bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Rho associated coiled kinase 2 (ROCK2) inhibitor reduces steatosis and fibrosis in mice model of liver disease
Authors: Ali Quaisar , Dalapati Sony , Prakash Natalia , Narayan Prakash , Paka Latha , Li Jingsong , Hu Zhijian , Goldberg Itzhak 
Published: April 2020

Background and AimsRho‐associated coiled kinases 1 (ROCK1) and 2 (ROCK2) are involved in variety of cellular processes including tissue fibrosis. Recently, we demonstrated that hepatic ROCK2 and not ROCK1 is upregulated in hepatic stellate cells in mice model of non‐alcoholic fatty liver disease. The present study was done to investigate if selective blocking of ROCK2 reduces hepatic steatosis, fibrosis and improves liver function.MethodUsing core hopping and structure based drug design approach, we synthesized highly selective (>200 fold selective for ROCK2 vs ROCK1), orally bioavailable ROCK2 inhibitor (ANG4201). We evaluated the antisteatotic and antifibrotic effects of ANG4201 in mice model (C57Bl/6) of NASH. Adult male mice were subjected to high fatty diet (FFD) for three months and CCl4 (0.2 μl (0.3μg/g)) of body wt (once a week, ip)) was administered to accelerate the progression of liver fibrosis. After three months, mice were randomized to vehicle or ANG4201 (10, 25 & 50 mg/kg body wt, PO, BID) for two and half months. Liver injury was analysed by measuring serum transaminases (AST/ALT). RNA was isolated from livers and panel of fibrotic and inflammatory markers were assessed by RT‐PCR. H&E staining was done to analyse NASH and scoring was done to determine NAFLD activity.ResultsCompared to control, ANG4201 treatment dose dependently reduced TAA+FFD induced liver fibrosis in mice, demonstrated by significant reduction in hepatic hydroxyproline as well as alpha‐SMA, collagen I mRNA expression. The reduction in fibrosis was associated with significant improvement in the ratio of liver wt and body wt and reduction in serum transaminases (AST/ALT) and cholesterol. ANG4201 treatment overall reduced NAFLD activity score. ANG4201 also reduced TGF‐β induced gel contraction in hepatic stellate cells which was also associated with significant decrease in connective tissue growth factor (CTGF) mRNA, a marker elevated in fibrotic condition.ConclusionThis study demonstrates that ROCK2 contributes to hepatic fibrosis and hepatic dysfunction in FFD+ CCl4 mice model. ANG4201 is a highly promising orally bioavailable therapeutic ROCK2 inhibitor with potential use in liver fibrosis.Support or Funding Information
1 R43 DK121642 01
Avoiding Drug Resistance by Substrate Envelope Guided Design: Toward Potent and Robust HCV NS3/4A Protease Inhibitors
Authors: Zephyr Jacqueto , Matthew Ashley N. , Rao Desaboini Nageswara , Henes Mina , Kamran Wasih , Hedger Adam K. , Lockbaum Gordon J. , Timm Jennifer , Akbar Ali , Yilmaz Nese Kurt , Schiffer Celia A. 
Published: April 2020

Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct‐acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV‐infected patients. However, selection of resistance‐associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance‐associated substitutions at D168. Our design strategy achieved better resistance profiles over both FDA‐approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.Support or Funding InformationThis work was supported by a grant from the National Institute of Allergy and Infectious Diseases of the NIH (R01 AI085051). JZ and ANM were also supported by the National Institute of General Medical Sciences of the NIH (F31 GM119345 and F31 GM131635 respectively). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.
Investigation of Cell Envelope‐Associated Proteins in Rumen Methanogens as Targets for Understanding Host‐Microbe Interactions
Authors: Yeung Juliana H. , Shu Dairu , Ferguson Timothy , Altermann Eric , Khanum Sofia , Gupta Sandeep , Loo Trevor S. , Sutherland-Smith Andrew J. , Heiser Axel , Janssen Peter H. , Wedlock D. Neil 
Published: April 2020

The rumen microbiota is complex and almost totally composed of anaerobic organisms, including methanogenic archaea (methanogens). The latter occupy a unique niche in the rumen, using hydrogen, formate and methyl compounds to produce methane.Many of the proteins predicted from analysis of genome sequences of rumen‐dwelling methanogens, in particular cell envelope‐associated proteins, have no known biological function. To elucidate the role of these proteins in methanogen activity in the rumen and in the interaction of these archaea with other ruminal microbes, we have produced antibodies against selected methanogen proteins. The proteins were expressed in a standard bacterial expression system. An important first step was to determine if recombinantly produced proteins can generate antibodies that bind and recognise the native methanogens proteins.Bioinformatic analysis of the genome of Methanobrevibacter ruminantium M1 identified suitable cell surface‐located proteins for the studies. Predicted extracellular domains of these proteins were synthesised in Escherichia coli and circular dichroism analysis was performed to check that each recombinant protein had an ordered structure. Antibodies were produced against each recombinant protein and used in ELISA and Western blotting on lysates and membrane protein enriched fractions prepared from M1 cells. Proteins in the fractions were further separated by 2D gel electrophoresis and specific binding of the antibodies to the native targets was confirmed by LC‐MS/MS. Binding of the antibodies to methanogen cells was demonstrated using ELISA and flow cytometry.Our results to date suggest that expression of some of the cell surface proteins in bacteria produced recombinant proteins that sufficiently mimic the native proteins, with cross‐reactivity of antibodies to methanogen cell extracts. Our immunological studies, together with new information on gene expression from transcriptomic analysis of M1 in cultures, is being used to further understand the biological function of methanogen cell surface proteins and how methanogens interact with other microbes in the rumen.Support or Funding InformationThis research was funded jointly by Pastoral Greenhouse Gas Research Consortium (PGgRc) and New Zealand Agricultural Greenhouse Gas Research Centre (NZAGRC)
Neuroprotective effects of 2‐aminoethoxydiphenyl borate (2‐APB) in β‐amyloid‐induced memory dysfunction is mediated through inhibition of transient receptor potential melastatin 2 (TRPM2) channels
Authors: Thapak Pavan , Khare Pragyanshu , Bishnoi Mahendra , Sharma Shyam Sunder 
Published: April 2020

β‐amyloid (Aβ) peptide is a hallmark of Alzheimer’s disease (AD) and associated with a progressive decline of cognition, a characteristic feature of AD. Aggregation of Aβ initiates the dysregulation of calcium ion homeostasis. Recently, it has been shown that transient receptor potential melastatin 2 (TRPM2), a Ca2+‐permeable non‐selective cation channel plays a crucial role in the Aβ‐induced neuronal cell death. However, the therapeutic potential of 2‐aminoethoxydiphenyl borate (2‐APB), a TRPM2 inhibitor has not been investigated in the Aβ‐induced cognitive deficits in rats yet. In the present study, I.C.V. administration of Aβ (Aβ25‐35, 10 μg) markedly induced cognitive impairment which shown in cognitive behavioral parameters. Moreover, mRNA and protein expression of TRPM2 were upregulated in the hippocampus of Aβ‐treated animals. AchE activity was also increased in the cortex of Aβ‐treated animals. Three‐week treatment with 2‐APB led to the down‐regulation of TRPM2 mRNA and protein expression in the hippocampus and also improved the cognitive functions. Moreover, the 2‐APB treatment also increased the calcium and memory associated proteins namely p‐CaMKII, p‐GSK3β, p‐CREB and PSD‐95 in the hippocampus and reduced the mRNA level of calcium buffering protein and calcineurin A (PPP3CA) in the hippocampus. Furthermore, 2‐APB significantly reduced the AchE activity in the cortex. Thus, our findings suggest that 2‐APB depicted potential neuroprotective effect against Aβ‐induced cognitive impairment.Support or Funding InformationNational Institute of Pharmaceutical Education and Research and National Agri‐Food Biotechnology Institute
Inhibition of ricin A‐chain (RTA) catalytic activity by a viral genome‐linked protein (VPg).
Authors: Domashevskiy Artem Victor 
Published: April 2020

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A‐chain (RTA) and a viral genome‐linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N‐terminal truncated variant, VPg1‐110, bind to RTA and abolish ricin’s catalytic depurination of 28S rRNA in vitro and in a cell‐free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten‐fold as temperature elevates (5 °C to 37 °C). RTA‐VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA‐VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.Support or Funding InformationNational Institutes of Health (NIH) and National Institute of Allergy and Infectious Disease (NIAID): 1R15AI124044
Variation of Glycosylation Patterns Revealed by PGC‐LC‐ and Gated‐TIMS‐EED Tandem Mass Spectrometry
Authors: Wei Juan , Tang Yang , Zaia Joseph , Hong Pengyu , Costello Catherine E. , Lin Cheng 
Published: April 2020

IntroductionGlycans are the most abundant biopolymers found in nature; they play crucial roles in a wide range of biological activities. Glycan biosynthesis does not follow genetic blueprints, and the resultant glycome is often a mixture of glycoforms with varying compositions, branching patterns, linkages, and stereochemical configurations. Subtle changes in the structure of a glycan can have a profound impact on its biological functions. Recently, we have demonstrated the potential of porous graphitic carbon liquid chromatography electronic excitation dissociation tandem mass spectrometry (PGC‐LC EED MS/MS) and gated‐trapped ion mobility spectrometry (TIMS) negative electron transfer dissociation (NETD) MS/MS in characterizing glycan mixtures, including the separation and identification of isomeric structures. Here, by studying N‐glycans released from different batches of glycoproteins, the glycoform variations at the isomer level were observed. The detailed structure of each isomer could be determined by EED MS/MS and their relative quantification may be achieved by high‐resolution LC or ion mobility separation.MethodsThe released N‐linked glycans were reduced and permethylated before analysis to improve the ionization efficiency and to facilitate spectral interpretation. All experiments were carried out on a 12T solariXTM Fourier‐transform ion cyclotron resonance mass spectrometer equipped with a prototype TIMS device (Bruker Daltonics) or a nanoACQUITY UPLC system (Waters).ResultsRibonuclease B (RNase B) is the glycosylated form of RNase A. With the attachment of a glycan at Asn‐34, the glycosylated ribonuclease showed an increased structural flexibility in the RNA binding region and a much higher activity on double‐stranded RNAs. Previously, application of PGC‐EED MS/MS to the analysis of N‐glycans released from RNase B showed the presence of 18 high‐mannose structures. Here, by comparing the PGC‐LC MS/MS results from two batches of RNase B purchased from different manufacturers, we observed variations in the abundance of each glycoform. Additionally, we have implemented EED MS/MS with gated‐TIMS separation, and showed its ability to resolve and identify isomeric structures. Using gated‐TIMS EED MS/MS, we were able to determine the batch‐to‐batch variation of chicken ovalbumin N‐glycans along with the conformational information.ConclusionsThe PGC‐ and gated‐TIMS‐ EED MS/MS approaches showed advanced performance in separation, characterization, and relative quantification of isomeric glycans, as well as in applications to determine the batch‐to‐batch glycoform variations of glycoproteins. These approaches hold great potential for comprehensive and confident glycan characterization.Support or Funding InformationThis research is supported by the NIH grants P41 GM104603, 703 R21 GM122635, U01CA221234, R01 GM132675, and S10 704 RR025082.
Discovery of survival predictor and early monitoring biomarkers for EGFR tyrosine kinase inhibitor therapy in lung adenocarcinoma by integrative omics analysis
Authors: Hsiao Ting-Feng , Wang Chih-Liang , Wu Yi-Cheng , Feng Hsiang-Pu , Chiu Yen-Chuan , Lin Hao-Yu , Liu Ko-Jiunn , Chang Gee-Chen , Chien Kun-Yi , Yu Jau-Song , Yu Chia-Jung 
Published: April 2020

Epidermal growth factor receptor (EGFR) mutation is a predictor of the initial treatment efficacy of tyrosine kinase inhibitors (TKIs) in advanced lung adenocarcinoma (ADC). Unfortunately, neither EGFR mutations nor molecular markers can accurately monitor cancer progression or predict patient survival. EGFR‐TKI susceptibility‐associated biomarkers to benefit lung ADC patients are in high demand. Plural effusion (PE) has been recognized as a promising source for biomarker discovery. By integrating PE proteomic and tissue genomic analyses, we herein identified fifteen proteins as potential biomarkers of EGFR‐TKI resistance. Among these candidates, cadherin‐3 (CDH3) attracted our attention, as we observed that the CDH3 protein expression level was indeed positively correlated with malignancy as well as EGFR‐TKI resistance in our verified cancerous tissues, PEs and cell lines. Importantly, altered soluble CDH3 (sCDH3) levels in the serum could be used to monitor the efficacy of EGFR‐TKIs at one month after treatment. Furthermore, the baseline serum sCDH3 level was associated with progression‐free survival in ADC patients with mutant but not wild‐type EGFR. Finally, we observed that the baseline serum sCDH3 level was positively associated with tumor stage and overall survival in advanced non‐small cell lung cancer patients. Our results collectively establish a biomarker dataset for EGFR‐TKI resistance and sCDH3 is identified as a survival predictor and real‐time indicator of treatment efficacy in ADC patients received EGFR‐TKI therapy.Support or Funding InformationMinistry of Science and Technology, Taiwan, R.O.C. (108‐2320‐B‐182‐007‐MY3) and Chang Gung Medical Research Fund (CORPD1J0091, CMRPD1H0081‐3, CMRPD1H0641‐2, CMRPD1H0082 and BMRP894).
Modulation of Gene Expression in Muscle of Hibernating Arctic Ground Squirrels (Urocitellus parryii) and Attenuation of Disuse Muscle Atrophy
Authors: Fedorov Vadim B. , Barnes Brian M. , Goropashnaya Anna V. 
Published: April 2020

Physical inactivity generates muscle atrophy in most mammalian species. In contrast, hibernating ground squirrels demonstrate limited muscle loss over the prolonged periods of immobility during winter suggesting that they have adaptive mechanisms to reduce disuse muscle atrophy. To identify transcriptional programs that underlie molecular mechanisms attenuating muscle loss, we conducted a large‐scale gene expression profiling in quadriceps muscle comparing hibernating ((late in a torpor and during torpor re‐entry after arousal) and summer active arctic ground squirrels using the next generation sequencing of transcriptome. Gene set enrichment analysis showed a coordinated up‐regulation of genes involved in all stages of protein biosynthesis and ribosome during hibernation at late torpor and entering torpor after arousal that suggests induction of translation in interbout arousals. Elevated proportion of down‐regulated gene involved in apoptosis as well as significant underexpression of atrogenes, upstream regulators (FOXO1, FOXO3, NFKB1A) and key components of the ubiquitin proteasome pathway (FBXO32, TRIM63, CBLB), and overexpression of PPARGC1B inhibiting proteolysis imply suppression of protein degradation in muscle during arousals. Coordinated underexpression of multiple genes involved in muscle contraction and up‐regulation of genes in the fatty acid β oxidation pathway are consistent with low muscle loading and major role of lipids as fuel in muscle metabolism during hibernation. The induction of protein biosynthesis and decrease in protein catabolism likely contribute to the attenuation of disuse muscle atrophy through prolonged periods of immobility of hibernation.Support or Funding InformationNIH [R21AR064995]; NIH Alaska INBRE [P20GM103395]; NIH COBRE [P20GM130443]
IL‐1β inhibits cGMP signaling enzyme expression in perinatal fibroblasts via NF‐κB signaling
Authors: Roberts Jesse , Bry Kristina , Zhong Ying 
Published: April 2020

Lung injury causes important morbidity and mortality in newborns by disrupting pulmonary vascular function and alveolar development. Recent evidence suggests that IL‐1β signaling plays a primary role in the pathogenesis of newborn lung disease. But the mechanisms are incompletely understood. Previously we showed that nitric oxide (NO) and cGMP signaling regulate pulmonary blood flow and development in newborns. Furthermore, we determined that disruption of cGMP signaling during newborn lung injury accentuates pulmonary disease. Accordingly, here we determined whether IL‐1β inhibits cGMP signaling in the newborn lung.We used a human fetal lung fibroblast cell line (IMR‐90 cells) and primary mouse pup lung fibroblasts to test whether IL‐1β down‐regulates the expression of soluble guanylate cyclase α1 subunit (sGCα1). This is because fibroblasts have an important role in regulating newborn lung function and alveolar development and sGCα1 is a prime initiator of cGMP signaling upon NO stimulation. We determined that physiologic levels of recombinant IL‐1β rapidly decreased sGCα1 mRNA expression in the IMR‐90 cells and protein levels in the primary lung fibroblasts. This inhibition of sGCα1 expression appeared to be at a transcriptional level. This is because IL‐1β treatment did not further decrease sGCα1 mRNA levels in the fetal lung fibroblasts treated with actinomycin D. TGFβ‐activated kinase 1 (TAK1) was determined to mediate IL‐1β’s regulation of sGCα1 expression; TAK1 knock‐down protected sGCα1 mRNA expression in IL‐1β‐treated IMR‐90 cells. Moreover, heterologous expression of TAK1 in these cells revealed that this kinase is sufficient to decrease sGCα1 mRNA levels. Nuclear factor‐kappaB (NF‐κB) signaling played a critical role in the IL‐1β‐TAK1‐sGCα1 regulatory pathway; NF‐κB signaling inhibition by an IKK2 inhibitor PHA‐408 was determined to protect sGCα1 mRNA and protein expression in IL‐1β‐treated perinatal lung fibroblasts. Lastly, using tissue samples and soluble proteins obtained from transgenic mice in which active IL‐1β was conditionally expressed in lung epithelial cells, we established that IL‐1β expression is sufficient to stimulate TAK1 activation and decrease sGCα1 protein expression in the newborn lung. Together these results indicate that IL‐1β has a direct role in decreasing cGMP signaling in the newborn lung. We speculate that targeting IL‐1β and NF‐κB signaling might protect cGMP signaling in injured newborn lungs.Support or Funding InformationNIH HL096779 and HL147863 and the Swedish Government’s Grants for Medical Research supported this work.
A novel CLK1‐THRAP3‐PPARγ axis is involved in the regulation of insulin sensitivity
Authors: Chang Lin , Wang Zhenguo , Zhao Xiangjie , Garcia-Barrio Minerva , Zhang Jifeng , Chen Yuqing Eugene , Zeng Rong , Wu Jiarui 
Published: April 2020

Increasing energy expenditure by promoting “browning” in adipose tissues is a promising strategy to prevent obesity and associated diabetes. To explore potential targets of cold exposure, which induces energy expenditure, we performed phosphoproteomics profiling in brown adipose tissue of mice that were housed in mild cold exposure at 16°C. We identified that CLK1 (CDC2‐like kinase 1) is one of the kinases that were significantly downregulated by mild cold exposure. In addition, chemical inhibition or genetic knockout of CLK1 in mice prevented diet‐induced obesity and insulin resistance at 22°C. By proteomics approach, we uncovered a protein, THRAP3 (thyroid hormone receptor‐associated protein 3), as an interacting partner of CLK1, which was confirmed by co‐immunoprecipitation assays. We further identified that CLK1 phosphorylated THRAP3 at Ser243, required for interaction with phosphorylated PPARγ (peroxisome proliferator activated receptor gamma), resulting in downregulation of adipsin expression in adipose tissue and insulin sensitivity. These data suggest that CLK1 plays a critical role in controlling energy expenditure through the CLK1‐Thrap3‐PPARγ axis.Support or Funding InformationThe studies were supported by NIH grants HL122664 (to L. Chang), HL068878 (to Y.E. Chen)