bluuebunny/crossref_metadata_2025_split huggingface dataset:
HGF/Met Axis Protects Human Retinal Pigment Epithelial Cells from Cell Death
Authors: Zarnegar Arman , Ma Jihong , Tan XinPing , Kwon YongKook , Zarnegar Reza 
Published: April 2020

Demise of the retinal pigment epithelium (RPE) caused by etiologies such as age‐related macular degeneration is a major underlying factor leading to vision loss. Cell death can occur through two major pathways: apoptosis and necrosis/necroptosis. In the former, Fas/FasL death receptor signaling induces apoptosis, but its role in human RPE cell death is not thoroughly elucidated. In the latter, reactive oxygen species (ROS) can induce necrosis/necroptosis. Additionally, Hepatocyte Growth Factor (HGF) is a potent growth and survival factor for epithelial cells, but its functional role in RPE is not well characterized, especially its ability to protect RPE cells from death and to induce their growth. Accordingly, we hypothesized that HGF/Met protects human RPE cells from Fas‐promoted apoptosis and H2O2‐induced necrosis/necroptosis.We analyzed whether the HGF receptor (Met) is expressed in human and non‐human primate (NHP) eye tissue via western blot analysis and immunohistochemistry. To determine whether HGF/Met protects RPE cells against death induced by Fas/FasL and H2O2, we utilized gain‐ and loss‐of‐function models, cell survival assays and gene expression analysis (RNAseq). Human ARPE‐19 cell line was treated with various combinations of HGF, Met kinase inhibitor SU11274 (SU), FasL or H2O2. Cell growth and survival was determined via MTT assay. Met, Akt, and Erk activation were analyzed by western blot.Western blot analysis and immunohistochemistry confirmed Met activity and signaling in human and NHP eye tissue, including in the RPE cell layer. We found that HGF elicited Met stimulation and signaling culminating in activation of its downstream effectors Akt and Erk, proteins involved in two major pathways controlling cell survival and proliferation. We also uncovered that HGF inhibited FasL‐ and ROS‐induced killing of RPE. Conversely, inhibiting Met signaling rendered RPE susceptible to cell death induced by FasL and H2O2. Global gene expression analysis using an RNAseq approach revealed that HGF controls expression of numerous genes governing RPE cell growth, metabolism, and survival to name a few. In particular, HGF significantly upregulated 400 and repressed expression of 420 genes six hours post‐treatment; at 24 hours post‐treatment, HGF upregulated 291 and downregulated expression of 260 genes. Expression of 11,000 genes was unchanged.Our studies establish that HGF and Met play a major role in promoting the survival and homeostasis of RPE cells by inhibiting two major pathways of RPE cell death. Our findings suggest HGF/Met is a potential druggable target for therapeutic management of RPE‐related human ocular diseases.
The Role of Cancer Associated Fibroblasts in Maintaining Cancer Stem Cells
Authors: Lewis Shantae , Jones Dennis 
Published: April 2020

Breast cancer is one of the leading causes of cancer related deaths in females. Current treatment options for breast cancer ultimately fail, resulting in cancer relapse. One explanation for recurrence after treatment is the presence of cancer stem cells (CSCs). CSCs have high proliferative potential and are able to initiate tumor growth. In order to target CSCs, it is important to understand how CSCs are maintained. Recent studies have shown that cancer‐associated fibroblasts (CAFs) aid in the maintenance of CSCs. CAFs are an abundant cell type in the tumor microenvironment and have been found to promote tumor growth and confer resistance to anti‐cancer drugs by supporting cancer stem cells. We hypothesized that CAFs promote and maintain the growth of cancer stem cells in a novel tumor model (MCaP). Mammary carcinoma cells (MCaP) were propagated in culture from a tumor that developed spontaneously in an aged mouse. Cancer cells, along with CAFs were expanded from the isolated tumor. Cancer cells were grown as a mixed culture with CAFs or alone after enrichment. A colony growth assay was used to assess the formation of CSCs, which grow as colonies. Cultures were fixed and stained with cytokeratin and colonies were counted manually. Colony size was quantified using ImageJ. Flow cytometry was performed to detect and quantify surface expression of CSC markers CD44 and CD24 in mixed cultures and isolated cancer cells. To investigate whether CAFs enable cancer cell resistance to chemotherapy, we measured the dose response to cyclophosphamide or paclitaxel. Immunofluorescence was performed on tissue sections of metastatic lymph node and primary tumor. Tissues were stained with cytokeratin, aldehyde dehydrogenase (ALDH), and alpha‐sma (SMA). From the colony growth assay we noticed that colony formation and the size of colonies were both increased when MCaP cells were cultured with CAFs. Flow cytometry showed that MCaP cancer cells grown with CAFs resulted in the appearance of a CD44 high/CD24low population. This CSC population was not seen in cancer cells grown alone. After treating cells with paclitaxel or cyclophosphamide, we saw no notable difference in the viability of cancer cells in the presence or absence of CAFs. Immunofluorescence staining showed ALDH was expressed in SMA‐positive CAFs, but not in cytokeratin‐positive cancer cells. Although we did not find that CAFs protected cancer cells from chemotherapy, CAFs appear to have a positive effect on the size and quantity of CSC colonies. In addition, we detected a CD44 high, CD24 low CSC population in mixed cultures. CAFs were found to express the CSC marker ALDH, with expression increasing as the size of the tumor increased. This finding suggests that CAFs may provide a niche for CSCs. In conclusion, these results suggest that CAFs play an important role in maintaining CSCs in the MCaP mouse model.Support or Funding InformationThe University of the Virgin Islands MARC grant (GM008422)and The Jones lab ACS Pilot grant (K22CA230315)
Ameliorative Effect of Ocimum gratissimum Leaf extract Against Phenylhydrazine‐Induced Toxicity in Albino Rats
Authors: Ugbogu Eziuche Amadike , Akara Emmanuel Ugochukwu 
Published: April 2020

This study investigated the effect of aqueous leaf extract of Ocimum gratissimum on haematological and biochemical parameters of phenylhydrazine‐induced toxicity in rats. The experimental rats were divided into five (5) groups of six (12) rats each (6 males and 6 females placed in different cages). The test groups (Groups B to E) were induced intraperitoneally with 50 mg/kg phenylhydrazine each for two consecutive days. Group A served as normal control and received orally 0.25 ml/kg of distilled water as placebo, while group B (negative control) was left untreated. Groups C, D and E received orally 5 mg/kg ferrous sulphate, 200 mg/kg and 400 mg/kg aqueous extract of O. gratissimum respectively for 14 days. In the acute toxicity, the LD50 was above 5000 mg/kg body weight. There were significant decrease (p<0.05) in total cholesterol, triglycerides, low density lipoprotein (LDL) concentrations in the extract treated groups while aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, urea, creatinine, potassium ion (K+) significantly increased (p<0.05) in the negative control. High density lipoprotein (HDL) concentrations statistically increased (p<0.05) in the extract groups (C, D and E) when compared to the negative control. Red blood cell indices, packed cell volume (PCV) and haemoglobin concentrations (P< 0.05) significantly decreased in the negative control when compared to the extract treated groups in both male and female rats. The results show that aqueous leaf extract of Ocimum gratissimum could have the potentials of ameliorating the metabolic distortions in haematology, hepatorenal and lipid profile inflicted by phenylhydrazine. Thus, the extract could be of a good therapeutic use and safe for consumption at the tested doses.Support or Funding InformationI received funding from Tertiary Education Trust Fund (TETFUND) Nigeria to attend this conference
Parkin‐Mediated Ubiquitination of Mitofilin Mediates Dopaminergic Neuron Death in Response to PD Stressors
Authors: Aliagan Abdulhafiz Imam , Feng Yansheng , Tombo Nathalie , Bopassa Jean C. 
Published: April 2020

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by a gradual and preferential loss of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction plays a critical role in the pathophysiology of dopaminergic neuron death induced by parkinsonian neurotoxins (PD stressors) that cause Parkinson’s disease (PD). However, the exact mitochondrial‐dependent mechanisms resulting in PD stressor‐induced dopaminergic neuron death remains elusive. The inner mitochondrial membrane (IMM) protein, Mitofilin, has been shown to play a key role of controlling and maintaining mitochondrial cristae morphology and its dysregulation has deleterious effects. Here, we investigated the role of Parkin‐Mitofilin interaction in PD stressor‐induced neuron death.MethodsN27‐A dopaminergic neural cell line was used to determine the mitochondrial‐dependent mechanisms by which dopamine (Dopa) and Rotenone (Rot) induce cell death. Mitochondrial dysfunction was assessed by measuring mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production as well as mitochondrial integrity. Cell death was measured by flow cytometry using Annexin V/7AAD dyes and cell viability assessed by MTT assay. Protein quantification was measured using biochemical techniques including Western blot analysis, immunoprecipitation, and confocal microscopy.ResultsWe found that PD stressors treatment increased Parkin translocation in to mitochondria where it interacts with Mitofilin in the IMM. This interaction promotes Mitofilin degradation via ubiquitination, which was associated with reduced MMP and increased ROS production, effects that were concomitant with abnormal mitochondrial structure and increased neuron death including by apoptosis. Further, we found that Dopa treated cells did not activate mitophagy process, while Rot treated cells exhibited excessive mitophagy. In addition, overexpressing the mitochondrial deubiquitinase, USP30, attenuated cell death induced by Rot, but not Dopa, suggesting that Parkin mediates two distinctive mechanisms leading to neuron death. Conversely, we found that Dopa treatment triggered an increase in mitochondrial calpain activity, cleavage and release of AIF into the cytosol, as well as apoptosis via AIF‐PARP dependent mechanism.ConclusionsTogether, our study reveals a novel role for Parkin‐Mitofilin interaction in PD pathogenesis, which leads to the ubiquitination and degradation of Mitofilin. The degradation of Mitofilin results in mitochondrial structural damage and dysfunction that causes apoptosis via AIF‐PARP pathway.Support or Funding InformationNational Institutes of Health [R01 grant HL138093 (JCB)]; Perry and Ruby Stevens Parkinson’s Disease Center of Excellence pilot grant (JCB).
Post‐treatment With Qing‐Ying‐Tang, a Compound Chinese Medicine Relives Lipopolysaccharide‐Induced Cerebral Microcirculation Disturbance in Mice
Authors: Han Jingyan 
Published: April 2020

ObjectiveLipopolysaccharide (LPS) causes microvascular dysfunction, which is a key episode in the pathogenesis of endotoxemia. This work aimed to investigate the effect of Qing‐Ying‐Tang (QYT), a compound Chinese medicine in cerebral microcirculation disturbance and brain damage induced by LPS.MethodsMale C57/BL6 mice were continuously transfused with LPS (7.5 mg/kg/h) through the left femoral vein for 2 h. QYT (14.3 g/kg) was given orally 2 h after LPS administration. The dynamics of cerebral microcirculation were evaluated by intravital microscopy. Brain tissue edema was assessed by brain water content and Evans Blue leakage. Cytokines in plasma and brain were evaluated by flow cytometry. Confocal microscopy and Western blot were applied to detect the expression of junction and adhesion proteins, and signaling proteins concerned in mouse brain tissue.ResultsPost‐treatment with QYT significantly ameliorated LPS‐induced leukocyte adhesion to microvascular wall and albumin leakage from cerebral venules and brain tissue edema, attenuated the increase of MCP‐1, MIP‐1α, IL‐1α, IL‐6, and VCAM‐1 in brain tissue and the activation of NF‐κB and expression of MMP‐9 in brain.QYT ameliorated the downregulation of claudin‐5, occludin, JAM‐1, ZO‐1, collagen IV as well as the expression and phosphorylation of VE‐cadherin in mouse brain.ConclusionsThis study demonstrated that QYT protected cerebral microvascular barrier from disruption after LPS by acting on the transcellular pathway mediated by caveolae and paracellular pathway mediated by junction proteins. This result suggests QYT as a potential strategy to deal with endotoxemia.
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 InformationNIH 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.
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

BackgroundCardiomyocyte 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.MethodsRat 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.ResultsH/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).ConclusionsIt 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 InformationFunding: 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.ConclusionsEzrin 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.