. MCP1 also stimulated F‐actin stress fiber formation as well as the migration and proliferation of these cells and inhibition of Pak1 reversed these effects. MCP1 also induced cyclin D1 expression and CDK6/4 activities, and these effects were dependent on NFATc1 activation. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1‐induced Pak1 phosphorylation/activation resulting in decreased HASMC F‐actin stress fiber formation, migration and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. Depletion/inhibition of Rac1 levels/activation abrogated MCP1‐induced NFATc1‐cyclin D1‐CDK6‐CDK4‐Pak1 signaling, and thereby, decreased HASMC F‐actin stress fiber formation, migration, and proliferation. In addition, smooth muscle‐specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1‐dependent NFATc1‐mediated cyclin D1 expression and CDK6 activity mediate this effect in the modulation of MCP1‐induced HASMC migration and proliferation in vitro and vascular wall remodeling in vivo.

Grant Funding Source: NHLBI
Maternal deposition of ApoA‐I‐mCherry transgene indicates an important role in early zebrafish development (1161.1)
Authors: Reece Takisha , Otis Jessica , Farber Steven
Published: June 2021

Our goal is to investigate the function of high‐density lipoprotein (HDL) particles of hepatic origin through in vivo visualization in the optically clear larval zebrafish (Danio rerio). We generated zebrafish stably expressing fluorescently tagged human apolipoprotein A‐I (ApoA‐I), a marker of HDL, through a liver‐specific promoter (LFABP:ApoA‐I‐mCherry; 6 lines). At 6‐days post fertilization (dpf), transgenic larvae show variable ApoA‐I‐mCherry localization: a subset of larvae show fluorescence in the liver, circulation, pronephric kidney and yolk syncytial layer, as expected; a second subset shows fluorescence in all of these regions except the liver. We hypothesized that variable ApoA‐I‐mCherry expression results from the first subset of larvae containing LFABP:ApoA‐I‐mCherry in their genome and that the second subset of larvae lack the transgene and are displaying maternally deposited ApoA‐I‐mCherry. Maternal RNA products deposited in embryos are degraded within hours, but the duration of maternal protein expression is less characterized. We pairwise crossed wild type and transgenic LFABP:ApoA‐I‐mCherry zebrafish and characterized ApoA‐I‐mCherry expression and localization. Only the progeny of LFABP:ApoA‐I‐mCherry females showed absence of ApoA‐I‐mCherry in the liver (n=12 experiments in 5 lines), supporting the conclusion that maternal deposition of ApoA‐I‐mCherry in non‐transgenic larvae created this phenotype. On‐going studies include Western Blot and PCR analyses to confirm and investigate the function of maternally deposited ApoA‐I in early development.

Grant Funding Source: APS 2013 Frontiers in Physiology Research Teacher Fellowship
Changes in macrophage phenotype and induction of epithelial‐to‐mesenchymal transition genes following acute Achilles tenotomy and repair (1166.3)
Authors: Sugg Kristoffer , Gumucio Jonathan , Lubardic Jovan , Mendias Christopher
Published: June 2021

Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. Fibroblasts have a well‐described role in synthesizing and remodeling extracellular matrix (ECM) after tissue injury. In many organ systems, epithelial cells can transdifferentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial‐to‐mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon regeneration, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM‐ and EMT‐related genes over a four week time course. The results from this study suggest that changes in macrophage phenotype and activation of canonical EMT programs likely contribute to the degradation and subsequent repair of injured tendon tissue.

Grant Funding Source: NIH grants R01‐AR063649, T32‐GM008616 and T32‐GM008322
Acute environmental hypoxia activates autophagy in human skeletal muscle (1167.2)
Authors: Deldicque Louise , Masschelein Evi , Van Thienen Ruud , D'Hulst Gommaar , Hespel Peter , Thomis Martine
Published: June 2021

Hypoxia‐induced muscle wasting is a phenomenon often described at high altitude, which has been attributed to an altered protein metabolism. We hypothesized that acute normobaric hypoxia would favour a negative net protein balance by repressing anabolic and activating proteolytic signaling pathways at rest and post‐exercise. Twenty‐two subjects participated in an experimental trial in normoxia and hypoxia (10.7% O2). Muscle biopsies were obtained before and after a 20‐min moderate exercise. In hypoxia, autophagic flux was increased as indicated by an increased LC3‐II/I ratio and LC3‐II expression and decreased p62/SQSTM1 expression. Following exercise in hypoxia a decrease in LC3‐I, LC3‐II and LC3‐II/I as well as in p62/SQSTM1 expressions was found. Bnip3 mRNA expression, a marker of mitophagy, was also increased in hypoxia. MAFbx mRNA expression, a muscle‐specific E3‐ligase, was increased in hypoxia and returned to levels similar to normoxia after exercise, while MuRF‐1 mRNA was not altered. In hypoxia, REDD1 expression and AMPK phosphorylation, two potential inhibitors of the PKB/mTOR pathway, were increased although phosphorylation of PKB, S6K1 and 4E‐BP1 was not modified. Our results indicate that environmental hypoxia modulates protein metabolism at rest and after moderate exercise by primarily altering protein breakdown, and more specifically the autophagy‐lysosomal system.
A new algorithm for in‐band noise removal and HRV analysis in mouse ECG recordings (1169.7)
Authors: Brockway Marina , Brockway Brian , Stiedl Oliver
Published: June 2021

Fast and accurate analysis of ECG signals from awake and behaving mice can be challenging due to muscle noise, movement artifact, ECG morphology changes, and rapid changes in heart rate variability in response to experimental stimuli. A new patented technology, Multi‐Domain Signal Processing (MDSP)TM, has been shown to reduce in‐band noise in ECG recordings from non‐human primates by up to 21 dB without distorting ECG morphology (M. Brockway and R. Hamlin, Journal of Pharmacological and Toxicological Methods, 2011). In this study, we evaluated MDSP performance on ECG signals from active telemetered (ETA‐F20, DSI, St. Paul, MN) C57BL/6 mice with signal‐to‐noise ratio ranging from ‐9 to 30 dB (per ANSI/AAMI EC‐57 standard). In‐band noise removal was evaluated by contaminating a clean ECG strip with varying levels of white noise per ANSI/AAMI EC‐57 methodology. In‐band noise was reduced by up to 15 dB (82% amplitude reduction) with virtually no ECG waveform distortion. The QRS detection accuracy achieved by MDSP required screening of < 0.5% of beats with no need to preselect clean ECG segments prior to HRV analysis. HRV analysis was performed using traditional frequency domain and a novel time‐frequency domain wavelet‐based analysis and the discriminatory power of these two techniques was compared. Results show that MDSP demonstrates significant potential for efficient and accurate analysis of ECGs from freely moving mice, especially where it is important to discriminate rapid changes in HRV.
Calcium signaling and glucose response is different in adult compared to neonatal rat astrocytes (1177.7)
Authors: Rarick Kevin , Eisenhauer Jessica , Harder David
Published: June 2021

Much of our mechanistic understanding of astrocytes comes from neonatal cells which may not represent the mature glial phenotype. We sought to determine if neonatal and adult astrocytes respond similarly when challenged with high glucose. Neonatal and adult rat astrocytes were cultured in 5.5 mM low glucose DMEM (LG) and then maintained in LG or switched to 25 mM high glucose DMEM (HG) for two weeks. A developmental decrease was observed in expression of the inositol trisphosphate receptor (IP3R) and the metabotropic glutamate 5 receptor (mGluR5). The L‐type calcium channel (CaV1.2) was only expressed in adult astrocytes. Adult and neonatal astrocytes both responded to HG with increased mGluR5 and decreased IP3R protein expression. Adult astrocytes also increased CaV1.2 expression following HG. Neonatal astrocytes had a greater magnitude and rate of rise of [Ca2+]i in response to ATP and glutamate compared to adult astrocytes. The [Ca2+]i responses to ATP and glutamate were similarly affected by HG, although adult astrocytes demonstrated minimal response to glutamate in all conditions. Cell viability decreased only in adult astrocytes following HG. These results suggest HG alters calcium signaling in astrocytes and developmental differences may exist in downstream pro‐survival signaling. The response of adult glia in specific disease paradigms may not be accurately reflected when using neonatal cells.

Grant Funding Source: Supported by T32 HL007792, R01 HL033833, R01 HL 092105, and R01 HL105997