bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Histone Methyltransferase G9a Promotes Cholangiocarcinogenesis through Regulation of the Hippo Pathway Kinase LATS2 and YAP Signaling Pathway
Authors: Ma Wenbo , Han Chang , Zhang Jinqiang , Song Kyoungsub , Chen Weina , Kwon Hyunjoo , Wu Tong 
Published: April 2020

BackgroundCholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. A better understanding of the molecular mechanisms of cholangiocarcinogenesis is of paramount importance to develop more effective target therapy.MethodsqRT‐PCR and Western blotting analyses were used to determine the expression of specific mRNAs and proteins. ChIP‐PCR was performed to analyze the enrichment of G9a and H3K9me2 in the promoter region of LATS2 gene. Dual‐Luciferase reporter assay system was used to evaluate YAP luciferase activity. CCA xenograft was developed by inoculation of human CCA cells with or without G9a depletion into SCID mice. CCA tumor induction was developed by SB transposase‐mediated integration of oncogenes (NICD/Akt) to mice via hydrodynamic tail vein (HDTV) injection.ResultsWe present novel evidence that the histone‐lysine methyltransferase G9a is upregulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase LATS2 and YAP signaling pathway. Kaplan–Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by siRNA/shRNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0638) significantly inhibited human CCA cell growth in vitro and in SCID mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of NICD and myr‐Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt‐injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a‐derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2 and this effect led to subsequent activation of oncogenic YAP. Accordingly, out data showed that G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition.ConclusionOur findings provide novel evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2‐YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.
Functional analysis of mosquito and Drosophila Na+‐dependent cation‐chloride cotransporters
Authors: Gillen Christopher M. , Holmes Heather L. , Piermarini Peter M. , Romero Michael F. 
Published: April 2020

Dipteran Na+‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na+‐K+‐Cl− cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na+ conductances than controls. Moreover, in flux assays using Li+ as a Na+ tracer, Li+ transport by aeCCC2 was not dependent upon extracellular Cl− and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl−‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na+ replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na+‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na+ with saturable Michaelis‐Menton kinetics. The apparent affinity for Na+ was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li+ and Rb+ were also detectable when added to the bathing saline as tracers for Na+ and K+. In w118‐control flies, fluid secreted by MT measured using these procedures had a [K+] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na+] of 61.2 ± 7.8 mM. In preliminary experiments, [K+] was 112.7 ± 1.3 mM (n=4) and [Na+] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.Support or Funding InformationFunding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.
Expression of genes related to autophagy and protein breakdown are positively correlated with protein synthesis and protein breakdown in skeletal muscle of healthy adults after a bout of resistance exercise
Authors: Wu Hexirui , Kim Il-Young , Jang Ji-Woong , Ferrando Arny , Wolfe Robert , Baum Jamie 
Published: April 2020

While it is well‐established that exercise increases muscle protein synthesis rate, it is still unclear that what role of protein breakdown plays in muscle after exercise. Therefore, the objective of this study was to determine if rates of protein synthesis and breakdown correlate with expression of genes related autophagy and protein degradation in skeletal muscle of healthy adults after a bout of resistance exercise. This is a secondary analysis of a previous published study (
Kim et al., 2016). In total, twenty‐three subjects were divided into two groups, rest (n = 12) and exercised (n = 11). Real‐time PCR was used for analyzing gene expression of baseline muscle samples from both groups, and student t test was used to compare significance (p < 0.05) between two groups. Relationships between variables were assessed using Pearson’s Product‐Moment Correlation, and p value lower than 0.05 was considered as significant. Autophagy‐related genes p62, p53, ATG3, ATG5, LC3B, Beclin1, ATF4, and GADD54A, were significantly upregulated (p < 0.05) in skeletal muscle from exercised subjects versus subjects at rest. Two genes involved in ubiquitin‐proteasome pathway, MuRF1 and Atrogin1, were also upregulated (p < 0.05) after exercise. Neither protein synthesis nor protein breakdown rate was correlated with gene expression in skeletal muscle from subjects at rest. However, protein synthesis rate was positively correlated (p < 0.05) with increased expression of p62, p53, ATG3, ATG5, ATG7, LC3B, Beclin1, ATF4, GADD54A, Atrogin‐1 and MuRF1 in exercised muscle. Protein breakdown rates were positively associated (p < 0.05) with increased expression of ATF4, ATG5, and p62 in exercised skeletal muscle. In conclusion, the results provide preliminary evidence that genes related to autophagy and protein breakdown are upregulated after exercise in skeletal muscle from healthy adults and are positively correlated with muscle protein synthesis rate.Support or Funding InformationThis study is funded by an Arkansas Biosciences Institute grant, we would like to thank thegenerous contribution of muscle samples by Dr. Wolfe.
Leptin Receptor Blockade Abolished Hypertension and Decreased Hypoxic Ventilatory Response in a Model of Obesity‐Related Hypertension
Authors: Kim Lenise Jihe , Shin Mi-Kyung , Pho Huy , Otvos Laszlo , Tufik Sergio , Andersen Monica Levy , Polotsky Vsevolod Y. 
Published: April 2020

INTRODUCTIONObesity causes hypertension and exacerbates sleep disordered breathing. Leptin is an adipocyte produced hormone which is implicated in regulation of metabolic rate, food intake, control of breathing and blood pressure. Obese individuals are resistant to metabolic and respiratory effects of leptin, but appeared to be sensitive to hypertensive effects of the hormone. We hypothesized that New Zealand obese mice (NZO), which are resistant to metabolic effects of leptin, remain susceptible to cardiovascular, but not respiratory, effects of this hormone.METHODSMale NZO mice of 12–13 weeks‐old were treated with leptin receptor blockers Allo‐aca (n=5) or control peptide Gly11 (n=3) for 9 consecutive days. Doses of 0.2mg/kg were administered 2x/day, at 10AM and 6PM subcutaneously. Blood pressure was measured by telemetry. Ventilation was assessed by whole‐body barometric plethysmography and control of breathing was examined by assessing the hypoxic ventilatory response (HVR). Sleep studies were conducted by full‐polysomnography.RESULTSSystemic leptin receptor blocker Allo‐aca did not affect body weight and food intake in NZO mice. In contrast, it induced a decrease in the mean arterial blood pressure from 132.9±5.9 mmHg to 121.2±11.3 mmHg during the light phase (P<0.001). Allo‐aca did not affect baseline minute ventilation in awake mice, but decreased HVR (P<0.05). There was a trend for systemic blockade of leptin receptors with Allo‐aca to suppress minute ventilation during NREM and REM sleep, whereas the control peptide Gly11 had no effect.CONCLUSIONSNZO mice were resistant to metabolic effects of leptin, but they were sensitive to the hypertensive and respiratory effects of the hormone. Thus, leptin receptor blockers can be used for pharmacotherapy for obesity‐associated hypertension, but sleep‐related hypoventilation should be considered as a potential side effect.Support or Funding InformationNIH R01s HL128970 and HL133100 to Dr. Vsevolod Y. Polotsky. Dr. Tufik and Dr. Andersen are CNPq fellowship (Brazil) recipients. Dr. Lenise Jihe Kim was supported by Associação Fundo de Incentivo à Pesquisa (AFIP, Brazil).
Regulation of the Mannitol Locus by MtlR in Vibrio cholerae
Authors: George Micayla , Liu Jane 
Published: April 2020

The intake of mannitol, a sugar produced by plants and algae, by Vibrio cholerae increases biofilm formation and thereby persistence of the pathogenic bacterium. The process of mannitol uptake is mediated by the mannitol phosphotransferase system (PTS). Within this system, the protein MtlA is directly responsible for the intracellular transport of mannitol, and is encoded by the mtlA gene located within a group of genes (mtlA, D, and R) on the mannitol (mtl) locus. While it is known that protein MtlR represses the expression of mtlA at the transcriptional level, the mechanism of this repression is unknown. The aim of this project is to better understand the dynamics between MtlR and other protein regulators of the mtl locus, such as the CRP activator protein, to gain insight into the mechanism of MtlR regulation of mtlA expression. This was accomplished through assessment of MtlR and CRP levels across V. cholerae via Western Blots, as well as through measuring mtlA expression in the absence of CRP and/or MtlR via LacZ assays. Previous work has shown that MtlR levels fluctuate depending on available carbon source, but we found CRP levels to be relatively unaffected. We also found that while deletion of the mtlR gene increases mtlA expression, this is not observed when the crp gene is also deleted, suggesting that MtlR repression is secondary to CRP activation of mtlA expression. This work helps shed light on possible mechanisms by which MtlR might be negatively regulating the expression of mtlA and overall intracellular mannitol intake by V. cholerae.Support or Funding InformationNational Institutes of Health; The Camille and Henry Dreyfus Foundation
After salt: ENaC in self‐sustaining hypertension
Authors: Arkhipov Sergey N. , Potter D’Anna L. , Pavlov Tengis S. 
Published: April 2020

BackgroundEvery year thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. However, most of the studies report a cohort of patients who does not significantly respond to sodium restriction. Generally, most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying self‐sustaining character of hypertension. Unfortunately, hypertension causes multiple abnormalities in the body which stays even when the trigger, caused the initial blood pressure raise, is gone. We hypothesize that chronic hypertension leads to a significant renal damage and abnormally high sodium reabsorption.MethodsWe used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4% NaCl diet for 3 weeks to induce hypertension and then diet was switched back to normal (0.4% NaCl). Patch‐clamp analysis was performed on freshly isolated split‐open cortical collecting ducts and CHO cells to characterize activity of the epithelial sodium channel (ENaC), responsible for the sodium reabsorption in the aldosterone‐sensitive distal nephron.ResultsWe found that 4% sodium diet significantly increases blood pressure within 3 weeks (from 111±0.9 to 138±3.5 mmHg) and switching back to 0.4% NaCl diet slightly reduces blood pressure (to 133.6±7.6). Patch clamp analysis reveals that development of hypertension was accompanied with elevated ENaC activity which also stayed high (NPo=1.22±0.23, n=8) after the salt challenge. Subsequent blockage of ENaC with a selective inhibitor, benzamil, caused a dramatic drop in blood pressure indicating that ENaC activity contributes to maintain sustaining hypertension in absence of the high salt consumption. We also found significant renal damage caused by hypertension. As earlier studies report that inflammation and reactive oxygen species production are involved in improper renal function and setting high blood pressure, we performed a subset of whole‐cell patch clamp experiments on CHO cells overexpressing mENaC to study effect of hydrogen peroxide on ENaC activity. We found that treatment with 100μM H2O2 within a few seconds to 2 minutes stimulated the ENaC current of the cells (−144±12 pA/pF vs −190±22 pA/pF before and after treatment respectively, n=8, p=0.0078).Based on our earlier publications and the current data we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge and reactive oxygen species production can keep ENaC active.Support or Funding InformationAcknowledgementASN Carl W. Gottschalk award; R00 HL116603
A Mathematical Model of the Rat Kidney. II. Antidiuresis
Authors: Weinstein Alan M. 
Published: April 2020

Water conservation by the kidney requires a hypertonic medullary interstitium, NaCl in outer medulla (OM) and both NaCl and urea in inner medulla (IM), along with a vascular configuration that protects against washout. A multi‐solute mathematical model of the rat kidney, which includes the important acid‐base species, was developed and used first to examine K+‐induced natriuresis (Am. J. Physiol. 312:F925, 2017). In the present work, model computation has been streamlined by parallelizing the calculation of the model Jacobian, and this has allowed finer medullary spatial resolution, along with more extensive examination of model parameters. Tubule compliance is now represented along the whole nephron, and this attenuates pressure swings due to variations in urine flow. OM NaCl is modestly increased when transporter density in ascending Henle limbs (AHL) from juxtamedullary nephrons (JM), is scaled to match the greater JM nephron solute flow. However, higher NaCl transport produces greater CO2 generation, and by virtue of countercurrent vascular flows, establishment of high medullary pCO2. This CO2 gradient can be mitigated, by assuming that a fraction of medullary AHL and collecting duct transport is powered anaerobically. Reduction of vascular flows or increasing vessel permeabilities does little to further increase OM solute gradients. In contrast to the medullary models of others, vessels in this model all have solute reflection coefficients close to zero; increasing these coefficients provides no substantive enhancement of solute profiles, but does generate high interstitial hydrostatic pressures, which distort tubule architecture. As others have noted, increasing medullary urea delivery via entering vasa recta, enhances IM urea, although not nearly to levels found in rats. In sum, (1) the magnitudes of medullary gradients of Na+, urea, and ammonia are still not captured by the model; and (2) the countercurrent architecture that provides antidiuresis also produces exaggerated pCO2 profiles and appears to be an unappreciated constraint on models of medullary function.Support or Funding InformationNIH RO1‐DK‐29857
Therapeutic Role of Dietary Nitrates on Cardiorespiratory Function in Cancer Survivors
Authors: Turpin Vanessa-Rose Garcia , Parr Shannon K. , Hammond Stephen T. , Colburn Trenton D. , Ade Carl J. 
Published: April 2020

IntroductionThe adverse physiological consequences of anticancer therapy include direct injury to the cardiovascular‐skeletal muscle axis. As such, cancer patients are at an increased risk of both cancer therapy‐related and age‐related pathological outcomes, including primary cardiovascular disease, exercise intolerance, and cancer‐related fatigue. To date, however, therapeutic strategies that mitigate these negative effects within the human body have yet to be established. Previous work has demonstrated that dietary nitrate (NO3−) supplementation can improve cardiac, vascular, and cardiorespiratory exercise parameters, highlighting its potential therapeutic use in clinical populations. Therefore, we hypothesized that NO3− supplementation improves both cardiac performance and exercise capacity. We also investigated the effects of NO3− supplementation on estimates of components of the convective oxygen transport pathway to obtain insight into potential mechanisms of action.MethodsTo date, 5 cancer survivors (58.6 ± 11.3 years) with a history of anticancer therapy completed a randomized, double‐blind, crossover study with an acute, single‐dose administration of NO3− or placebo (PL) [140 mL]. Transthoracic echocardiographic measures at rest were made to obtain left ventricular stroke volume. Patients performed a supine‐cycling steady‐state exercise test (30W) and a maximal‐effort cardiopulmonary exercise test, with measurements of arterial blood pressure, stroke volume, and cardiac output.ResultsThere was a statistically significant increase in plasma nitrite during the NO3− condition compared to PL (NO3− 1300 ± 963 μM vs. PL 111 ± 49 μM, respectively; P = 0.02). Additionally, we observed a decrease in oxygen uptake (VO2) during steady‐state exercise with NO3− compared to PL (NO3− 8.2 ± 2.4 vs. PL 8.6 ± 2.5 ml/kg/min; P = 0.03), indicating an improved exercise efficiency. Resting and steady‐state arterial blood pressure, stroke volume, and cardiac output were not different between conditions. Furthermore, we did not observe any differences between conditions for VO2 peak (NO3− 22.87 ± 4.14 vs. PL 23.45 ± 4.4 ml/kg/min; P = 0.31) or total work done (NO3− 75.73 ± 29.9 vs. PL 75.82 ± 31.3 kJ; P = 0.48) during the maximal‐effort cardiopulmonary exercise test. Similarly, there was no difference is peak cardiac output, convective O2 transport, or the arterial‐venous O2 difference.ConclusionsA single‐dose of inorganic nitrate supplementation in cancer survivors with a history of anticancer therapy enhanced steady‐state exercise efficacy, but had no effect on exercise cardiac performance or peak exercise capacity.
Effects of Prior Anatomy Coursework on Assessment Performance and Study Habits
Authors: Wehrman Gregory , Ryan Katharine , Auerbach Benjamin 
Published: April 2020

Students entering health science fields often take multiple human anatomy courses throughout their education, including settings such as high school, community colleges/technical schools, and 4‐year undergraduate universities. Additionally, it is not uncommon for students to retake a human anatomy course during their undergraduate careers if they are not satisfied with their initial performance. While research has shown that final grade performance improves for those retaking the same undergraduate course, it is unclear whether previous anatomy coursework in high school, community college, or other undergraduate university settings confers the same advantage on course performance or influences study methods employed in the course. In other words, students who have had experience in anatomical coursework may have developed effective study approaches before taking a more advanced anatomy course. The purpose of this study is to assess the effect of previous human anatomy coursework on lecture and lab exam performance and study habits in an undergraduate human anatomy course at a large state university in Tennessee for the 2018–2019 and 2019–2020 academic years (N = 442).The course enrollment consists of nursing, kinesiology, or pre‐professional undergraduate majors, of which more than half (53%) are sophomores, and the remainder juniors (17.5%) and seniors (29.5%). We collected data from students at midterm through an anonymous survey. Students self‐reported their performance on assessments for the first major examination in the course, which has a written lecture component and a practical lab component. The survey also included questions about previous anatomy coursework, prior enrollment in the current course, study habits through a range of modalities, and use of extracurricular resources. We performed nonparametric Kruskal‐Wallis tests to examine the relationship between prior anatomy experience and performance on initial lecture and lab assessments, as well as study methods. Results indicate that no significant difference in first lecture or lab assessment performance is associated with prior anatomy coursework, regardless of prior course setting (high school versus college). Additionally, prior coursework in anatomy is not associated with a significant difference in when students began studying for their first lecture/lab assessments. There is a trend for students with prior coursework in anatomy to perform slightly better on assessments than those who had not. In addition, preliminary analysis of study modalities shows that students with prior anatomical experience do not engage in study methods different from those who have not taken an anatomy course before.