bluuebunny/crossref_metadata_2025 huggingface dataset:
AER‐Related FGFs Upregulate LHX2 Through MEK in the RAS‐Associated Signaling Pathway During the Maintenance of ZPA‐Related SHH Expression
Authors: Damoah Ruth-Love , Shah Neera M. , Watson Billy A. , Pira Charmaine U. , Oberg Kerby C.
Published: April 2020

Fibroblast growth factors (FGFs) and sonic hedgehog (SHH) are mitogens known to affect limb growth and patterning during embryonic development. While FGFs establish the proximal‐distal axis from the apical ectodermal ridge (AER), SHH directs anterior‐posterior patterning from the zone of polarizing activity (ZPA). In addition, FGFs and SHH act in an autoregulatory loop by maintaining each other’s expression to coordinate limb patterning during limb outgrowth. The LIM Homeobox 2 (LHX2) transcription factor, which acts in the progress zone (PZ), was identified as an intermediary in the FGF to SHH arm of the maintenance loop. FGF mediates its action on cells through multiple intracellular signaling pathways including the JAK‐STAT self‐renewal pathway, PKC cell motility pathway, PDK1‐AKT cell survival pathway, and RAS‐associated cell proliferation pathway. We wondered whether one or more of these pathways directed FGF‐mediated upregulation of LHX2 in the FGF‐SHH regulatory loop. To evaluate a specific FGF signaling pathway, we implanted a bead soaked in a selective pathway‐specific inhibitor adjacent to an FGF2‐soaked bead in the posterior margin of Hamburger‐Hamilton stage 23–24 (HH23‐HH24) chicken limbs, a region known to induce LHX2 expression in response to FGF. The embryos were harvested after 4 hours and assayed for LHX2 and SHH expression by whole mount in situ hybridization. Ectopic LHX2 and SHH expression levels were reduced in the presence of the RAS and MEK inhibitors, indicating that FGF requires functional RAS‐associated pathways that utilize MEK to upregulate LHX2 expression.
S369 phosphorylation of RIPK3 by PLK1 during G2/M phases enables its apoptotic function in the ripoptosome independent of necroptosis
Authors: Gupta Kartik , Liu Bo
Published: April 2020

Receptor interacting protein kinase 3 (RIPK3) regulates a form of regulated necrosis called necroptosis. When an altered conformation is induced by via mutations in its kinase site or using chemical inhibitors, RIPK3 associates with an ensemble of proteins called the ripoptosome, containing FADD, RIPK1, Caspase 8 and cFLIP. RIPK3 now becomes decisive in the execution of apoptosis. Paradoxically, in contexts not completely understood, the ripoptosome cleaves RIPK3, highlighting a knowledge gap on how RIPK3 fulfills its role via the ripoptosome which is responsible for its own degradation. Recently, ripoptosome assembly was shown to occur physiologically during mitosis. We found RIPK3 levels to be elevated during the G2/M phases of the cell‐cycle, allowing us to study the mechanism by which RIPK3 resists degradation by the ripoptosome. We now report that polo‐like kinase 1 (PLK1) associates with RIPK3 and phosphorylates it at serine 369 before cells enter mitosis. RIPK3 in this phase has pro‐apoptotic activity but when released from ripoptosome, can elicit necroptosis. Disruption of the PLK1 site via T368A and S369A mutations causes degradation of RIPK3. RIPK3‐degradation can be prevented by the addition of pan‐caspase inhibitor zVAD.fmk, via a cis‐mutation in caspase 8 cleavage site i.e. D333A, but not by PLK1 over‐expression, suggesting that the S369 site regulates the apposing caspase cleavage site (D333) in cis‐manner. Additionally, S369D mutation on RIPK3 prevents its degradation via the ripoptosome whereas TS>>AA version of T368 and S369 is degraded. Together, phosphorylation of RIPK3 at S369 prevents its cleavage in the ripoptosome, thereby retaining its pro‐death activity during G2/M phases. The shift of cell‐death towards RIPK3 may have evolved as a mechanism by which the responsibility of balancing necroptosis vs apoptosis in mitotic cells converges on RIPK3.

Support or Funding Information

This study was supported by the National Institute of Health R01HL088447 (BL) and R01HL122562 (BL), and American Heart Association 17PRE33670082 (KG).
Systems Glycoengineering: Integrated Analyses of Glycomics, Transcriptomics and Phenotypic Data Reveal Complex Cellular Response to Glycoengineering in CHO Cells
Authors: Chiang Wan-Tien , Gazestani Vahid H. , Bao Bokan , Liang Chenguang , Sorrention James T. , Kellman Benjamin P , Ménard Patrice , Arnsdorf Jonnhy , Sukhova Zulfiya , Bjørn Sara Petersen , Brøndum Karen Kathrine , Hansen Anders Holmgaard , Yang Zhang , Joshi Hiren , Clausen Henrik , Voldborg Bjørn G. , Lewis Nathan E.
Published: April 2020

Glycosylation is fundamentally important to the activity, stability and pharmacokinetics of biopharmaceuticals. Move on to the future, glycoengineering is a promising way to improve protein drug efficacy. Recently, several major advances in engineering efforts have successfully led to desired protein glycoforms, but some desirable glycoengineering designs have proven toxic to the host cells (e.g., CHO cell lines). Up to date, the critical barrier in controlling the glycan structures is the diversity and complexity of glycosylation. Moreover, there has not been a systematic study unraveling how engineering efforts impact the host cell and their protein secretion system. There is a great need to bridge the knowledge gap that enables rational glycoengineering and elucidate how such strategies impact the host cell. To accomplish this, we have comprehensively studied the impact of glycoengineering on a library of more than 180 CHO cell clones, wherein dozens of glycosyltransfersases were knocked out or knocked in. We integrate data that glycoprofiled all these clones, quantified the impact of different glycosyltransferase knockouts on the bioprocessing phenotypes of the CHO cells (e.g., cell size, growth, viability, and metabolism), and expression profiling these cells using RNA‐Seq data to identify dysregulated pathways and genes following glycoengineering. Here, we deployed an innovative systems biology approach to rapidly study the changes in glycosylation across all glycoengineered mutants and to study the molecular basis of the phenotypic changes. Our key results are (1) we identified dominant glycosyltransferses responsible for the N‐linked glycosylation in CHO cells, and the glycoengineered cells differentially expressed isozymes in response to a knockout; (2) we identified groups of glycosyltransferases whose perturbations led to a more severe impact on cell glycosylation and phenotypes; (3) we also identified key metabolic and signaling pathways are modulated when different glycosyltransferase families were perturbed. This research is the first study for gaining a comprehensive view of the complex cellular response to glycoengineering on the host cells producing the recombinant protein drugs. The insights from this study provides valuable knowledge that enables the rational engineering of glycosylation to be able to control this critical quality attribute on diverse recombinant protein drugs.

Support or Funding Information

This work was supported by generous funding from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark (grant no. NNF10CC1016517).
Amino acids‐induced cardiac protection by promoting mitochondrial function via mTOR and Opa‐1 signaling
Authors: Morio Atsushi , Satomi Shiho , Toyota Yukari , Watanabe Tomoyuki , Tsutsumi Yasuo M
Published: April 2020

Objects

Amino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs.

Methods

Cardiac myocytes were isolated by cardiac retrograde aortic perfusion and collagenase and were subjected to simulated ischemia and cell death was quantified. Immunoblot and real time PCR were performed as biochemical assay and mitochondrial function was assessed using Seahorse XF cell Mito test.

Results

In myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA‐induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). To elucidate the mechanism, we assessed changes in the mitochondrial fusion regulators Opa‐1, Mfn‐1, and Mfn‐2. BCAA treatment increased expression of Opa‐1, but not Mfn‐1 or Mfn‐2. Enhanced mitochondrial fusion also improved mitochondrial function including oxygen consumption rate.

Conclusions

BCAA acutely regulates mitochondrial metabolism in cardiomyocytes and increased mitochondrial fusion, Opa‐1, and the Akt‐mTOR pathway, offering a novel target for prevention and treatment of coronary disease.

Support or Funding Information

This study was supported by Japan Society of Promotion Science #19K09353 to YMT.
Manipulating Levels of Stress‐Response Proteins in a Drosophila Model of Myosin‐Based Inclusion Body Myopathy 3 Worsens Muscle Dysfunction
Authors: Suggs Jennifer A. , Manalo Kimberley , Melkani Girish C. , Melkani Anju , Foster D. Brian , Bernstein Sanford I.
Published: April 2020

Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with >1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.

Support or Funding Information

This work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.
Manganese‐Induced Central Precocious Puberty (MnPP) Increases Hormone Receptor Expression (HR+) in Proliferating Mammary Epithelial Cells in Adult Female Rats
Authors: Dearth Robert K. , Hamilton Alina M. , Srivastava Vinod , Hiney Jill K. , Dees W. Les
Published: April 2020

In the developing mammary gland (MG), hormone receptor expressing (HR+) cells very rarely proliferate. The actions of estrogen (E2) and progesterone (P4) on ductal morphogenesis are paracrine and require mediators to stimulate growth of HR− adjacent cells. However, in premalignant and invasive human breast lesions, proliferating cells are HR+. Thus, a dramatic change occurs in MG cell fate with a suspected increased sensitivity to steroid mediated growth. Studies have theorized that early pubertal onset alters MG cell fate increasing the risk of breast cancer later in life. Yet, it is not known if early puberty can cause this conversion to HR+ proliferating MG cells and if this phenotype is sustained in the adult gland. We previously demonstrated that manganese‐induced precocious puberty (MnPP) accelerates E2‐regulated MG development, resulting in sustained proliferation of MG epithelial cells and adult hyperplasia. In the current study, we characterized the normal distribution of HR+ proliferative cells in the MG of prepubertal and adult female rats, determined if MnPP could increase this population and identify cellular proteins that may be involved. Sprague Dawley female rats were exposed daily to 10mg/kg manganese chloride (MnCl2) or saline (control) via gastric gavage from post‐natal day (PND) 12 to PND 30. MGs were collected on PNDs 30 and 120. Using dual labeling immunofluorescence (IF), only 2% of total MG epithelial cells co‐express HRs (E2 or P4 receptor) and the proliferative marker PCNA in the prepubertal MG. As expected, MnPP significantly (p<0.01) increased the percentage of proliferating (PCNA+) MG cells. The percentage of proliferating cells co‐expressing progesterone receptor (PR+/PCNA+) increased by more than 2‐fold in MnPP females relative to controls at PND30 (p<0.01). The proportion of proliferating HR+ cells (ER+/PCNA+ or PR+/PCNA+) remained elevated in PND120 glands of MnPP females compared to controls (p<0.001). Western blot analysis revealed a significant switch in expression levels of the cell cycle regulator, p27, and the mitotic stimulator EREG in MGs that corroborated changes in PR+ proliferation between PND 30 and 120. Furthermore, histologically normal human breast tissue adjacent to ductal carcinoma in situ (DCIS) from The Human Protein Atlas show ER and PR expression similar to expression levels observed in our MnPP rat MGs at PND120. Collectively, these data show that MnPP results in persistent HR expression in proliferating MG cells. Thereby possibly sensitizing it to the hormonal milieu and making the MG more responsive to steroid mediated growth.

Support or Funding Information

This work was supported by NIEHS grant ESO13143 awarded to WLD and NIGMS grant 1R25GM100866 awarded to RKD.
Pharyngeal Pumping Variability Assessment Reveals Subtle Mutant and Treatment Effects in the Nematode C. elegans
Authors: Harris Michael B. , Barrientos Christopher , Bui Andy , Co Mary , Gill Sunny , Lim Shelby , Ly Lily , Nguyen Micayla , Ortiz Brianna , Parabo Ashley , Phillips Derek , Rieger Iris , Shilleh Ahmad , Suree Nathan , Tran Adrianna , Valdez Vanessa , Vu Tien , Taylor Barbara E. , Berlemont Renaud
Published: April 2020

In C. elegans, feeding is achieved through a well characterized behavior, pharyngeal pumping. This pumping is controlled by two pairs of pharyngeal motor neurons (MC and M3). Each pump cycle corresponds to the propagation of a single pharyngeal muscle action potential, initiated by MC and transmitted across a single cholinergic neuromuscular junction. The eat‐2 gene is required for nicotinic neurotransmission at the pharynx. We assessed pumping patterns exhibited by wild type (N2) and a series of eat‐2 mutant strains, each identified as possessing deficits in pumping, including: (Strain: genotype) DA465: eat‐2(ad465) II; DA1113: eat‐2(ad1113) II; DA1116: eat‐2(ad1116) II. We also assessed pumping patterns in N2 strain animals incubated in the anthelmintic and acetylcholine receptor antagonist Levamisole ((−)‐Tetramisole hydrochloride; 0 – 50 uM). All studies we done in animals incubated in 10mM serotonin in M9 buffer. We recorded electropharyngeograms (EPG) using the recently developed ScreenChip system (NemaMetrix) to detect individual pump events, and analyzed recordings using an algorithm (WormBeat) to distinguish and quantify neuromuscular fidelity of the pharynx and pharyngeal pumping pace variation. As expected, mutant strains and Levamisole‐exposed N2 worms each illustrated pump pattern defects. Results show that the WormBeat algorithm can identify subtle treatment effects and distinguish phenotypic variation between mutant strains. Pump pattern assessment distinguished individual strains and allowed comparison of defect to the Levamisole dose‐responses of N2s.

Support or Funding Information

Work reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers 1R15HL126105, 1SC2GM112570, UL1GM118979, TL4GM118980, and RL5GM118978. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.
The Evolution of High‐fidelity Patient Simulation in Medical Education: A Literature Review
Authors: Klein Barbie Ann
Published: April 2020

INTRODUCTION

The use of patient simulation in healthcare education is extensive. This literature review identified and discussed the origins and current state of high‐fidelity patient simulation (HFPS) in medical education.

AIM

The aim of this research was to piece together a comprehensive history of clinical simulation and to compile results from past and present HFPS investigations in order to develop evidence‐based recommendations for best practices when incorporating HFPS into medical curricula.

METHODS

Data was obtained from an iterative review of historical literature, primary resources and references, as well as personal communication with authors of articles and texts related to the clinical application of simulation. Databases consulted included PubMed, Web of Science, ERIC, ProQuest Dissertations & Theses, EBSCOhost, Google Scholar, and the Indiana University library archive collections. As human subjects were not directly involved in this study, ethical approval was not applicable.

RESULTS

Over 110 different sources were reviewed and synthesized for data regarding the history, fidelity, advantages, challenges, and learning theories associated with HFPS in medical education. A review of current clinical HFPS research is organized by discipline and includes undergraduate medical education, continuing medical education, nursing education, and undergraduate pre‐professional education.

DISCUSSION AND CONCLUSION

This review spanned historical applications of clinical simulation dating back to Mesopotamia between the fourth and first millennia BCE and concludes with a review of computer‐controlled high‐fidelity simulators available today to train healthcare providers. Recommendations based on the literature include: formally integrating HFPS throughout the medical curriculum; providing authentic interprofessional education (IPE) opportunities for medical and other allied health professionals to develop essential team skills; and investing in the initial and continued training for those delivering the simulated experience and post‐simulation debrief.
G‐protein coupled receptor kinases’ (GRKs) role in APC/PAR1‐induced endothelial cells
Authors: Ramirez Monica Lizeth Gonzalez , Lin Ying , Molinar-Inglis Olivia , Birch Cierra , Trejo JoAnn
Published: April 2020

Endothelial dysfunction is a hallmark of inflammation and prevalent in vascular diseases. Recent efforts are focused on developing new strategies to enhance endothelial resistance to inflammation. One promising candidate is activated Protein C (APC), which has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses. APC enhances endothelial cytoprotection by activation of PAR1 through cleavage at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. G‐protein coupled receptor kinase (GRK) family plays a role in phosphorylating PAR1 and recruiting b‐arrestins, however, the contribution of GRKs to b‐arrestin driven cytoprotection is unknown. Our study focuses on determining the role and mechanisms by which GRKs regulate APC/PAR1‐induced endothelial cytoprotection to delineate their function in this pathway. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.

Support or Funding Information

This work is funded by NIH R01 GM090689, NIH R35GM127121, and IRACDA K12 GM068524.
Expression and Characterization of the Drosophila melanogaster (Dm)IKKβ:γ complex
Authors: Cohen Samantha , Wu Sheri , Huxford Tom
Published: April 2020

In Drosophila, the IMD pathway is indispensable for proper innate immune responses. Infection by gram‐negative bacteria elicits a signaling cascade culminating in the rapid induction of antimicrobial peptide gene expression by the NF‐κB transcription factor Relish. Signal‐dependent activation of Relish is an essential component in the IMD pathway and is regulated by the Drosophila melanogaster IκB Kinase (DmIKK) complex. DmIKK is composed of two subunits: the catalytic subunit IKKβ and non‐catalytic subunit IKKγ. Like many protein kinases, DmIKK must be activated for proper catalysis. Although there has been extensive research into the signaling pathways of the Drosophila innate immune response, little is known about the molecular mechanisms that lead to DmIKK activation. Here we report expression, purification, and in vitro characterization of a catalytically active multi‐subunit DmIKK complex.

Support or Funding Information

This work is supported in part by the California Metabolic Research Foundation. S. Cohen is a recipient of the Arne N. Wick predoctoral fellowship.
Analyzing the Localization of SAUL1 and AtPUB43 Proteins in Nicotiana benthamiana
Authors: Alcantara Michelle A. , Tyler Brett
Published: April 2020

Plants have evolved elaborate immune systems to resist infection, and thus are resistant to most microbes. In response to this resistance, pathogens have evolved in order to successfully overcome the immune systems of their specific hosts. Over the last few years, a new mechanism used both by plants for defense and by microbes for attack has been uncovered. This mechanism involves the secretion of membranous vesicles called exosomes. Exosomes transport small RNAs, proteins, and toxic chemicals from one organism to another during infection. However, little is known about the mechanisms by which plants and pathogens produce and release exosomes. Multivesicular bodies have been identified as a potential source of exosomes through fusion with the plasma membrane, but the mechanism of fusion is unknown. Recently the Tyler lab documented that, in plants, MVBs could be tethered to the plasma membrane by artificial protein fusions and by certain E3 ubiquitin ligase proteins that contain ARM (armadillo) domains, namely the Arabidopsis proteins SAUL1 and AtPUB43. They showed that the ARM domains of SAUL1 and AtPUB43 could trigger tethering of MVBs to the plasma membrane. Furthermore, the full‐length proteins could bind the plasma membrane and could trigger tethering during infection of plants by Phytophthora. This research project aims to test which parts of the SAUL1 and AtPUB43 proteins are involved in tethering using agroinfiltration. Different pieces of the proteins will be fused to Venus fluorescent protein and then expressed in Nicotiana benthamiana leaves. The leaves, examined by confocal microscopy, will observe whether tethering has occurred. Proteins will be expressed independently in Nicotiana benthamiana leaves to verify their localization. Once verified, proteins will then be coexpressed and analyzed to see if any tethering had occurred. In this study, we have found that the artificial infusion of the proteins, Vam7p and ACBP1 coexpressed, triggered the fusion of MVBs to the plasma membrane. The tethering of MVBs seen from these proteins in Nicotiana benthamiana leaves, can further test the hypothesis that Vam7p and ACBP1 can trigger the tethering of MVBs to the plasma membrane in the pathogen Phytopthora sojae. This can further explore the hypothesis that tethering might be involved in the release of exosomes of Phytophthora during infections.
New insights from The Cancer Genome Atlas: Implications for GPCR expression in cell compartments in solid tumors
Authors: Sriram Krishna , Moyung Kevin , Insel Paul A.
Published: April 2020

Assessment of the transcriptome of tumors via high‐throughout methods such as RNA‐sequencing (RNA‐seq) has yielded insights that enable understanding of tumor biology and facilitate drug discovery efforts. A challenge with using such “omics” methods is the presence of technical variation between groups of samples (often termed ‘batch effects’) which arise from differences in sample collection, handling and processing, especially with large, multi‐center, consortium‐based efforts such as The Cancer Genome Atlas (TCGA). Approaches to correct for technical artifacts should seek to minimize variance introduced by known technical factors, while preserving the biological variability within the dataset. We present a workflow for assessing the magnitude of identifiable technical artifacts within RNA‐seq data in TCGA and to identify the most relevant sources of variation are most relevant and steps to correct for such artifacts using bioinformatics tools. We show that certain TCGA datasets are highly impacted by technical variation and that correction for these artifacts can alter downstream analyses such as association studies and survival analysis. Correction for these effects using batch correction tools removes the influence of technical artifacts, while preserving biological variation and the overall structure and integrity of the data. We use these corrected data to establish an updated list of significant genes that impact survival in a range of solid tumor types in TCGA . In addition, we use methods to identify networks of co‐expressed and co‐regulated genes and identify genes that are sensitive to molecular features of tumors, such as the presence of specific driver mutations.

Using these “tidied‐up” datasets, we analyzed the expression of GPCRs (G protein‐coupled receptors) in tumors to identify the cellular compartments within the tumor microenvironment that express particular GPCRs. Due to their widespread utility as pharmacological targets and numerous recent results re‐emphasizing a role for GPCRs in cancer, such analyses provide a valuable tool for drug discovery. We document a number of GPCRs that are widely expressed in immune cells in a range of solid tumors and that whose functions may impact the phenotype of immune cells, including in cancer. Such widely expressed immune‐associated GPCRs are examples of novel putative targets in the rapidly growing field of immuno‐oncology. These analyses for GPCRs can be expanded to other classes of targets as well, providing a general tool for using large databases of gene expression to identify and evaluate genes that are likely to be expressed in cell types within tumors in vivo, thereby avoiding pitfalls that arise when working with isolated cells or single‐cell based approaches.

Support or Funding Information

ASPET David Lehr Award
Butyrate Priming Improves Gut Health by Modulating Host Protease Activity in Murine Non‐Alcoholic Fatty Liver Disease
Authors: Mondal Ayan , Seth Ratanesh , Bose Dipro , Nagarkatti Mitzi , Nagarkatti Prakash , Chatterjee Saurabh
Published: April 2020

Reports from clinical investigations implicate a strong association of non‐alcoholic fatty liver disease (NAFLD) and inflammatory bowel diseases (IBD). Recently we reported a rapid alteration of gut microbiome in murine NAFLD model that resulted in gut leaching, tight junction protein alteration and intestinal inflammation. Advancing our present understanding, we show that altered gut microbiome in murine NAFLD significantly decreased microbial proteases content in the host gut micro‐environment. Dysbiosis significantly increased expression of host proteases derived from gut immune cells which were associated with intestinal inflammation and gut leaching. Results also showed significant upregulated expression of a disintegrin and metalloproteinase 17 (ADAM17) and serine proteases Granzyme M, Cathepsin G and Proteinase 3 in intestines of NAFLD mice. Increased expression of these proteases were associated with activation of protease activated receptor 1 (PAR1), significant over production of pro inflammatory cytokines TNFα and IL‐1β, decreased expression of Occludin, and increased expression of Claudin2. Most interestingly administration of butyrate to NAFLD mouse group significantly increased microbial protease content in gut, decreased expression of ADAM17, Granzyme M and Cathepsin G, and increased expression of protective matrix metalloproteinases 7 and 2 (MMP7 and MMP2). Decreased expression of ADAM17, Granzyme M and Cathepsin G improved gut leaching by upregulation of Occludin and down regulation of Claudin 2 expression, decreased PAR1 expression, and decreased TNFα and IL‐1β secretion. Furthermore, increased expression of MMP 2 and MMP 7 showed elevated expression of anti‐inflammatory cytokines IL‐4 and IL‐10 in small intestine. Thus, in conclusion our present study demonstrates that butyrate plays a crucial role in gut homeostasis by a previously unknown function of modulating host protease activity in NAFLD and can be of significant therapeutic value.

Support or Funding Information

This project has been supported by NIH grants # 2P01AT003961 (Project 4) and #P20GM103641‐07(Project3) to Dr. Saurabh Chatterjee
Protein structure, a new constraint for glycosyltransferase specificity: Template‐like association between the glycoprotein surface on glycan structure
Authors: Kellman Benjamin P. , Lewis Nathan E.
Published: April 2020

The multitude of glycans on any cell surface can appear homogeneous overall, but a more site‐specific perspective suggests some deliberate diversity. Many have argued that site‐specific microheterogeneity arises from the influence of protein structure on glycan synthesis, yet, the prevailing consensus is that glycan synthesis is non‐template driven. We examine the possibility that the diversity of site‐specific glycoprofiles is a result of the affinity for glycosyltransferases to various regions of that protein. In this study, we examine an empirical glycoprotein database containing evidence of site‐specific glycosylation events. Each glycan substructures was correlated to site‐specific protein structure features (e.g. secondary structure, proximal amino acids). By characterizing correlations between glycan substructures and local protein chemistry, we have produced a draft template which we have applied to further our understanding of the HIV envelope mannosylation, predict temporospatial changes in flu glycosylation, explain discrepancies in amino acid substitutions matrices, predict TAPBP dependence in HLA, and predict secretion rates. In an attempt to share our advance, we are currently predicting glycosylation throughout PDB and SWISSMOD and identifying mutations in ClinVar most likely to be glycan‐modulated. By determining occlusion of GTs on structurally annotated sites, we can learn about the structural specificities of these impactful proteins thereby increasing our capacity to understand and predict their behavior.

Support or Funding Information

NIH 1 R21 HD088953, NNF10CC1016517
Proteolytic activation of the epithelial sodium channel: role of pro‐protein convertases and prostasin
Authors: Svenningsen Per , Granhoj Jeff , Dimke Henrik
Published: April 2020

The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro‐protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC.

We used the M‐1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro‐protein convertase inhibitor decanoyl‐RVKR‐chloromethyl ketone (dec‐RVKR‐cmk) and serine protease inhibitor camostat mesilate (CM). Knockout cell lines of prostasin and three PCSKs were generated with the CRISPR/Cas9 system in M‐1 cells. As an index of ENaC activity, amiloride‐sensitive short‐circuit current (ISC) and transepithelial voltage (VTE) was measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane levels and cleavage were tested by western blotting of surface biotinylated M‐1 cells with an antibody against the C‐terminal of γENaC. All values are means SE, compared with students t‐test or ANOVA as appropriate.

Pro‐protein convertase inhibition decreased the amiloride‐sensitive ISC in M‐1 cells compared with controls (25.9 1.9 μA, n=5, vs. 45.7 7.0 μA, n=6, p<0.05). Similarly, serine protease inhibition decreased VTE (−34.5 7.8 mV, n=3, vs. −74.2 0.9 mV, n=3, p<0.05) but ISC was not significantly lower (68.0 8.5, n=3, vs. 74.0 2.3, n=3, n.s.). Preliminary evidence suggests that although both inhibitors decreased membrane expression of γENaC, only serine protease inhibitor treatment reduced γENaC cleavage (n=1). Knockout of the Psck3 (furin) gene by CRISPR completely abolished ENaC activity, while knockout of Pcsk4, Pcsk7, and prostasin all showed decreased ENaC activity compared to wild‐type M‐1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin reduced γENaC cleavage.

We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.

Support or Funding Information The Danish Medical Research Council
Insulin Similar to Activator of KCNQ1 Channel Recovers the Prolonged Repolarization of Ventricular Cardiomyocytes from Insulin Resistant Aged Rats
Authors: Turan Belma , Olgar Yusuf , Durak Aysegul , Tuncay Erkan
Published: April 2020

Both cardiac dysfunction and systemic insulin resistance mostly observed together with advancing aging or metabolic syndrome (MetS), while the exact underlying mechanisms of heart‐specific insulin resistance and cardiac dysfunction still remain unclear. There also are relations between delayed ventricular repolarization, increased risk in arrhythmia and QT‐prolongation in ECG with diabetes, aging and MetS. They also showed that the mutations/functional alterations in QT‐prolongation related K+‐channels such as KCNQ1‐channels might be responsible for these changes. In addition, most of recent observations that insulin sensitivity of glucose metabolism are affected by a new molecule KCNQ1, which brings up a thought on possible relation of development of insulin resistance, changes in this channel and QT‐prolongations in senescent mammalian cardiomyocytes. Therefore, we aimed to clarify a possible association between prolonged action potential duration and alterations in KCNQ1‐channel function as well as insulin application in ventricular cardiomyocytes from either insulin resistant aged or MetS rats. Our data showed that there is a significant prolongation in action potential repolarization phase in isolated cardiomyocytes from insulin‐resistant aged or MetS rats, while those animals have a significant prolonged‐QT in ECG and can respond to insulin application. Besides, application of KCNQ1‐channel activator reduced the prolonged action potential repolarizations and this indicates the contribution of KCNQ1‐currents and possible benefits of insulin on action potential prolongation of both aged and MetS cardiomyocytes. Our present study also showed that the protein expression level of KCNQ1 and KCNE1 remained unchanged. Overall, our present results can reveal important information on the contributions of these parameters on the aging‐related or metabolic syndrome‐related cardiac dysfunction and the effect of insulin on these alterations. Therefore, our findings will contribute to the literature and the clinical applications with a new therapeutic approach by revealing the molecular mechanisms of insulin treatment on insulin resistant aged or MetS mammalian cardiac dysfunction.

Support or Funding Information

Supported through TUBITAK SBAG‐119S661
Identification of Critical MCUR1 Domains in the Mitochondrial Calcium Uniporter Complex that Regulates Cellular Metabolism
Authors: Enslow Benjamin Troy , Madaris Travis , Ravichandran Joel , Jacob Reggie , Srikantan Subramanya , Stathopulos Peter , Muniswamy Madesh
Published: April 2020

Mitochondria are multivariate signal processors that decode diverse cellular inputs into various bioenergetic outputs. One such signal is the calcium (Ca2+) flux between the cytosol and mitochondria which modulates the organelle’s function. Under physiologic cytosolic [Ca2+], energy output (ATP) can be adjusted to meet cellular demands. To maintain the Ca2+ homeostatic circuit, a selective mitochondrial Ca2+ Uniporter (MCU) complex promotes Ca2+ uptake. MCUR1 is an integral mitochondrial inner membrane protein which serves as a scaffold factor for the MCU complex and regulates its functional activity. The resolved NMR structure of the human MCUR1 protein revealed a high degree of conservation for 3 residues within the “head domain” of the protein, A170, L174, V203, and Y204.

Objective

We hypothesized that alterations of these critical residues would affect MCUR1 function, and thereby, MCU complex activity.

Methods

To test this hypothesis, MCU‐mediated mitochondrial calcium (mCa2+) uptake, resting matrix mCa2+ levels, and mitochondrial bioenergetics such as NAD(P)H production and oxygen consumption rate (OCR) were evaluated.

Results

Remarkably, cells expressing the MCUR1A170R/L174R mutant demonstrated near complete ablation of MCU‐mediated mCa2+ uptake, NAD(P)H production and OCR. Conversely, cells expressing MCURWT, or the MCURV203R/Y204R mutant demonstrated similar effects on MCU activity and mitochondrial bioenergetics suggesting a nominal change at the structural level.

Conclusion

Collectively, our results indicate that the MCUR1 head domain is necessary for proper MCU complex activity, and that MCU‐mediated mCa2+ uptake modulates mitochondrial bioenergetics and cell function.

Support or Funding Information

This research was funded by the National Institutes of Health R25R25DK078381 program. (NIHR01GM109882, R01HL086699, R01HL142673, DOD/CDMRP PR181598P and NIH1S10RR027327 to M. Madesh and R25DK078381 to W.B.R. B.T.E. is supported by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award Number TL1TR002647. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
CAROGENIC MICROORGANISMS IN CHILDREN WITH PRIMARY AND MIXED DENTITION
Authors: Rodriguez Patricia Noemi , Mateo Maria Teresa , Iglesias Maria Veronica , Alonso Maria Gabriela , Lifshitz Fima , Molgatini Susana , Friedman Silvia Maria , Canga Liliana
Published: April 2020

The oral cavity is one of the most important and complex habitats of microorganisms, housing a great diversity of communities, which colonize the mucous membranes and form the dental biofilm. The acidification of biofilm by carbohydrate intake is an important factor for the onset and progression of carious lesions, where the microorganisms involved produce acids and tolerate a low pH medium.

Objective

Determine in children with and without carious lesions the composition of the supragingival biofilm according to the type of dentition.

Materials and methods

The study population included 49 children (24 with primary dentition PD, 36–72 months of age and 25 with mixed dentition MD, 74 to 110 months of age), with and without carious lesions. Presence of caries was determined by the visual method ICDAS and dietary history was recorded. To perform microbiological study of acidogenic microorganisms (SM: Streptococcus spp, L: Lactobacillus spp, C: Candida spp. and V: Veillonella spp), a sample of supragingival biofilm plaque was collected from molar.

Agar mitis salivarius with bacitracin, MRS agar (Britany®Argentina), selective and differential medium CHROMagar Candida and selective medium for Veillonella with the addition of vancomycin and lacquered blood were inoculated. All were incubated under atmospheric conditions according to the requirements of each of the studied microorganisms. Results were statistically analyzed using ANOVA or Fisher, when appropriate. The study was approved by the Ethics Committee of the School of Dentistry‐UBA.

Results

SM and L counts was higher in PD, regardless of the presence of caries, p< 0.0001. In PD the percentage of positive C cultures was higher in children with caries, differences that were not observed in MD (p=0.0478). With respect to V, only a higher percentage of positive crops were observed in children with caries in MD (p=0.0119).

Conclusion

the count of SM and L and the presence of C and V vary with the type of dentition, suggesting a change in the composition of the microbiota, even in teeth without caries.

Support or Funding Information

Granted by UBACYT 20720170100005BA.
Subcutaneous grafting of ovaries from newborn rabbits into adult ovariectomized mice does not result in normal folliculogenesis
Authors: Valdez Dominik , Ruiz Saul , Foster Robert , Rodriguez-Sosa Jose Rafael
Published: April 2020

About 10% (6.1 million) of women in the United States ages 15–44 have difficulty becoming pregnant or staying pregnant. About 1/3 are fertility problems in women. Abnormal ovarian function is a major contributor. A greater understanding of ovarian function and especially follicular development would improve treatment. Follicles form when germ cells arrive in the primitive sex cords, which are derived from the coelomic epithelium. In most species, these cords fragment and the germ cells become covered by precursors of the granulosa cells. Thus primordial follicles form. Pools of these follicles are eventually recruited and undergo maturation; a series of functional and morphological transformations that lead to ovulatory follicles. In species like humans, cattle and pigs, follicles form during the embryonic phase of gestation, while maturation takes place after birth. In contrast, both phases occur postnatally in rabbits and makes them an excellent model to study folliculogenesis. Laboratory studies using non‐rodent species are logistically more difficult and even prohibitive. One alternative is ovarian grafting into immunocompromised mice. When adult ovarian tissue is transplanted from large animals into mice, some primordial follicles survive and undergo maturation. We investigated the rabbit model by transplantation of immature rabbit ovaries under the dorsal skin of adult ovariectomized NCr N
ude mice (1 ovary / mouse). At 4 – 5 weeks, growth of the grafted ovaries was evident. Macroscopically, 50% of the grafts (5/10) had one or two follicle‐like structures on their surface. Histologically the structures were cysts lined by cuboidal cells and contained cells with the germ cell phenotype. In conclusion, ovaries grafted subcutaneously from newborn rabbits into ovariectomized adult mice developed cystic primordial follicles. Normal follicle formation and maturation did not occur.

Support or Funding Information

Midwestern University intramural funds
A study on Morphometric Features of Esophagus
Authors: Alimohammadi Majid , Doroudi Majid , Zilabi Golsan , Ebrahimi Azarnoush
Published: April 2020

Certain data such as weight and dimensions of anatomical structures are important in dosimetry for treating cancer patients using radiotherapy.

The objective of this study is to determine the morphometric features of esophagus and discuss the possible correlation between the height and weight of esophagus with other parameters such as height, weight and age of patients. 23 samples were measured using measuring tape and digital scale. The results of measurements showed significant correlation between the age of individuals and the weight and length of esophagus. However, no significant correlation was found between the length and weight of esophagus with either gender, height or weight of individuals.
SPERMIDINE DOES NOT INFLUENCE LIMB MUSCLE MASS FOLLOWING ANDROGEN DEPLETION
Authors: Rossetti Michael L. , Casero Robert A. , Gordon Bradley
Published: April 2020

Skeletal muscle atrophy increases the risk of morbidity and mortality during various pathological conditions. In males, a decrease in the production and/or bioavailability of androgens (termed hypogonadism) directly contributes to muscle atrophy during such conditions. While it is known that androgens prevent muscle atrophy, the mechanism(s) by which androgens mediate this effect are largely undefined. To identify potential androgen sensitive pathways regulating limb muscle mass, we subjected total RNA from the tibialis anterior (TA) from castrated mice and total RNA from the TA of sham mice to microarray analysis. This approach showed that androgen depletion significant altered the expression of genes involved in the polyamine metabolic pathway. The polyamines, spermidine (Spd), spermine (Spm) and their diamine precursor putrescine, are naturally occurring, polycationic alkylamines that are absolutely required for mammalian cell growth, differentiation, and survival. Accordingly, we confirmed that androgen depletion reduced muscle expression of ornithine decarboxylase (ODC1), S‐adenosylmethionine decarboxylase (AMD1), spermine oxidase (SMOX), and spermidine/spermine N‐1acetyltrasferase (SAT1), which are genes involved with polyamine biosynthesis (ODC1, AMD1) and catabolism (SMOX, SAT1). Changes in the expression of these polyamine metabolic genes following androgen depletion coincided with a preferential decrease in Spd concentration in the TA, and this reduction was strongly related to the degree of muscle atrophy induced by the androgen depletion. To assess the role of Spd in the regulation of limb muscle mass, we treated previously castrated mice with Spd in the drinking water for 8 weeks. Even though Spd treatment completely prevented the decrease in muscle Spd concentrations following androgen depletion, mass of the TA or other limb skeletal muscles was not affected. Moreover, treating previously castrated mice with nandrolone decanoate increased muscle mass and restored expression of the polyamine metabolic genes without restoring Spd values. Thus, the decrease in Spd concentration in the limb muscle following androgen depletion likely mediates processes that are independent of muscle mass regulation.

Support or Funding Information

NIH Grant CA204345 to (RAC) Institute for Successful Longevity to (BSG)