bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Glucose Control is Associated with Caloric Intake, but not Body Weight in Diet‐Induced Obese Mice
Authors: Henderson Chloe G. , Swoap Steven John
Published: April 2020

Alternate day fasting (ADF) and caloric restriction are common and effective diets for weight loss in both rodents and humans. Weight loss is associated with improved circulating glucose control. However, the improvement in glucose homeostasis is associated with total caloric intake, but not body weight in genetically obese mice on an ADF diet. We therefore tested the hypothesis that improved glucose control similarly would be related to caloric intake, but not body weight, in diet‐induced obese mice. At the experiment onset, high fat fed male mice (n=25) weighed (41.0±3.3 g, mean±SD) significantly greater, p<0.001, than low fat fed mice (24.2±2.6 g (n=11)). To induce weight loss, diet‐induced obese mice were assigned to either 1) low fat food ADF, 2) low fat ad libitum food availability, 3) high fat caloric restriction (CR=75%), or 4) high fat food ADF (n=5 for each group). A group of mice (n=5) continued on a high fat ad libitum diet. After six weeks of diet manipulation, low fat ADF mice lost ~11g, low fat ad lib mice lost ~8.5g, while the high fat CR group and high fat ADF groups both lost ~5 grams. Glucose tolerance tests were performed after six weeks of diet manipulation, and area under the curve (AUC) was calculated over a two‐hour period. All diet manipulations led to increased glucose tolerance compared to high fat ad libitum mice (p<0.001 for all comparisons). We found that AUC positively correlated with caloric intake (r2=0.7361, p<0.001), but not with body weight (r2=0.06, p=0.169), or food fat content. There were no significant differences in glucose tolerance among the ADF and CR groups; however, the ADF groups had significantly lower AUCs than all other groups (p<0.05 for all comparisons). In total, these data suggest that 1) these diet manipulations lead to both increased glucose control and weight loss, and 2) body weight is not a good predictor of glucose control.
The Role of BCAT1 on Pediatric Dilated Cardiomyopathy
Authors: PIRES DA SILVA Julie , MIYANO Carissa , GARCIA Anastacia , SUCHAROV Carmen
Published: April 2020

Background

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondria function are dysregulated in pediatric hearts. One potential factor which may be implicated in pediatric cardiac dysfunction is the Branched‐Chain Amino Acid Transaminase 1 (BCAT1), an enzyme highly expressed in pediatric non‐failing hearts and down‐regulated in response to DCM‐induced heart failure. While BCAT1’s role in cardiac dysfunction is unclear, in cancer cells and macrophages, BCAT1 is important for mitochondria function. The objective of this study is to investigate the role of BCAT1 in the heart, which may result in age‐specific therapies.

Methods

We measured metabolite composition of the pediatric DCM and non‐failing (NF) hearts. Activity and level of BCAT1 by enzymatic assay, RT‐qPCR and Western Blot in adult and pediatric and DCM and NF hearts was also measured. Pathologic changes in gene expression and mitochondrial function were assessed in Neonatal Rat Ventricular Myocytes (NRVMs) by RT‐qPCR and Seahorse assay in the presence or absence of BCAT1 inhibitor.

Results

Analysis of metabolite content showed a dramatic accumulation of branched‐chain amino acids (BCAAs) in pediatric DCM hearts. BCAT1 mRNA and protein levels are increased in pediatric NF hearts compared to adult NF hearts, and decreased in pediatric DCM hearts. In addition, inhibition of BCAT1 in NRVMs results in pathologic gene expression and inhibition of pro‐inflammatory cytokines. Finally, Seahorse assay showed a decrease of mitochondrial respiration in NRVMs treated with BCAT1 inhibitor.

Conclusions

Decreased levels of BCAT1 are detrimental to the pediatric failing heart. These findings suggest that BCAT1 could be a specific therapeutic target for the pediatric population.

Support or Funding Information

R01 HL139968 and HL126928
The Personalized Precise Exercise Training Holistic Integrative Protocol (PPET‐HIP) for Patient with Myocardial Incompetence and Catecholamine‐Sensitive Ventricular Arrhythmia after her Sudden Cardiac Arrest
Authors: Song Ya , Sun Xingguo
Published: April 2020

INTRODUCTIONS

The patient was a 23yrs female nurse in Fuwai‐hospital. After 2d overtime work, she had suddenly cardiac arrested followed by successful CPR and 5hr unconsciousness. Afterward she was diagnosed as catecholamine‐sensitive ventricular tachycardia, myocardial insufficiency and etc; and some physicians suggested installing of cardiac pacemaker with ICD. However, based on HIPM, we believe her main problem was the mismatching of supply‐demand and poor integration among systems, and Personalized Precise exercise Training Holistic Integrative Protocol (PPET‐HIP)can reverse those. We suggested give us ~100d PPET‐HIP and then decide for pacemaker‐ICD.

METHODS

After performing symptom limited maximum cardiopulmonary exercise testing (CPET) for objectively quantitative evaluation, we generated her PPET‐HIPΔ50% Power for intensity titration. According to results of continuous beat‐by‐beat blood pressure and continuous blood glucose monitoring during 30min Δ50% Power intensity titration we did select qid, 6d/w intensive management.

RESULTS

After 1m of intensive management, her continuation duration of PPET‐HIP was increased from 1–2min to 6–7min. The total exercise duration was increased from 17 to 24min, and resting was decreased from 8–10 to 3–4 times/30min. In 1st CPET, ventricular premature (PVC) began from loading intensity over 75w/min until peak loading127.5W/min , and total PVC was 119beat/130s.After three‐month PPET‐HIP,CPET showed PVC began from loading intensity over 100w/min until peak loading 162.5W/min , and total PVC was 68beat/170s. Comparing with the 1st one, three‐month CPET’s key parameters of peakVO2, AT, peak loading, peak CO and oxygen uptake efficiency plateau (OUEP) were increased by 20.1%, 15.2%, 21.5% and 22.5%, 21.7% and 5.7% respectively, only VE/VCO2 was decreased by 5.4%; all indicated improvement of functional status. The she has been believed that she couldn’t be nursing work again. However, after leaving Fuwai, she has become a clinical nurse again and working in cardiac rehabilitation, which is very meaningful for her.

CONCLUSION

Under the guidance of HIPM, we could safely and effectively manage patients by PPET‐HIP.

Increased functional capacity parameters in CPET by 5–23%.

Increased threshold of PVC and decreased rate of PVC during CPET.

Decreased number of PVC and decreased rate of PVC in 30min training of PPET‐HIP.

Support or Funding Information

National Natural Science Foundation of China (81470204);

Fuwai Hospital, National Cardiovascular Institute of Chinese Academy of Medical Sciences (2012‐YJR02);

Research on Clinical Characteristics of the Capital (Z141107002514084);

Research and Outcome Promotion of Clinical Characteristics in the Capital (Project No. Z161100000516127)
Gene regulation networks in muscle differentiation in lamin A associated muscular dystrophy
Authors: Dutta Subarna
Published: April 2020

A type lamin has came into the limelight due to it’s involvement in the different plethora of diseases, collectively known as laminopathies. According to the previous reports, lamin A has involvement in disrupting differentiation of overexpressed R453W mutant of lamin A associated autosomal EDMD. In our immunofluorescence studies, we have ventured myoblast cells expressing a severe skeletal muscular dystrophy mutated lamin A, had a significant lower capacity to differentiate than wild type one. Anomalies in the reulations of protein expression levels of MyHC, MyoD were also checked by western blot analysis. These convey that, A type lamins play a crucial role by influencing expression of genes necessary for muscle cells differentiation .The abnormalities due to muscular dystrophy may lead to impairment of gene circuitry ,which are related to myoblast fusion necessary for the formation of mature myotubes. In our RNA sequencing study, we evaluated a thorogh gene expression profiling in the context of complex, highly coordinated process of muscle differentiation in stably transfection of wild type and mutant in C2C12 myoblast cells, which were differentiated for a specific time period. In the expression profiling of seven day time course, the genes which are showing highest scores in the disputes between wild type versus mutants ,were analysed further to get eloquent idea on cell signaling pathways were involved in. We identified dysfunctional pathways to compare wild type versus mutant of muscular dystrophy during differentiation of myoblast to myotube transition. These data provide a model in the muscular dystrophy pathogenesis to interlace the impacts of genetic modifiers in the differentiation potentiality.

Support or Funding Information

This work was funded by DAE, SERB‐DST.
Propofol Postconditioning Ameliorates Hypoxia/Reoxygenation induced H9c2 Cells Apoptosis and Autophagy via upregulating Forkhead Transcription Factors under Hyperglycemia
Authors: Han Ronghui , Han Hong , Xie Xiang , Chen Hao , Liu Danyong , Zhang Liangqing , Xia Zhengyuan
Published: April 2020

Administration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post‐ischemic reperfusion (propofol postconditioning, P‐PostC) has been shown to confer cardioprotection against ischemia‐reperfusion injury , while the underlying mechanism remains incompletely understood. Also, whether or not P‐PostC can confer cardioprotection in diabetes or under hyperglycemic condition has yet to be explored. The FoxO transcription factors were reported to play critical roles in promoting cardiomyocyte survival during conditions of oxidative stress through induction of antioxidants and cell survival pathways (J Biol Chem. 2011;286(9):7468–78) and was also involved in hypoxic postconditioning mediated neuroprotection. However, the role of Foxo in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown. We hypothesized that P‐PostC may attenuate hypoxia/reoxygenation (H/R) injury in cardiac origin H9c2 cells by attenuating apoptosis and autophagy through upregulating FoxO1 and FoxO3a under hyperglycemia. Rat heart‐derived H9c2 cells were exposed to high glucose (HG) for 48 hours, then subjected to H/R (composed of 8 hours of hypoxia followed by 12 hours of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol at the onset of reoxygenation. Our data showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P‐PostC), especially at the concentration of 25μM. Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were associated with increased FoxO1 and FoxO3a expression. The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a. Taken together, these results indicated that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

Support or Funding Information

Supported by National Natural Science Foundation of China (NSFC) grant (81970247)
Increased Levels of Cortisol in Patients Undergoing Total Joint Arthroplasty may be Associated with Stress and Inflammatory Processes
Authors: Haddad Sandra , Khanna Rajan , Schmitt Daniel , Slovacek Hannah , Siddiqui Fakiha , Hoppensteadt Debra , Liles Jeffrey , Wanderling Christopher , Fareed Jawed , Hopkinson William
Published: April 2020

Introduction

Osteoarthritis is a condition in which joint cartilage and its adjacent bone degenerate progressively over time due to inflammatory processes involving activation of matrix degrading enzymes. Total joint arthroplasty (TJA) is a common surgical treatment for osteoarthritis of the hip or knee. Cortisol is a steroid hormone in the class of glucocorticoids which is produced in the adrenal gland. Cortisol is generated in response to inflammation and physical or emotional stress. This study aims to investigate the cortisol levels in patients undergoing TJA.

Materials and Methods

Plasma samples were prospectively collected from 71 TJA patients at baseline (pre‐surgery), 24 hours post‐operation, and 5 days post‐operation. Normal controls represented plasma samples from drug free health individuals (n=50). Cortisol and TNFa levels were measured in each sample using a commercially available ELISA kit. All results were compiled as group means ± SD.

Results

The plasma cortisol level in normal individuals were 67 ± 11 ng/mL. In contrast, the pre‐surgical and 24‐hour post‐surgical samples showed a marked increase in cortisol levels (> 200ng/mL). The blood samples drawn at the 5th day after surgery showed a downward trend reaching near normal levels (74 ± 12 ng/mL). There was no significant difference between cortisol levels pre‐operatively and within 24 hours post‐TJA, however there was a significant difference in comparison to the levels on the 5th day and normal. The TNFa levels were elevated in TJA patients in the pre‐surgical (18.6 ±3.2 pg/ml), at 24 hours post‐surgical (24.2 ± 7.6 pg/ml) and on the 5th day (11.3 ± 3.1 pg/ml) in comparison to normals (5.1 ± 2.1 pg/ml).

Discussion and Conclusion

At 5 days post‐operation, cortisol levels were significantly lower than at baseline or 24 hours post‐operation. This finding differs from previous findings, which reported increased levels post‐operatively relative to at baseline. The TNFa levels also showed elevated levels in the pre‐ and post‐surgical samples which followed a downward trend on the 5th day. These results are consistent with the previous reports that such biomarkers as IL‐6 and TNFa are upregulated in TJA patients prior to surgery suggesting a role of inflammatory process. Furthermore the increased level of cortisol may be related to not only the inflammation but pre‐surgical emotional stress in TJA patients. The near normalization of the cortisol levels on the 5th post‐surgical day is suggestive of differential down‐regulation of the inflammatory process and stress levels in TJA patients.
Ozanimod Attenuates Ischemia‐Induced Inflammation and Dysfunction in Human Brain Microvascular Endothelial Cells
Authors: Wendt Trevor S. , Li Yu Jing , Liu Qiang , Shi Fu-Dong , Gonzales Rayna J.
Published: April 2020

Sphingosine‐1‐phosphate receptor (S1PR) modulators elicit neuroprotection during stroke, however the mechanism(s) associated with beneficial outcome remains unknown. Endothelial dysfunction is a key pathophysiological contributor in acute ischemic stroke. Here, we researched the vascular specific role of the S1PR1 ligand, ozanimod, on endothelial function and health following hypoxia plus glucose deprivation (HGD), an in vitro ischemic‐like model. Specifically, we investigated whether ozanimod (0.5 nM) alters human male brain microvascular endothelial cell (HBMEC) proliferation, morphology, and integrity. HBMECs were exposed to HGD (1% O2) or normoxia (21% O2) for 3h at passage 7 using a humidified chamber (BioSpherix®) housed within a 5% CO2 incubator. For the proliferation studies, cells were treated with bromodeoxyuridine and incorporation of the synthetic nucleoside analog during normoxic or HGD exposure was measured via flow cytometric analysis. Morphology was assessed post normoxic or HGD exposure in HBMECs stained with crystal violet and visualized with brightfield microscopy. Trans endothelial electrical resistance was measured following HGD or normoxia exposure using polyethylene terephthalate 8 well plates and electric cell‐substrate impedance sensing (ECIS® Z Theta). Additionally, we investigated the impact of HGD on S1PR1 and cyclooxygenase‐2 levels using standard immunoblotting techniques. In some experiments, selective blockade with the antagonist, W146, verified S1PR1 activation involvement. We observed that S1PR1 was basally expressed in HBMECs and HGD increased expression suggesting S1PR1 as a potential target during acute ischemic injury. Ozanimod attenuated HGD‐mediated decreases in HBMEC proliferation and W146 reversed this response. S1PR1 blockade had no effect on cell proliferation following normoxia. Ozanimod also preserved HGD‐induced morphological changes. During electric cell‐substrate impedance assessment, ozanimod attenuated HGD mediated reduction in endothelial cell resistance and W146 abolished this effect suggesting S1PR1 dependency. Concomitantly, we observed that HGD reduced expression levels of zonula occludens (ZO)‐1 and claudin‐5. Ozanimod also decreased HGD‐mediated increases in proinflammatory mediator expression of cyclooxygenase‐2. In conclusion, ozanimod elicited protection against HGD in HBMECs and may play an important beneficial role against acute ischemic damage by improving brain endothelial integrity and viability via selective S1PR1 activation.

Support or Funding Information

University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG, FS), American Heart Association 19AIREA34480018 (RJG).
Slow Molecular Recognition by RNA
Authors: Sengupta Raghuvir N. , Herschlag Daniel
Published: April 2020

Molecular recognition is fundamental to life as demonstrated by the remarkable specificity observed in biological processes mediated by proteins and RNAs. Nevertheless, the most accurate process is of no value to biology if it cannot be carried out rapidly. Proteins associate with ligands with a wide range of association rate constants, with maximal values matching the theoretical limit set by the rate of diffusional collision. As less is known about RNA association, we recently compiled association rate constants for all RNA/ligand complexes that we could find in the literature. Our analysis indicates that RNAs, like proteins, exhibit a wide range of association rate constants. However, the fastest RNA association rates are considerably slower than those of the fastest protein associations and fall well below the diffusional limit. The apparently general observation of slow association with RNAs presumably reflects basic physical properties of RNA and has implications for evolution and for modern‐day biology. We also discuss potential strategies for increasing RNA association kinetics, such as the use of electrostatic forces to accelerate binding, which may provide broadly applicable strategies for developing fast‐binding RNA ligands as well as RNA‐targeted therapeutics.
DNA Repair in Colon Cancer Cell Lines.
Authors: O’Donoghue Tristan , Podlutsky Andrej
Published: April 2020

Colorectal cancer remains the third most common cancer world‐wide, despite recent advances in early diagnosis and following treatment strategies. Genome stability plays important role in cancer’s origin and progression. The fidelity of DNA repair is of most importance for the colorectal cancer, which characterized by accumulation of mutations in oncogenes and tumor suppressor genes, and dozens of so called “passenger” mutations in genes that are not driving cancer progression. Studying of cell cultures established from colon‐cancer patients are important tool for understanding molecular mechanisms behind cancerous transformation and drug development. Two colon‐cancer cell lines are widely used in such research are HT29 and HCT116. We analyzed the nucleotide excision repair (NER) in these cells by monitoring DNA repair following UV‐C exposure. The NER pathway is one of several fundamental repair mechanisms that is guarding genome from accumulation of DNA damage. Following the single gel electrophoresis technique, we determine that HT29 cells are slower at repairing UV‐C induced damage compared to HCT116 cells or normal human fibroblasts, HDFa cells. This is an important funding, because it shows that colon‐cancer cell lines are different in stress response capacity. Such difference might be important for the drug development and our understanding of basic mechanisms of genome stability in cancer cell lines.

Support or Funding Information

1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual:
www.alaska.edu/titleIXcompliance/nondiscrimination. 2) NASA EPSCoR, NNH15ZHA003C
Tango1 Coordinates the Formation of ER/Golgi Docking Sites to Mediate Secretory Granule Formation
Authors: Reynolds Hayley , Zhang Liping , Tran Duy , Hagen Kelly Ten
Published: April 2020

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Here, through high‐resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, we found that Tango1 forms ring‐like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis‐Golgi. Moreover, we observed nascent secretory mucins emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis‐Golgi, mucin O‐glycosylation and secretory granule biosynthesis. Additionally, we identified a Tango1 self‐association domain that is essential for formation of this structure. Our results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues.
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 Aims

Rho‐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.

Method

Using 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.

Results

Compared 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.

Conclusion

This 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 Information

This 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 Information

This 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 Information

National 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 Information

National 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

Introduction

Glycans 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.

Methods

The 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).

Results

Ribonuclease 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.

Conclusions

The 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 Information

This 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 Information

Ministry 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 Information

NIH [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 Information

NIH 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 Information

The studies were supported by NIH grants HL122664 (to L. Chang), HL068878 (to Y.E. Chen)