bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of UBR5 on Mitogen‐activated protein kinase (MAPK) signalling and muscle mass regulation in mice
Authors: Hughes David C. , Baehr Leslie M. , Sharples Adam P. , Bodine Sue C.
Published: April 2020

E3 ubiquitin ligases are commonly studied in skeletal muscle under atrophy‐inducing conditions. However, we recently identified a novel E3 ubiquitin ligase, UBR5 to be involved in skeletal muscle hypertrophy and recovery from atrophy (
Seaborne et al., 2019). Previous literature suggests an interaction between UBR5 and the MAPK signalling cascade (ERK 1/2, p90RSK) in various other tissues. Thus, we sought to investigate the role of UBR5 on MAPK signalling in basal skeletal muscle.

Male C57BL/6 mice (12‐week old) were subjected to in vivo electroporation with a mouse specific UBR5 RNAi plasmid (20ug) in the tibialis anterior (TA) muscle with the contralateral leg serving as a vector control (EV). After 7 days, TA muscles were collected, weighed and sectioned for cross‐sectional area (CSA) and GFP visualization. Western blotting was performed to determine phosphorylation of ERK 1/2, p90RSK, MEK1/2 and MSK1 signalling levels in the transfected TA muscles.

After 7 days, muscles transfected with the UBR5 RNAi displayed significant increases in muscle mass. Alongside a 50% reduction in UBR5 protein, we observed a significant reduction in phosphorylation of ERK 1/2 and ~2‐fold increase in p90RSK phosphorylation in RNAi transfected muscles compared to the EV. There were no differences observed in the phosphorylation status of MEK1/2 or MSK1 levels.

Gene manipulation of UBR5 in basal skeletal muscle lead to alterations in muscle mass and activation of the MAPK signalling pathway. Future studies will seek to explore the effect of chronic UBR5 knockdown on muscle mass regulation.
Revealing the sequence determinants of SMYD1 lysine methyltransferase substrate selectivity with lysine‐oriented peptide libraries
Authors: Spinetti Eric
Published: April 2020

Lysine methylation is a protein post‐translational modification (PTMs) that can affect protein stability, activity, subcellular localization, and molecular interactions. Revealing protein substrates for lysine methyltransferases (KMTs) will help connect enzymatic activity to cellular functions and pathologies associated with these enzymes. Toward this goal, we developed a high‐throughput screening assay to define the sequence determinants of KMT substrate selectivity. This assay queries a highly degenerate lysine‐oriented peptide library (K‐OPL) to identify amino acid preferences at three resides N‐ and C‐terminal to a fixed central lysine. Here, we demonstrate the utility of this approach to facilitate identification of several novel substrates of KMT activity, including for SMYD1 (SET and MYND domain‐containing protein 1), a KMT expressed in cardiac and skeletal muscle that is essential for heart development and organ homeostasis. We further demonstrate the utility of this tool, in conjunction with in silico and biophysical approaches, for revealing the impact of a cardiac disease‐associated mutation of SMYD1 on its KMT function. Collectively, our studies reveal new hypotheses toward a better understanding of SMYD1 function in cardiac and skeletal muscle development and implicate its aberrant KMT activity in heart disease.

Support or Funding Information

NIH R356M14736
A Structure‐and‐Function‐Based Approach to Target Ryanodine Receptor 2 for Heart Failure
Authors: Sun Qing , Roosild Tarmo , Bobkov Andrey , Ma Chen-Ting , Holleran John , Sergienko Eduard
Published: April 2020

Ryanodine receptor 2 (RYR2) controls transient calcium release (calcium spark) from the sarcoplasmic reticulum (SR) into the cytoplasm in cardiomyocytes. It is an essential part of the calcium handling machinery which drives cardiac contraction. In pathological conditions caused by hyperphosphorylation or mutation, RYR2 can become leaky, resulting in cytosolic calcium overload and abnormal calcium sparks, manifesting as loss of cardiac contractility, arrhythmia and cardiac hypertrophy, leading to cardiomyocyte injury and death. This is a key dysfunction underlying heart failure, a disease that has a five‐year mortality rate of over 50% and affects 26 million people worldwide. Current standard‐of‐care drugs alleviate symptoms, but do not directly address pathophysiology of the disease. Efforts in targeting RYR2 in the past have met considerable challenges, given the extremely large size of this tetrameric calcium channel, its complex structure and regulatory mechanisms involving a multitude of binding partners and modifiers. There is a clear unmet need for direct modulators of RYR2 able to suppress excessive calcium transport caused by hyperphosphorylation or mutations.

Here we search for modulators of RYR2, which can reduce calcium leak from the SR, restore calcium homeostasis and normalize afterdepolarization in the cardiomyocyte. We utilized a twotier approach, taking advantage of protein thermal shift (PTS) assays to identify initial hits that bind to the protein and relying on cell‐based assays to establish the functional outcome of the binding. To overcome the large size of RYR2, we designed protein constructs corresponding to various domains with hotspots of pathogenic mutations and hyperphosphorylation, as well as binding sites of key regulatory proteins. We successfully produced six soluble domains or domain complexes and developed PTS assays for five of them. In a proof of concept study, four of these domains were utilized for pilot screening with a twenty‐thousand compound collection. The hits for each domain were profiled in PTS assays on all four domains, and select representatives were reconfirmed in isothermal titration calorimetry assays.

The second tier of the approach utilized RYR2‐expressing cells. We built cell‐based assays to monitor cytosolic and endoplasmic reticulum (ER) calcium basal levels and transients using the HL‐1 mouse cardiomyocyte cell line and an RYR2‐expressing HEK‐293 cell line. We then validated the assays using known RYR2 modulators. Pathophysiological cell models are being established that carry hyperphosphorylation on S2814 and S2808 on the RYR2 protein, which are the key abnormalities causing SR calcium leakage in diseased cardiomyocytes. Our goal is to identify RYR2‐binding compounds that restores intracellular calcium in these hyperphosphorylated cells to wildtype levels.
Branch‐Chain Interpenetration of the Mesothelial Glycocalyx with Naturally‐Occurring Biopolymers
Authors: Zheng Yifan , Pierce Aidan , Wagner Willi , Servais Andrew , Ackermann Maximilian , Mentzer Steven
Published: April 2020

The mesothelial surface layer of the lung, heart, bowel, and liver is a dynamic epithelial tissue that regulates a variety of physiologic processes including the healing of visceral organ injuries. A major barrier to addressing these injuries is the slippery and nonadhesive surface of visceral organs, which consists of a carbohydrate‐rich glycocalyx. The glycocalyx is difficult to study, however, because traditional fixation methods destroy this carbohydrate layer. To define the mesothelial glycocalyx, we utilized a high pressure (2100 bar) and rapid freezing technique of the pleura. Examining the glycocalyx with scanning electron microscopy, we demonstrated that the pleural glycocalyx is more than 100‐times thicker than previously realized (more than 12 um) and is notable for its branch‐chain polysaccharide structure. The unique branch‐chain structure of the mesothelial glycocalyx is reminiscent of the plant structural heteropolysaccharide called pectin. Pectin has a branch‐chain polysaccharide structure that appears to mirror the glycocalyx. When 35 mm circular pectin films were applied to porcine mesothelium, the pectin films appeared to fuse with the mesothelial surface. Our hypothesis is that pectin adhesion to mesothelium is via branch‐chain entanglement. Quantitative adhesive strength testing demonstrated that pectin was significantly more adhesive to the mesothelial surface than linear‐chain polysaccharide polymers (p<0.0001). The work of adhesion between pectin and mesothelial glycocalyx was measured to be 80 to 90 N*sec while the work of adhesion measured between linear‐chain polymers and mesothelium was 10 to 20 N*sec. Transillumination stereomicroscopy of the interaction between two branch‐chain polymers demonstrated entanglement of the polymer chains, while stereomicroscopy of the branch‐chain polymer with the linear‐chain polymer or two linear‐chain polymers demonstrated no entanglement. We conclude that the effective binding of pectin to the mesothelial glycocalyx is the result of reciprocal branch‐chain interactions between these structural polysaccharide polymers. We anticipate that the adhesivity between branch‐chain polymers will provide important experimental and therapeutic opportunities in the study of the mesothelium.
Simulated remote ischemic preconditioning inhibits Smad2 and enhances post‐hypoxic autophagy and survival of H9c2 cells
Authors: Xie Xiang , Xue Jianjun , Pan Qixian , Xu Ziqing , Zhang Dengwen , Ge Zhidong , Lin Han , Cai Yin , Wu Lianpin , Xia Zhengyuan , Wang Sheng
Published: April 2020

Background

Cardiomyocyte apoptosis is considered as one the major forms of cell deaths of myocardial ischemia/reperfusion (I/R) injury. Remote ischemic preconditioning (RIPC) protects the cardiomyocytes from I/R injury that involves the enhancement of autophagy, but the underlying mechanism and especially the relationship with apoptosis has not been delineated. Smad2 is an important autophagy regulatory protein, but its role in RIPC is unknown.

Methods

Rat cardiac myoblast H9c2 cells hypoxia/reoxygenation (H/R) injury model was established with 6 hours of hypoxia followed by 12 hours of normoxia reoxygenation to mimic I/R injury. The cells in the conditioning group were exposed to 3 cycles of 10 minutes of alternating nitrogen‐flushed hypoxia and reoxygenation as stimulated RIPC (sRIPC) 24 hours before inducing prolonged H/R. The cell viability, oxidative damage, inflammatory markers, expressions of apoptosis and autophagy‐related proteins were assessed.

Results

H/R caused significant cell injury in untreated control H9c2 cells that was associated reduced autophagy (reduction in the ratio of LC3‐II to LC3‐I) and sRIPC enhanced post‐hypoxic cell survival, reduced reactive oxygen species formation and oxidative damage manifested as increased lipid peroxidation product Malondialdehyde (MDA), inhibited Smad2 transcriptional repressor activity, activated Beclin‐1 and autophagy but reduced apoptotic cell death (all p < 0.05).

Conclusions

It is concluded that inhibition of Smad2 and the subsequent activation of autophagy may represent a mechanism by which sRIPC attenuate post‐hypoxic cardiomyocyte injury.

Support or Funding Information

Funding: This study was supported by grants NSFC(81800245 and 81670770), and in part by Health and Medical Research Fund (HMRF, 05161826)
p‐Ezrin (Thr567) regulates Hippo pathway and Yap in liver
Authors: Xue Yuhua , Bhushan Bharat , Mars Wendy M. , Bowen William , Tao Junyan , Orr Anne , Stoops John , Yu Yanping , Luo Jianhua , Duncan Andrew , Michalopoulos George K.
Published: April 2020

It has been shown that up ligation of CD81 (the portal of entry of Hepatitis C virus) by agonistic antibody results in phosphorylation of Ezrin. We have previously shown that in liver, Ezrin phosphorylation occurs via Syk kinase, causing suppression of Hippo pathway, therefore increasing Yap activity. The opposite occurs when Glypican‐3 (GPC3) or E2 protein of HCV bind to CD81. Mice over‐expressing GPC3 in hepatocytes have decreased p‐Ezrin(Thr567) and Yap, increased Hippo activity and suppressed liver regeneration. The role of Ezrin in these processes has been speculated, but not proven. We now provide dynamic picture of how Ezrin regulates Hippo pathway and Yap. Forced expression of plasmids expressing mutant Ezrin (T567D) (which mimics p‐Ezrin(Thr567)) suppressed Hippo activity and activated Yap signaling. And this mutant Ezrin drive more cell proliferation to cell division through up regulated Yap activity in vitro and in vivo. CD81 loses expression, while p‐Ezrin(Thr567) increases in JM1 and JM2 hepatocellular carcinoma (HCC) cells. Administration of compound NSC668394, a characterized p‐Ezrin(Thr567) antagonist, caused significant decrease in HCC cell proliferation. We additionally present evidence that pEzrin(T567) is also controlled by EGFR and MET.

Conclusions

Ezrin phosphorylation, mediated by CD81 associated Syk kinase, is directly involved in regulation of Hippo pathway, Yap levels and growth rates of normal and neoplastic hepatocytes. The finding has mechanistic and potentially therapeutic applications in understanding and regulating growth of hepatocytes and HCC and HCV pathogenesis.
MET and EGFR signaling pathways play different roles in bile duct ligation (BDL) mice
Authors: Xue Yuhua , Mars Wendy M. , Bowen William , Orr Anne , Stoops John , Michalopoulos George K.
Published: April 2020

Liver failure is the common ending of most irreversible liver diseases. We have identified that shutdown of both MET and EGFR signaling simultaneously in the murine resting or regenerating liver contributes to this process. In this study, we explore the role of MET and EGFR signaling pathways in biliary injury repair after common bile duct ligation. Mice with combined disruption of MET and EGFR signaling became very ill by 24 hours and died at 1 to 2 days after bile duct ligation. Mice with elimination of EGFR signaling alone showed decreased hepatocyte proliferation, cholangiocyte proliferation, and ductular reaction at 7 days. However, Met knockout mice showed increased proliferation of cholangiocytes and hepatocytes. Mice with elimination MET signaling alone became very ill since 7 days. Furthermore, either Met or EGFR single inhibition mice showed less liver to body ratio at 5 and 7 days time points. We also observed p‐Ezrin (Thr567) is down regulated in the either Met or EGFR deletion mice, while the total Ezrin expression did not change.

Conclusions

Our study demonstrates that, in addition to maintaining the baseline liver homeostasis together, MET and EGFR differentially control liver injury response in BDL model, and their combined disruption leads to acute liver failure and early death following severe biliary injury. The receptor tyrosine kinases MET and EGFR appear to play different roles in the biliary injury‐repair responses.
Maternal dexamethasone exposure suppresses placental iron transport through GR‐mediated TFR1 pathway in rats
Authors: Guo Shihui , Yan Kai , Jiang Shuxia , Ni Yingdong , Zhao Ruqian , Ma Wenqiang
Published: April 2020

Iron is one of the essential trace elements for almost all living organisms, and of course it is especially important for pregnancy and fetal development. However, inappropriately low or high levels of iron during pregnancy are detrimental and contribute to serious and lasting effects on the fetus, such as fetal growth and development disorders, thereby increasing the risk of adulthood. Here we stress pregnant rats by drinking water with 10−7 mol/L dexamethasone, and then explore its effects on iron metabolism of placenta and fetal rats. The results showed that dexamethasone treatment inhibited placental development, down‐regulated the expression of placental iron transporter TFR1, and decreased iron levels in fetal rats. Furthermore, we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking inhibit of TFR1 under Dex challenge, our results demonstrate that the decrease in TFR1 caused by dexamethasone is alleviated by the addition of dexamethasone inhibitor RU486. Taken together, our results show that dexamethasone downregulate TFR1 expression adjust iron metabolism via GR‐mediated.

Support or Funding Information

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0500502), the National Natural Science Foundation of China (31872439), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.
Leukocytic Thy‐1 Expression Differs in Kidney Cortex and Medulla in Spontaneously Hypertensive Rats
Authors: Meariman Jacob K. , Sutphen Jane , Denys Ian , Harrison David G. , Kapusta Daniel R. , Gao Juan
Published: April 2020

Sympathoexcitation and increased inflammation are both involved in mediating hypertension, and decreasing sympathetic nerve activity is vital for reducing inflammation in this disease. However, the mechanism(s) by which the nervous system communicates with the immune system in hypertension is still largely unknown. To examine this interaction in this study, twenty‐week‐old spontaneously hypertensive rats (SHR) underwent renal denervation (RDN) via radiofrequency ablation of the renal artery. After two months, kidneys were harvested and separated into cortex and medulla, and lymphocytes were detected by flow cytometry. RDN significantly decreased CD4+ T cells, CD8+ T cells, and B cells in the renal medulla but had no effect on these immune cells in the cortex. In a separate group of intact SHR, Thy‐1, a reported activator of T cells, was measured in leukocytes isolated from the renal cortex and medulla. Thy‐1 mRNA expression was significantly increased in T cells, non‐T cells, CD45+ cells, and CD45− cells from the leukocytes isolated from the cortex compared to those isolated from the medulla. These findings suggest that Thy‐1 in cortical leukocytes might activate the immune system in the renal cortex independent of the renal nerves. Downregulation of Thy‐1 in the renal cortex could increase the benefit of RDN and decrease renal inflammation in hypertension.

Support or Funding Information

NIH NIGMS P30 GM106392
Structural defect leads to human severe (Class I) loss of function in glucose‐6‐phosphate dehydrogenase
Authors: Horikoshi Naoki , Hwang Sunhee , Gati Cornelius , Matsui Tsutomu , Castillo-Orellana Carlos , Garcia Adriana A. , Raub Andrew G , Jabbarpour Fatemeh , Mochly-Rosen Daria , Wakatsuki Soichi , Vöhringer-Martinez Estevan
Published: April 2020

Oxidative stress caused by infection, medication, food, and imbalance of metabolic cycles damages DNA and organelles, which may lead to cancer, blood disorders, and other serious diseases. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate‐limiting enzyme in the pentose phosphate pathway, which is essential for nucleotide, fatty acid, cholesterol, and hormone synthesis. In addition to those roles, G6PD reduces NADP+ to NADPH, which is crucial for reducing reactive oxygen species. Dysfunction of G6PD increases susceptibility to oxidative stress, especially in erythrocyte due to the lack of mitochondria, which is another source of NADPH. Interestingly, 400 million people worldwide have mutations on the g6pd gene, and the World Health Organization (WHO) has classified more than 160 missense mutations into five classes. The Class I G6PD deficiency is the most severe form; patients have less than 10% enzymatic activity of wild‐type G6PD and suffer from chronic non‐spherocytic hemolytic anemia. Here, we provide a new insight into the loss of activity in G6PD deficiency, based on the structure of the Class I pathogenic mutants of G6PD. We will discuss the structural basis of the Class I mutants of G6PD and its implications to various symptoms in G6PD deficiency.

Support or Funding Information

National Institutes of Health, R01 grant, HD084422; Japan Society for the Promotion of Science KAKENHI, Grant‐in Aid for Research Activity Start‐up, JP19K23713; University of Tsukuba; Stanford University; SLAC National Accelerator Laboratory
The Effect of Habitual Cannabis Smoking on Arterial Stiffness and Vascular Endothelial Function: A Preliminary Analysis
Authors: Cheung Christian P. , Millar Philip J. , Burr Jamie F.
Published: April 2020

Cannabis ranks globally as the most commonly used recreational drug, and both recreational and medicinal use have recently been legalized in numerous states and countries. The most common means by which cannabis is consumed recreationally is via smoking. Despite its prominent global use, a thorough understanding of the chronic cardiovascular effects of habitual consumption via smoking is lacking. Previous investigations have identified an association between cannabis use and accelerated vascular aging, and rodent models demonstrate impairments in vascular endothelial function following exposure to second‐hand cannabis smoke. Given these findings, it is likely that habitual cannabis smoking by humans results in central artery stiffening and/or decrements in vascular endothelial function; outcomes which are established risk factors for cardiovascular disease. The present work compared the carotid‐femoral pulse wave velocity (cfPWV) and brachial artery reactive hyperemia flow‐mediated dilation (FMD) of habitual cannabis users (CU) and age/anthropometrically matched non‐users (NU). CU were defined by cannabis use a minimum of once per week for ≥3yr. cfPWV was measured using arterial pulse wave tonometry. The FMD protocol included a 1‐minute baseline period, a 5‐minute occlusion of the forearm, and a 3‐minute reperfusion period. FMD is reported as peak change in vessel diameter. CU (n=10, 7M, 3F) and NU (n=10, 6M, 4F) did not differ in age (CU: 21.7±0.4, NU: 21.5±1; p=0.9) nor BMI (CU: 24.2±0.6kg/m2, NU: 23.4±0.4kg/m2, p=0.6). Systolic blood pressure (CU: 111.8±6.8mmHg, NU: 113.3±2.5mmHg, p=0.7), diastolic blood pressure (CU: 68.3±1.4mmHg, NU: 70.3±2.4mmHg, p=0.5), and supine resting heart rate (CU: 65.6±1.8bpm, NU: 66.0±2.8bpm; p=0.9) were similar between groups. Peak FMD (CU: 5.9±0.9%, NU: 6.8±1.1%; p=0.6) was also similar between groups, as was 60 second shear rate area‐under‐curve (CU: 4547±510, NU:4450±572; p=0.9). Peak change in blood velocity (CU: 38.1±3.6cm/s, NU: 36.7±2.0cm/s; p=0.7) and peak change in blood flow (CU: 307±51mL/min, NU: 253±33mL/min; p=0.4) during the FMD test were similar between groups, indicating no differences in microvascular dilatory capacity. cfPWV was higher in CU than in NU (CU: 6.0±0.2m/s, NU: 5.4±0.2m/s; p=0.05). Although no differences in vascular endothelial function were observed in this analysis, an increased sample size will be necessary to confirm these findings. The present data does indicate that habitual cannabis smoking may be associated with increased arterial stiffness.

Support or Funding Information

This work was supported by the Natural Sciences and Engineering Research Council of Canada under Grant 03974.
A novel imaging tool; fluorescent organosilica nanoparticles
Authors: Fujiki Yuki , Yuasa Noriyuki , Nomura Eri , Hashino Kimikazu
Published: April 2020

The new type of silica nanoparticles as a novel imaging tool, organosilica nanoparticles, has been developed by Nakamura and colleagues. The nanoparticles can be synthesized in a few of simple reactions and have a good size and dispersibility. Organosilica nanoparticles contain fluorescent dyes and are superior in fluorescence intensity to the conventional dye. These nanoparticles have SH groups on their surface, which makes it possible to conjugate them with antibodies and other protein molecules. We synthesized organosilica nanoparticles whose diameter is 100 nm including fluorescein isothiocyanate (FITC) or Rhodamine B and then conjugate these nanoparticles with an anti‐mouse IgG antibody and so on. We applied these conjugated antibodies to immunofluorescence staining to mouse brain sections. We also discuss this novel imaging tool to detect many biomolecules.
The Photoreceptor Cells in Retina and Pineal Gland of Amphibian Hoplobatrachus rugulosus
Authors: Liao Meng-Lin , Lin Tzu-Cheun , Chien Chung-Liang
Published: April 2020

Hoplobatrachus rugulosus, commonly referred to as tiger‐skinned frog, is to be chosen the animal model in this study. It has been found that both chicken α‐internexin and zebrafish inaa (an ortholog of mammalian α‐internexin) were developmentally distributed not only in photoreceptors of retina but also in the photoreceptor‐like cells of the pineal gland from our previous studies. However, from an evolutionary point of view, there is still lack of evidence to illustrate the temporal and spatial distribution of frog α‐internexin protein in the developing pineal gland. According to other studies about Xenopus laevis, the animal model that is usually used for amphibian developmental research, the mRNA sequence which encodes Xenopus α‐internexin had been identified as “xefiltin”. Although H. rugulosus is not the same species with X. laevis, both of them are classified in the same order. The aims of this study is to characterize the expression pattern of α‐internexin‐like proteins, internexin neuronal intermediate filament protein alpha S homeolog (ina.S) and low molecular weight neuronal intermediate filament S homeolog (nif.S) which might be expressed in the developing retina and the pineal gland of the tiger‐skinned frog. Thus, the specific antibodies were generated to detect the frog α‐internexin‐like proteins and to confirm the distribution of these proteins in the developing frog retina as well as the pineal gland by western blot and immunohistochemistry. Furthermore, the markers applied to detect the photoreceptor cell in the retina and the pineal gland are recoverin and XAP1, both of them are reported to be specific to photoreceptors. Based on the distribution patterns in frog retina, immunopositive staining of anti‐nif.S could be observed in the outer segment of photoreceptors, but anti‐ina.S could only be found in retinal neurons. In the pineal gland, both photoreceptor markers, recoverin and XAP1, could be detected. The α‐internexin‐like protein, ina.S and nif.S, could be detected only in a few photoreceptor‐like cells at the early stage of the frog pineal gland. In summary, the distribution patterns of photoreceptor cells in retina and in pineal gland of developing tiger‐skinned frogs has been characterized in this study. It can be concluded that XAP1 could be a good marker to identify the photoreceptor cells in the developing pineal gland of frogs.

Support or Funding Information

Grant Sponsor

Ministry of Science and Technology, Taiwan; Grant Number: MOST 108‐2312‐B‐002‐030‐MY2.
Role of Carbonic Anhydrase in the Regulation of Na‐H exchanger‐1 in Experimental Colitis
Authors: Khan Islam
Published: April 2020

A role of carbonic anhydrase (CA) was investigated in the regulation of Na–H exchanger‐1 (NHE‐1) in experimental colitis. Trinitrobenzenesulphonic acid (TNBS) was administered intra‐rectally to induce colitis in rats, and the animals were sacrificed on day 6 post TNBS. The isoforms CA‐I and CA‐II were the abundant isoforms in the colon but expressed as minor ones in the ileum. Both CA isoforms, on co‐immunoprecipitation and confocal microscopy, appeared to be colocalized with the NHE‐1 isoform in the colon. Importantly, inflammation reduced the expression of these proteins in the colon, but not in the ileum. The reduction in the level of co‐immunoprecipitated CA isoforms was more pronounced as compared to their level of expression in the inflamed tissue. Ex‐vivo treatment of uninflamed colonic strips by TNF‐α reduced the expression of CA isoforms. The binding site of the CA‐II isoform was identified in a peptide (83 aa) selected from the NHE‐1 C‐terminus. However, the CA‐I isoform did not bind with the same peptide. Since the tissues used in this study expressed inflammatory markers, the present data suggest that the uncoupling of CA and NHE‐1 is due to inflammation. This uncoupling would lead to a reduction in the NHE‐1 activity and an accumulation of H+ intracellularly, which together cause acidosis and necrosis in the inflamed colon in the present model.

Support or Funding Information

Research Sector, Kuwait University, Kuwait (Grant # MB03/15)
Blood Brain Barrier Permeability and Brain Capillary Endothelial Cell Tight Junctions in the Dahl S Model of Spontaneous Superimposed Preeclampsia
Authors: Maeda Kenji J. , Hoskins Glenn A. , Bidwell Gene L. , Garrett Michael R. , Sasser Jennifer M.
Published: April 2020

Previous studies in our lab have shown increases in brain water content and Evan’s Blue extravasation in the brains of pregnant Dahl salt‐sensitive (Dahl S) rats compared to Sprague Dawley (SD) rats. The goal of this study was to test the hypothesis that pregnant Dahl S rats display increased blood brain barrier (BBB) permeability and disruption of brain capillary endothelial cell tight junctions.

Female (20 week old) Dahl S and SD virgin and pregnant rats were used. Female rats were mated with males of the same strain, and gestational day 1 (G1) of pregnancy was defined by the detection of sperm histologically from a vaginal swab. On G20, rats were perfused with a 1.0% paraformaldehyde/2.0% glutaraldehyde solution and brains were collected for electron microscopy. Transmission electron microscopic images were used to assess the following parameters of brain capillaries: basement membrane status (% dissolved), basement membrane thickness, vesicle formation, tight junction status (% open), luminal membrane protrusions, and vessel diameter. In a separate group, rats were perfused with 5mg/kg of 3kDa Texas Red dextran (1 hour circulation) followed by 5mg/kg injection of 500kDa FITC (3 minute circulation) on G20, brains were collected, and 5μm sections from the posterior cortex were used for confocal microscopy. A ratio of extra‐:intra‐vascular Texas Red mean intensity values was determined to portray a percentage of Texas Red extravasation. All image analyses were conducted blindly.

Dahl S pregnant rats showed significant differences in basement membrane status (23% increase of dissolution) and vesicle formation (11±1 vs. 4±1) compared to SD pregnant rats (p < 0.01, 2way ANOVA for multiple comparisons – Tukey post‐hoc). Significant differences were observed in tight junction status among the groups (% open: SD Virgin = 3.03, SD Pregnant = 11.77, Dahl S Virgin = 11.77, Dahl S Pregnant = 26.47, p = 0.04, Fisher’s Exact Test of Independence). No statistical differences were found for measurements of basement membrane thickness, luminal membrane protrusions, or vessel diameter. Dahl S pregnant rats displayed a 17% increase in Texas Red extravasation compared to Dahl S virgin rats (p = 0.01, unpaired t‐test with Welch’s correction).

Our results indicate that pregnant Dahl S rats display increases in BBB permeability to low molecular weight fluorescent dextran and increased disruption of brain capillary endothelial cell tight junctions as well as decreased basement membrane integrity. In addition, the increase in vesicle formation may suggest increases in BBB permeability as it relates to transcellular transport across the BBB.

Support or Funding Information

KJM was supported by 5T32HL105324‐10 and 20PRE352000021. Work was supported by 1R01HL137673 to MRG and R01HL134711 to JMS.
Using substrate envelope to develop better HIV‐1 protease inhibitors.
Authors: Kerr Evelyn , Bhakta Meera , Hunter Jesi , Hernandez John , Stelluti Carolyn
Published: April 2020

Human Immunodeficiency Virus (HIV) is a latent virus which attacks the immune system. Inhibitors disrupting HIV‐1 protease, a reproductive enzyme of the HIV virus, are a vital part of the cocktail of drugs necessary for treatment. Protease often accumulates mutations which do not affect enzyme function yet repel the inhibitor. New protease inhibitors (PI), engineered to fit within the substrate envelope, approximate the structure of the protease’s natural substrates. This approach limits the inhibitor’s contact with enzyme moieties that can cause resistance. Saquinavir is an outdated PI which protrudes beyond the substrate envelope at several locations. When HIV‐1 protease at mutations Gly48Val, Ile84Val, or Val82Ala occur, the drug becomes completely ineffective. Darunavir, a modern PI which fits almost perfectly within the substrate envelope, binds to the enzyme 1000 times more tightly than Saquinavir. Seven enzyme mutations must accumulate to repel this inhibitor completely. Protease residues with the potential to confer resistance to Darunavir include Val32, Ile47, Ile50, and Ile84. The Snyder High School SMART Team has designed two models contrasting the interactions of two PIs, Saquinavir and Darunavir, with HIV‐1 protease. Using Jmol modeling software and 3D printing technology, the team render protease residues and inhibitor molecules to highlight similarities and differences between the drugs, thus presenting specific HIV‐1 protease structure‐function relationships. Understanding the molecular basis of protease resistance is critical to developing effective drugs for treatment of HIV and other diseases. Continued research on the structure and function of protease‐PI interactions may allow researchers to innovate more effective therapies.
Tumor cell survival in EYA4 knockdown cells exposed to cytotoxic drugs
Authors: Martinez Hannah , Dray Eloise V. , Sung Patrick M.
Published: April 2020

Eyes Absent 4 (EYA4) belongs to a family of protein phosphatases initially identified in Drosophila melanogaster as a compound of a network of transcriptional regulators associated with eye development. In the recent years, it has been demonstrated that EYA4 is deregulated in different types of cancer, acting as an oncogene in MPNST and as a tumor suppressor gene in esophageal adenocarcinoma, colon and colorectal cancer, hepatocellular carcinoma, lung cancer, acute myeloid leukemia and pancreatic cancer. Despite its association with different pathologies, the exact function of EYA4 is still unknown. A better understanding of the novel role of EYA4 in the maintenance of genome integrity and its implication in tumorigenesis and cancer development would help determining if EYA4 could be a new potential cancer therapeutic target. As part of this effort, colony formation assays have been performed after the exposure to different cytotoxic/genotoxic drugs, in order to study cell survival in EYA4 knocked down cells, and gain knowledge in sensitivity/resistant to chemotherapeutic agents. It was proven that EYA4 knockdowns and control has no resistance to MMC and MMS.
Microvascular Pachymeningeal Lymphatic Structures: Association with Blood Vessels and Cranial Bones
Authors: Glinskii Olga , Huxley Virginia , Xie Leike , Bunyak Filiz , Palaniappan Kannappan , Glinsky Vladislav
Published: April 2020

The role of cranial dura mater microvascular networks, as means for maintaining brain fluid movement and balance, and as the source of significant initiators and/or contributors to neurological disorders, has been underappreciated until recently. These networks consist of both blood and lymphatic vessels. The goal of this study was to uncover microvascular pachymeningeal lymphatic structures that are not associated with venous sinuses and investigate their interrelationship with the blood microvascular networks and cranial bones. Using markers specific for blood and lymphatic endothelia, we demonstrate the existence of the complex non‐sinus‐associated lymphatic microvasculature. We show the interrelationship and possible connections between lymphatic vessels and dural blood vessels. Furthermore, we unveil vascular connections between dural microvascular networks and cranial bone marrow sinusoids. Our novel findings reveal the presence of lymphatic‐like structures that exist on their own and/or in close proximity to microvessels. Of particular interest are sub‐sets of vascular complexes with dual (lymphatic and blood) vessel identity representing a unique microenvironment within the cranial dura. The close association of the systemic blood circulation and meningeal lymphatics achieved in these complexes could facilitate fluid exchange between the two compartments and constitute an alternative route for CSF drainage.

Support or Funding Information

This work was supported in parts by the NIH National Institute of Neurological Disorders and Stroke (R01NS110915), the NIH National Cancer Institute (R01 CA160461), and the U.S. Army Research Laboratory project W911NF‐18‐2‐0285.
Intestinal lncRNA H19 and miRNA‐675 expression Influenced by Metal Transporter ZIP14
Authors: Jimenez Felix R. , Ruggiero Courtney H. , Cousins Robert J.
Published: April 2020

Zinc has been shown to influence intestinal barrier function. To function properly, zinc must be placed at specific sites of action in cells. Previously, we demonstrated that the zinc transporter Zip14 (Slc39a14) is highly expressed in the small intestine and is localized to the basolateral membrane of enterocytes. Ablation of Zip14 in mice produces a phenotype that includes increased intestinal permeability and metabolic endotoxemia. To investigate the mechanism involved, we performed genome‐wide examination of gene expression in the small intestine from WT and Zip14 KO mice using Illumina RNA sequencing. This RNAseq analysis revealed that among 1079 long non‐coding (lnc) RNAs profiled, 36 annotated lncRNAs were identified as overexpressed in intestines of Zip14 KO mice. H19 RNA was the most highly overexpressed lncRNA. Read densities of other selected lncRNAs demonstrated specificity in the differential expression of H19 in the intestine of the KO mice. Upon confirmation using qPCR, we found that H19 was significantly up‐regulated in intestines of the Zip14 KO mice. Up‐regulation of miR‐675‐5p, a microRNA derived from H19, was also demonstrated. Intestinal epithelial cells (IEC) isolated by a chelation method revealed high expression of intestinal alkaline phosphatase and Zip14 mRNAs, indicative of substantial enterocyte‐enrichment. Expression of H19, but not miR‐675‐5p, was localized to the isolated IECs. Markedly enhanced expression of H19 was detected in IECs from the Zip14 KO mice compared to WT mice. The increased H19 expression in Zip14 KO mice was limited to IECs and skeletal muscle of the six tissues examined. IECs were also isolated from intestine‐specific Zip14 KO (I‐KO) mice. The up‐regulation of H19 was observed in IECs from the I‐KO mice compared to those from flox/flox controls, but miR‐675‐5p expression was not different between cells from the two genotypes. These data suggest that intestinal H19 is produced by IECs and is up‐regulated with Zip14 ablation. H19 overexpression suggests it is involved in a molecular defect in intestinal function of Zip14, perhaps including intestinal barrier dysfunction.

Support or Funding Information

NIH Grant R01 DK 094244
In‐silico Screening of ATP‐competitive Inhibitory Activities of Phytochemicals against Janus Kinase 3 and Its In‐vitro Evaluation
Authors: Kim Ryun-Hee , Lim Taehwan , Hwang Keum Taek
Published: April 2020

Janus kinases (JAKs) play a crucial role in development of alopecia areata (AA) by mediating cytokine signaling between hair follicular epithelial cells and T‐cells. Among JAKs, expression level of JAK3 is higher than those of the other JAKs in the skin of AA patients. Moreover, the amount of JAK3 increases during the time hair starts to fall out. Thus, inhibition of JAK3 can alleviate progression of AA. The purpose of this study was to predict in‐silico which phytochemicals were most likely to inhibit JAK3 and to evaluate inhibitory activities of the selected phytochemicals against JAK3 in‐vitro. By molecular docking with AutoDock Vina and sub‐databases (NuBBE, Biopurify Phytochemicals, and NPACT) from ZINC15, binding energies of 4975 phytochemicals against ATP‐binding sites of the JAKs were predicted. Only 24 out of 4975 phytochemicals were calculated to have lower binding energies against the ATP‐binding site of JAK3 than those of the other JAKs. The predicted binding energies of all these phytochemicals were also lower than that of tofacitinib (−8.7 kcal/mol), a well‐known JAK3 inhibitor. Among the 24 phytochemicals, poncirin (ZINC8234320) was calculated to have binding energy of −9.3 kcal/mol against the ATP‐binding site of JAK3. In addition, schisandrol A (ZINC3799118) was calculated to have higher binding energy (−4.7 kcal/mol) against the ATP‐binding site of JAK3 than ATP (−8.7 kcal/mol). Afterwards, inhibitory activities of tofacitinib, poncirin, and schisandrol A against JAK3 were tested by in‐vitro assay. Tofacitinib at 1 nM inhibited JAK3 activity by 40.3%, whereas poncirin at 1 nM inhibited JAK3 activity by 57.3%. Poncirin, which was predicted to have higher binding affinity toward JAK3 than tofacitinib, had higher inhibitory activity against JAK3 than tofacitinib at the same nanomolar level. Schisandrol A at 1 nM inhibited JAK3 activity by 7%. Schisandrol A inhibited JAK3 activity much less than tofacitinib at the same nanomolar level, which was coincident with the in‐silico result that schisandrol A was predicted to have lower binding affinity toward JAK3 than tofacitinib.
The Role of The DNA Damage Checkpoint in The Relocalization of CAG Trinucleotide Repeats to The Nuclear Pore Complex During S‐phase
Authors: Johnson Matthew , Whalen Jenna , Freudenreich Catherine
Published: April 2020

The instability of CAG trinucleotide repeats has been known to cause neurodegenerative diseases including Huntington’s disease and myotonic muscular dystrophy. Moreover, CAG trinucleotide repeats are capable of forming lesion inducing hairpin structures during the replication of DNA during S‐phase. These lesions must be repaired in order for DNA to be faithfully replicated and properly segregated during mitotic division. Failure to correct these lesions may result in deleterious mutations at these sequences. The DNA damage checkpoint elicits the first response to damage including double stranded breaks and collapsed replication forks resulting from CAG hairpin stalling. Notably, these collapsed forks are relocalized from the center of the nucleus to the nuclear pore complex (NPC) for repair. It is hypothesized that the DNA damage checkpoint plays a role in signaling relocalization of these lesions to the NPC during S‐phase.

To test this hypothesis, a GFP based zoning assay system was designed to assess CAG repeat relocalization to the NPC in several gene backgrounds defective in the DNA damage checkpoint response. These zoning assays have indicated that the DNA damage checkpoint does indeed play a role in the relocalization of CAG repeats during S‐phase. Several mutants tested also displayed abnormal nuclear arrangement. This research demonstrates that the DNA damage checkpoint is necessary for the relocalization of CAG tracts to the NPC which is known to be necessary for repair of persistent DNA lesions. Further studies will determine the mechanisms by which the checkpoint is involved in this relocalization and why abnormal nuclear arrangement is sometimes seen in mutants that are unable to elicit a proper checkpoint response.

Support or Funding Information

All support and funding is provided through the Tufts University Biology Department and specifically the Freudenreich Lab.
Competitive Counterflow (CCF) To Distinguish Between Transported And Non‐Transported Inhibitors Of MATE1 and MATE2‐K
Authors: Martinez-Guerrero Lucy J. , Wright Stephen H.
Published: April 2020

The MATEs (hMATE1 & hMATE‐2K) are membrane proteins expressed in the apical membrane of renal proximal tubule cells that mediate the flux of organic cations into the filtrate. In this study we set to test CCF as a method to identify substrates of pharmacological interest for both MATEs.

We first determined IC50 values for inhibition of MPP transport into CHO cells that stably expressed either MATE1 or MATE2‐K for several clinically important compounds: Amlodipine (IC50= 7‐6 μM), Atenolol (10‐4 μM), Dofetilidae (4‐0.8 μM), Entecavir (302‐140 μM), Lamivudine (336‐53 μM), Omeprazole (53‐8 μM), Propranolol, (16‐5 μM) and Trimethoprim (6‐0.4 μM). For CCF, cells were exposed to ~12 nM [3H]MPP for 40 min, sufficient to reach steady state. At time 0, the medium was removed and replaced with medium containing either ~12 nM [3H]MPP (control) or ~12 nM [3H]MPP plus one of the test compounds, or a known MATE substrate (e.g., Metformin); PAH (1mM) was used as a known non‐transported compound. After an additional 5 min incubation, radiolabeled MPP remaining in the cells was determined. If the extracellular test compound was transported, the associated turnover of the transporter should result in net loss of labeled MPP from the cells. Each test was performed (in sextuplicate) in 2–3 separate experiments.

Whereas exposure to 1mM PAH resulted in no loss of MPP from the cells (which was distributed to steady state), exposure to metformin resulted in net loss of radiolabeled substrate (70%). Of the test compounds, Atenolol, Amlodipine & Lamivudine supported a net loss of [3H]MPP (70‐30%) from both MATE1 & MATE2K‐expressing cells, whereas Entecavir reduced total accumulation of MPP only for MATE2K expressing cells. There was no loss (or gain) of MPP induced by Dofetilidae, Propranolol or Omeprazole, suggesting they are non‐transported inhibitors for both MATEs.

These data support the conclusion that the method of CCF is applicable for identification of MATE substrates, and identified a difference in the selectivity profile of hMATE1 & hMATE2K.

Support or Funding Information

Supported by NIH Award 5R01GM129777
Follicle Stimulating Hormone Receptor Expression In Histologically Normal And Neoplastic Canine Mammary Gland Tissues
Authors: Jelesijevic Tomislav , Nesic Vladimir , Aleksic-Kovacevic Sanja , Marinkovic Darko , Gledic Dusan , Knezevic Milijana , Jovanovic Milijan
Published: April 2020

The roles of Follicle Stimulating Hormone (FSH) in reproductive physiology have been recognized for a long time. The hormone achieves its functions by binding to the Follicle Stimulating Hormone Receptor (FSHRc) that is strongly expressed in both male and female gonads. Over the last 30 years FSH and its receptor have been discovered also in normal and neoplastic extragonadal tissues including: placenta, uterus, and most recently mammary glands. In neoplasias they contribute to cancer development, cancer cell migration, invasion and neovascularization. Canine mammary gland tumors (MGTs) are the most frequent tumors in intact bitches. Though frequently studied, their pathogenesis is still not completely understood and the presence of FSHRc has not been investigated. The purpose of this study was to evaluate histologically normal and neoplastic mammary glands for the presence of FSHRc using immunohistochemistry (IHC) and in situ hybridization (ISH). Positive immunohistochemical cytoplasmic staining is noticed in epithelial and the underlying myoepithelial cells of normal and neoplastic mammary glands and also in the cytoplasm of chondrocytes in mixed MGTs. In situ hybridization detected the expression of FSHRc in the nucleus and perinuclear cytoplasm of epithelial and myoepithelial cells of normal and neoplastic mammary glands. The signal was observed in the cytoplasm and, to a lesser degree, in the nucleus of chondrocytes in mixed MGTs. Further studies are necessary to elucidate the physiological and pathophysiological roles of FSH and FSHRc in histologically and neoplastic mammary glands and to investigate their potential use as targets for highly specific chemo‐ or immunotherapies of MGTs.