bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Method for Normalization in Western Blotting that is Superior to the Use of Housekeeping Proteins
Authors: Thompson Jordan , Diller Thomas , Steer Brian 
Published: April 2020

Western blotting workflows have long relied upon the use of housekeeping proteins (HKPs) as a control for sample normalization in order to account for differences between sample loads. Unfortunately, HKPs often show signal saturation, can vary in response to the treatments being studied, or show variation in expression between different cell types leading to erroneous results. In contrast, new methods of normalization that involve Total Protein Normalization (TPN) have been gaining support. In this study we investigate a new method for TPN that utilizes the Invitrogen No‐Stain Protein Labeling Reagent, which allows for all of the proteins on a membrane to be labeled uniformly and quickly while not interfering with downstream immunodetection methods. We compare this new method against common housekeeping proteins in four different cell backgrounds (HeLa, MCF‐7, Jurkat, and A431 cells). Our data illustrate how common housekeeping proteins exhibit signal saturation and yield incorrect normalization data. The No‐Stain TPN method shows superior protein normalization compared to the use of housekeeping proteins with decreased sample‐to‐sample variation (8% versus 48%). The No‐Stain TPN method provides an elegant alternative for achieving accurate quantitative western blots.Support or Funding InformationThis work was supported by Thermo Fisher Scientific
GPR120‐mediated Connexin43 Phosphorylation is Implicated in Antiarrhythmic Effects of Icosapent Ethyl in Post‐infarcted Rat Hearts
Authors: Chen Wei-Ting , Lee Chen-Che , Lee Tsung-Ming 
Published: April 2020

AimsThe w‐3 fatty acids exert as an antioxidant via the G protein‐coupled receptor 120 (GPR120). Icosapent ethyl, a highly purified ethyl ester of eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connnexin43 expression and whether the GPR120 signaling is involved in the protection.Methods and resultsMale Wistar rats after ligating coronary artery were randomized to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative‐nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial connnexin43 levels revealed a significant decrease in vehicle‐treated infarcted rats compared with sham. These changes of oxidative‐nitrosative stress and connnexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connnexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative‐nitrosative stress and connnexin43 phosphorylation were abolished by administering AH‐7614, an inhibitor of GPR120. SIN‐1 abolished the Cx43 phosphorylation of IPE without affecting GPR120 levels.ConclusionsChronic use of icosapent ethyl after infarction is associated with up‐regulation of connnexin43 phosphorylation through a GPR120‐dependent antioxidant pathway and thus plays a role in the beneficial effect on arrhythmogenic response to programmed electrical stimulation.Support or Funding InformationThis work was supported by the grants of China Medical University (DMR‐108‐035) and Ministry of Science and Technology (MOST‐108‐2622‐B‐039‐002‐CC3), Taiwan.
Skeletal Muscle Oxidative Capacity is Linked to Cardiovascular Health
Authors: Bubb Aleena M. , Blanks Anson , Looney Jacob , Blackburn Marsha , Bieck Maire Rose , Derella Cassandra C. , Sanchez Savanna , Pace Brooke , Thomas Jeffrey , Norland Kimberly , Ye Bingwei , Wang Xialong , Harris Ryan A. 
Published: April 2020

BackgroundCardiovascular disease (CVD) affects 48% of the adult population and is the leading cause of death in the United States. Vascular stiffness, endothelial dysfunction, and decreased skeletal muscle health are each independently associated with increased CVD risk. This study sought to test the hypothesis that indices of vascular health are associated with skeletal muscle function in the general population.Methods22 (16 women and 6 men) apparently healthy adults participated in this study. Dual energy x‐ray absorptiometry was performed to obtain body composition and skeletal muscle mass. Carotid‐femoral pulse wave velocity (PWVCF), augmentation index normalized for heart rate (AI@75bpm), and flow mediated dilation normalized for shear rate (FMD/Shear) were assessed to evaluate arterial stiffness and endothelial function. Handgrip strength was assessed using a handgrip dynamometer and skeletal muscle mitochondrial oxidative capacity (Kox cap) was assessed using near‐infrared spectroscopy (NIRS) to evaluate indices of skeletal muscle health and function, respectively.ResultsKox cap was inversely correlated with both carotid‐femoral pulse wave velocity PWVCF (r= −0.515; p= 0.014) and AI@75BPM (r= −0.453; p= 0.034). In addition, a trend in the relationship between FMD/Shear and Kox cap (r= 0.326; p= 0.139) was observed. Compared with women, men had significantly higher PWVCF (p= 0.008), handgrip strength (p< 0.001), and skeletal muscle mass (p= 0.006); however, muscle function was similar (p= 0.209) between sexes.ConclusionsThese findings demonstrate that improved muscle function can favorably impact vascular health. Additionally, skeletal muscle strength and size were significantly different between sexes; however, muscle function was similar. These data highlight the importance of assessing all three indices of muscle health. Future studies are warranted to investigate if increases in vascular endothelial function will improve skeletal muscle function and contribute to prevention of CVD.Support or Funding InformationThis project was supported in part by The Augusta University Medical Scholars Program (AB) and NIH R01DK117365 (RAH and XW).
Activation of Piezo1 Mechanosensitive Channels in Retinal Ganglion Cells Leads to Cell Death
Authors: Friemel Taylor , Grillo Stephanie , Stella Salvatore L. 
Published: April 2020

PurposePiezo1 mechanosensitive ion channels play an important role in mechanotransduction in all vertebrates and have been identified in a variety of tissues and cell types including neurons. Previously, we have identified Piezo1 mechanosensitive ion channels in zebrafish and mammalian retinal ganglion cells (RGCs). The principle aim of this study is to test whether activation of Piezo1 channels can produce neurodegeneration of zebrafish and mammalian RGCs.MethodsFor zebrafish Ca2+ imaging was performed using a genetically‐encoded Ca2+ indicator, GCaMP6f, and was targeted predominantly to RGCs using the Tg(elavl3: GCaMP6f) zebrafish line, and confirmed by backfilling the optic nerve with Texas Red hydrazide. In order to activate Piezo1 channels in RGCs, cytosolic [Ca2+]i changes were measured in response to application of the Piezo1 selective agonist, Yoda1. Immunoblotting and immunohistochemical localization for Piezo1 were used to determine the expression profile in rodent and zebrafish retina using retinal wholemounts and vertical sections. To examine apoptosis, a short‐term organotypic eyecup culture preparation was used. Eyecups were incubated in oxygenated Ringer’s solution (for 1–8 hrs) with various concentrations of Yoda1 (0.1–100μM). Following treatment with Yoda1, eyecups were fixed, sectioned, and prepared for immunohistochemistry. TUNEL assays was used to label apoptotic cells in fixed vertical retinal sections, and TUNEL‐positive cells were quantified using ImageJ software. All imaging was acquired on a Nikon C2+ confocal microscope.ResultsCalcium imaging experiments revealed that Yoda1 evokes a dose‐dependent activation of Piezo1 ion channels in GCAMP6f expressing RGCs, which was reduced in the presence of GsMTx‐4, a mechanosensitive channel antagonist. Immunohistochemical findings show strong Piezo1 immunoreactivity in RGCs and their axons in rodent and zebrafish retina. Moreover, Yoda1 evoked a concentration‐dependent increase in TUNEL‐positive labeling of RGCs in both zebrafish and rodent retina, implicating a common Ca2+‐mediated pathway for Piezo1 channel induced RGC death.ConclusionsThis study provides the first evidence for Piezo1 channel‐mediated RGC death and suggests that there is a potential link between Piezo1 channel activation on RGCs, increased [Ca2+]i influx and apoptosis. Therefore, changes in cell stiffness and pressure acting through Piezo1 mechanosensitive channels could contribute to the neurodegenerative effects observed in optic neuropathies, such as ocular injury and glaucoma.Support or Funding InformationHMC CURE Zebrafish Pilot Grant, Penn State University‐Hershey College of Medicine; Pennsylvania Department of Health, Tobacco CURE Funds
Yes‐associated protein (YAP) in Kupffer cells enhances the production of pro‐inflammatory cytokines and promotes the development of non‐alcoholic steatohepatitis
Authors: Song Kyoungsub , Kwon Hyunjoo , Han Chang , Zhang Jinqiang , Chen Weina , Ma Wenbo , Wu Tong 
Published: April 2020

Background & AimsYes‐associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non‐neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of pro‐inflammatory cytokines and promotes the development of non‐alcoholic steatohepatitis.MethodsWe generated mice with Kupffer cell (KC)‐specific deletion of YAP (YAPφKO) or TLR4 (TLR4φKO) and the animals were fed high fat diet (HFD) or treated with LPS. The liver tissues were harvested and subjected to histopathological and biochemical analyses. Kupffer cells were isolated for in vitro analyses. Chromatin immunoprecipitation, DNA pull‐down, qRT‐PCR and promoter reporter activity assays were performed to validate transcriptional regulation of YAP and inflammatory cytokine production in Kupffer cells/macrophages. Analyses of liver tissues from NASH patients were performed using immunofluorescence imaging and Gene Expression Omnibus (GEO) datasets.ResultsThe expression of YAP is significantly increased in KCs of wild type mice fed HFD. The YAPφKO mice fed HFD showed lower serum ALT/AST levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced the accumulation of YAP in KCs in vitro and in mice which was prevented by KC‐specific deletion of TLR4 (TLR4φKO). LPS transcriptionally activates YAP via AP‐1 in macrophages/KCs. The LPS‐induced YAP further enhances the expression of pro‐inflammatory cytokines (including MCP‐1, TNF‐α and IL‐6) through YAP association with the TEAD‐binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of pro‐inflammatory cytokines. Treatment of HFD‐fed mice with the YAP inhibitor, verteporfin, prevented Kupffer cell activation, reduced liver inflammation and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that the level of YAP in human liver tissues is positively correlated with the expression of pro‐inflammatory cytokines.ConclusionsThis study describes an important role of YAP in Kupffer cells for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent a novel and effective therapeutic strategy for NASH treatment.
Female Offspring of Hyperandrogenemic Female (HAF) Rat Model Exhibit Insulin Resistance and Increased Blood Pressure With Aging
Authors: Shawky Noha M. , Vinson Ruth M. , Zuchowski Yvonne P. , Reckelhoff Jane F. 
Published: April 2020

BackgroundPolycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia, oligo/an‐ovulation and polycystic ovaries. It is also associated with metabolic dysfunction and elevated blood pressure (BP). Whether daughters born to PCOS mothers exhibit a PCOS phenotype is controversial. In addition, whether daughters of PCOS women develop metabolic dysfunction and elevated BP with aging has not been reported.ObjectivesThe present study aimed to test the hypothesis that aging causes an increase in MAP and metabolic dysfunction in female offspring of HAF rats (model of PCOS).MethodsHyperandrogenemia was induced in Sprague Dawley (SD) females by implantation of 5α‐dihydrotestosterone pellets (DHT; 7.5 mg/90 days, subcutaneously) starting at 4 weeks of age. Rats were mated with SD males, and allowed to deliver and lactate. Female offspring (F1HAF) were weighed at birth, left untreated and studied at 4–6 (adult) or 16–18 (aging) months of age. Control dams were implanted with placebo pellets subcutaneously, allowed to mate, deliver and suckle their offspring (F1Placebo).ResultsDespite the similar litter size, body weight (BW) at birth was significantly lower in female F1HAF compared to F1Placebo (6.0 ± 0.1 vs 6.5 ± 0.1 g, n=13–15). As adults (4–6 months), BW, fat and lean masses, proteinuria and mean arterial pressure (MAP, radiotelemetry) of female F1HAF were not different compared to F1Placebo. With aging, female F1HAF rats showed increased MAP compared to F1Placebo (109 ± 1 versus 96 ± 1 mmHg, n=3–4). Fasting blood glucose was not different between groups (91 ± 3 vs 90 ± 2 mg/dl), but female F1HAF rats showed significant hyperinsulinemia (0.90 ± 0.05 vs 0.78 ± 0.03 ng/ml, p ≤ 0.05) and insulin resistance (­ HOMA‐IR index; 3.3 ± 0.2 vs 2.8 ± 0.1, p ≤ 0.05, (n = 5–6)). Despite the insulin resistance, lean mass (248.4 ± 8.5 vs 241.2 ± 3.9 g) and fat mass (41.2 ± 7.9 vs 39.7 ± 1.8 g) by Echo MRI, in addition to body weight (307.5 ± 16.2 vs 298.1 ± 6.4 g; (n = 2–4)) were also not different between groups.ConclusionAdult female offspring of hyperandrogenemic females do not show a PCOS phenotype (e.g. no hypertension, insulin resistance, metabolic phenotype), whereas with aging, they develop hypertension, hyperinsulinemia and insulin resistance with no changes in fasting glucose, lean/fat mass or body weight. These data suggest that women whose mothers had PCOS may be at increased risk for hypertension and metabolic dysfunction with aging, although the mechanisms for the hypertension are not clear.Support or Funding InformationR01HL135089, P01HL051971, P20GM121334, P20GM104357
Evidence for P2Y2 Receptor Facilitation of Hyperglycemia‐induced Insulin Resistance in Human Hepatocytes
Authors: Senfeld Jared I. , Shen Jianzhong 
Published: April 2020

It is known that deletion of the ectonucleotidase CD39 leads to liver insulin resistance in mice; however, it remains unknown whether this phenotype is due to a lack of extracellular adenosine generation or an excessive nucleotides accumulation and signaling. We hypothesized that the nucleotide P2Y2 receptor mediates insulin resistance in human hepatocytes. RT‐PCR showed that among the eight known P2Y receptors, only the P2Y2, P2Y6 and P2Y11 mRNAs were detected in cultured HepG2 cells, a human hepatocytes model; however, stimulation of the cells with P2Y6‐ and P2Y11‐selective agonists all failed to induce intracellular Ca2+ mobilization, whereas both ATP and UTP induced significant Ca2+ signaling in a dose‐dependent manner, which was abolished by pretreatment of the cells with ARC‐118925XX, a selective and competitive P2Y2 receptor antagonist, suggesting that the P2Y2 receptor is the only functional P2Y receptor expressed in HepG2 cells. In addition, we found that stimulation of the P2Y2 receptor induced MAPK signaling including phosphorylated ERK1/2, JNK and p38, but not the AKT pathway. Interestingly, stimulation of the cells with ATP or UTP dose‐dependently blocked insulin‐induced AKT phosphorylation, but potentiated ERK1/2 signaling, indicating a selective disruption of the insulin‐AKT signaling axis by P2Y2 receptor. Furthermore, we found that a notable release of ATP into the extracellular space was observed when exposing HepG2 cells to high glucose concentrations (25mM) as compared with normal glucose (6.1 mM). Consistent with this, we also found that extracellular nucleotides signaling through P2Y2 receptor induced a dose‐dependent reduction in hepatocyte glucose uptake. We conclude that the P2Y2 receptor mediates liver insulin resistance in response to hyperglycemia, possibly through activation of the JNK pathway and inhibition of insulin signaling to the AKT pathway, highlighting that P2Y2 receptor may be a new drug target for type‐2 diabetes.Support or Funding InformationSupported in part by NIH funding 1R01HL125279‐01A1 (JS)
Time Restricted Feeding Reduces Vascular Damage and Oxidative Stress in Male Mice on a Chronic High Fat Diet
Authors: Pati Paramita , Zhang Dingguo , Colson Jackson , Allan John Miller , Sedaka Randee , Gamble Karen L. , Bailey Shannon M. , Pollock David M. , Pollock Jennifer S. 
Published: April 2020

Irregular timing of food intake is known to influence risk for cardiometabolic disease. Studies in humans and animals demonstrate that a chronic high fat diet (HFD) leads to endothelial dysfunction and cardiovascular disease. We hypothesized that restricting food availability to the active period with a HFD leads to reduced vascular damage, increased nitric oxide synthase (NOS3) activation, and reduced oxidative stress. Mice (male 8‐week old, C57BL/6J) were given a normal diet (ND; 10% fat) or HFD (45% fat) for 20 weeks ad libitum. Time restricted feeding (TRF; food availability only during the active phase between ZT12 to ZT0 or 7 PM to 7 AM) was enforced in half of the HFD mice during the final 2 weeks of feeding. At the end of the study, aortae and plasma were collected at ZT5 and ZT17 in the middle of the inactive and active periods, respectively. 24 hour food intake was similar between ad lib and TRF mice. Aortic wall thickness was significantly increased in mice on HFD compared to ND (HFD: 76±5.1 μm; ND: 59±4.6 μm; n=6, p=0.03). Interestingly, aortic fibrosis was reduced by TRF in HFD mice (HFD: 18±2.3%; HFD+TRF: 11±1.9%; n=6, p=0.04). To assess NOS3 activation, Western blotting was used to measure pNOS3(S1177) and NOS3 abundance in aortae. At ZT5 pNOS3(S1177) abundance was not affected by TRF in HFD mice (HFD: 0.5±0.3 rdu, HFD+TRF: 0.7±0.1 rdu, n=4). TRF significantly increased pNOS3(S1177) abundance in HFD mice at ZT17 (HFD: 0.5±0.2 rdu, HFD+TRF: 2.1±0.6 rdu, n=4, p=0.06). Total NOS3 expression was unaffected by TRF at both time points. During the inactive period, endothelial‐dependent relaxation to acetylcholine was significantly impaired in HFD mice and improved by TRF (ND: 93±1.4%; HFD: 57±14%; HFD+TRF: 79±4.0%, n=5–11, ND vs HFD: p=0.01). Endothelial function was not different between groups in the active period. Endothelial‐independent relaxation was similar in all groups in both inactive and active periods. Plasma nitrite, a metabolite of NO, at ZT5 was unchanged between all groups. At ZT17 plasma nitrite was significantly decreased in HFD mice compared to ND mice and unaffected by TRF (HFD: 0.6±0.06 μM; HFD+TRF: 0.7±0.03 μM; ND: 1.1±0.2 μM; n=10–11, p=0.03). 8‐isoprostane, an indirect measure of oxidative stress, was significantly reduced by TRF in HFD mice at ZT5 (HFD: 159±31 ng/mL; HFD+TRF: 60±12 ng/mL, n=4–6, p=0.01), but not different between groups at ZT17. In conclusion, these results demonstrate that restricting timing of food intake improves vascular damage and decreases oxidative stress following diet‐induced obesity in a time‐dependent manner.Support or Funding InformationSupported by T32 DK007545, an AHA Postdoctoral Fellowship (18POST34070051) to PP, an AHA Predoctoral Fellowship (18PRE33990345) to DZ, P01 HL069999 to JSP and DMP, and P01 HL136267 to JSP, EWI, and DMP.
Cortical and trabecular bone microstructural parameters and dietary stable isotopes throughout ontogeny
Authors: Beresheim Amy Catherine , Singh Atul K. , Grynpas Marc D. 
Published: April 2020

IntroductionRecent research has revealed correlations between δ15N, an indicator of dietary protein sources, and measures of intracortical and endosteal bone remodeling in adults. Given that these isotopes are used to infer the prevalence and duration of breastfeeding in past populations, it may be possible to specifically target bone microstructural variation associated with early life nutrition. It is critical to develop a more robust understanding of skeletal responses to weaning and infant diet, as they ultimately influence final body size proportions, skeletal fragility, and osteoporosis risk.Materials and MethodsThe sample is 27 mid‐sternal rib bone sections, each representing one child (0–16 years of age) from the Wesleyan Methodist cemetery of Prospect Hill, Newmarket, ON (1824–1879 AD). All specimens were scanned using the SkyScan 1174 Micro‐CT system at the Lunenfeld‐Tanenbaum Research Institute, Toronto, ON. Quantitative parameters were derived using SkyScan CT‐analyser software. Carbon and nitrogen stable isotope ratios from bone collagen were previously reported (Katzenberg and Pfeiffer1995).Spearman’s rank order correlation coefficients were calculated to explore relationships between each bone microstructural variable and age‐at‐death. Partial corrections, controlling for age, were used to examine the relationships between bone microstructural variables and δ13C and δ15N.ResultsCortical bone variables relating to cross‐sectional size increase from birth to late adolescence. With age, bone volume fraction (BV/TV), trabecular number (Tb.N) and connectivity density (Conn.Dn) decrease, while trabecular spacing (Tb.Sp) increases. Relationships between bone microstructural variables and δ13C or δ15N values are not straightforward.Discussion and ConclusionOntogenetic patterns of microstructural variation in the rib are similar to those of other bones undergoing endochondral ossification. Linkage to characteristics of infant and childhood dietary protein may be mitigated by causes of death.
Academic Predictors of MCAT performance for Post‐baccalaureate Pre‐medical program students
Authors: Dobrydneva Yuliya , Ganjoo Rohini , Schwartz Lisa 
Published: April 2020

Career changers, i.e. students who did not major in the traditional pre‐medicine fields like chemistry and biology, presently constitute a large percentage of medical school applicants. Post‐baccalaureate pre‐medical programs (PBPMP) provide prerequisite coursework for non‐life science majors who aspire to become physicians. Curriculum includes physics, general and organic chemistry, biology and biochemistry. Students entering these programs generally do not have significant exposure to natural sciences, and are considered “scientifically naïve”. Therefore, post‐baccalaureate programs are searching for criteria for admission process to help identify applicants with potential to succeed in PBPMP and matriculate into medical schools.This study analyzed academic metrics of career changer pre‐medical students to predict their success on mandatory Medical College Admission Test (MCAT). Data analysis correlated MCAT scores with gender, high school Scholastic Aptitude Test (SAT) scores, undergraduate major or field of study, and undergraduate Grade Point Average (GPA). Undergraduate GPA is not correlated with performance on MCAT. Analysis yielded positive correlations between MCAT scores and SAT scores, and between MCAT and undergraduate non‐science major/field of study. The results provide insight into academic metrics that could predict future success on MCAT and matriculation into medical school. While positive correlations between certain sub‐scores on the SAT and MCAT were found, other holistic non‐quantitative characteristics should be considered for medical school admission.