bluuebunny/crossref_metadata_2025_split huggingface dataset:
Muscle biopsy in anti-neutrophil cytoplasmic antibody–associated vasculitis: diagnostic yield depends on anti-neutrophil cytoplasmic antibody type, sex and neutrophil count
Authors: Lacou Mathieu , Leroy Maxime , Le Lan Nowenn , Toquet Claire , Espitia-Thibault Alexandra , Graveleau Julie , Masseau Agathe , Agard Christian , Volteau Christelle , Mussini Jean-Marie , Hamidou Mohamed , Néel Antoine 
Published: April 2020

Abstract

Objectives
This study aimed to examine the sensitivity of muscle biopsy (MB) in ANCA-associated vasculitis (AAV), identify factors predicting MB positivity and assess the prognostic value of a positive MB.

Methods
We conducted a single-centre retrospective study of AAV with an MB performed at diagnosis. AAV classification [granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA)] followed the European Medicines Agency algorithm. A logistic regression model was used to identify the factors associated with MB positivity. Survival curves were generated using the Kaplan–Meier method.

Results
Among 276 AAV patients (1995–2018), 101 had an MB. Seventy-eight patients were included: 33 with GPA, 25 with MPA and 20 with EGPA. MB samples were positive in 45 cases (58%): 17 GPA, 16 MPA and 12 EGPA. Univariate analysis focussed on GPA and MPA, revealed that the MB yield was higher in females [22/31 (71%) vs 11/27 (41%); P = 0.02] and in anti-MPO patients [25/37 (68%) vs 6/19 (32%) for anti-PR3; P = 0.01]. By multivariate analysis, three factors predicted MB positivity: anti-MPO ANCA [odds ratio (OR) 10.67 (CI 2.09, 81.68)], female sex [OR 5.3 (CI 1.16, 32.35)] and neutrophil count [OR 1.33 (CI 1.07, 1.8)]. MB positivity had no impact on relapse, death or end-stage renal disease–free survival.

Conclusions
MB is a safe and efficient diagnostic tool for AAV. Predictors of MB yield include ANCA type, sex and neutrophil count. MB cannot substitute for kidney biopsy when indicated, but should be considered in other cases.
Mechanistic Dissection of Spatially Encoded GPCR Signaling Through High‐throughput CRISPR Screening
Authors: Tsvetanova Nikoleta 
Published: April 2020

Our recent work has overturned the prevailing textbook model for GPCR signaling, which held that GPCR activation at the plasma membrane is responsible for all downstream consequences of signaling. We have demonstrated that receptor/second messenger signaling on internal compartments but not plasma membrane drives all receptor‐dependent transcriptional responses, and the site of receptor activation can dictate how cells “interpret” different ligands, including medically relevant drugs. This phenomenon of functional specialization of signaling based on receptor localization has been gaining significant support in the last few years from us and other groups, yet not much is known about its underlying mechanisms and functional consequences. To begin to address these open questions, we set up a functional genomic screen aimed at identifying factors regulating ß2‐adrenergic receptor (ß2‐AR)‐dependent transcription‐ a location‐biased GPCR signaling response. We combined pooled CRISPR interference libraries targeting all annotated transcripts in the human genome and a novel fluorescent transcriptional reporter for GPCR/cAMP signaling that we have developed with fluorescence‐activated cell sorting and deep sequencing. Through this comprehensive screen, we have identified both known and novel mediators of the GPRC pathway. We are currently defining the biochemical mechanisms and specificity of the new regulators and their contributions to location‐biased receptor signaling, and have intriguing data linking a highly‐penetrant genetic risk factor for psychosis to these pathways.Support or Funding InformationNational Institute of Mental Health [Grant MH10963301]
The role of sympathetic activation and carbon dioxide tension in human neurovascular coupling
Authors: Rosentreter Ryan E , Mirzadzare Niloofar , Cho Woojin , Potemkin Michael , Scott Berkeley A , Soriano Jan Elaine , Fisher James P , Phillips Aaron A 
Published: April 2020

Neurovascular coupling describes a series of processes that serve to match blood supply to neuronal metabolism within the central nervous system. We know that the sympathetic nervous system plays a role in several cerebrovascular control pathways, and preclinical non‐human work demonstrates that the sympathetic nervous system may play a role in neurovascular coupling. These preclinical models utilize isolated cell preparations lacking key inputs to the neurovascular unit such as that of the autonomic nervous system, or utilize anesthetics which blunt cerebrovascular regulatory pathways.Here, our objective was to provide preliminary insight into the role of sympathetic nervous system activation in neurovascular coupling by using our non‐anesthetized, non‐stressed, human, truly in vivo model. We utilized moderate lower body negative pressure (−40 mmHg) to elevate sympathetic activity without affecting mean arterial pressure. Beat‐by‐beat blood pressure was recorded via finger photoplethysmography, while cerebral blood velocity in the middle and posterior cerebral arteries was measured via transcranial Doppler. Neurovascular coupling was elicited using our standardized visual stimulus protocol and data was analyzed using our custom software. As lower body negative pressure leads to sympatho‐excitation and hyperventilation, in a subset of individuals we maintained end‐tidal carbon dioxide levels identical to baseline during lower body negative pressure.Absolute cerebral blood velocity was influenced by arterial carbon dioxide levels. Neurovascular coupling was preserved during lower body negative pressure induced sympatho‐excitation and reduced arterial carbon dioxide levels.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Libin Cardiovascular Institute, Hotchkiss Brain Institute, Compute Canada
Static Magnetic Field accelerates diabetic wound healing by facilitating resolution of inflammation
Authors: Ren Huiwen , Shang Wenlong , Yu Ying 
Published: April 2020

Impaired wound healing is commonly encountered in patients with diabetes mellitus, which may lead to severe outcomes such as amputation, if untreated timely. Macrophage plays a critical role in the healing process including the resolution phase. Although magnetic therapy is known to improve microcirculation, its effect on wound healing remains uncertain.In the present study, the model of diabetic mice was used to prove the effect of static magnetic field (SMF) in wound healing process. The positive role of SMF in macrophage polarization was observed in wound areas of db/db mice by the immunofluorescence staining and flow cytometric analysis. In order to examine the molecular mechanism of SMF in the wound healing, peritoneal macrophages were isolated from db/db mice and treated by LPS or IL‐4.We found 0.6 T SMF significantly accelerated wound closure rate by 50%. Histological analyses revealed significantly shorter distances by 26.7% between the epithelial tips of punched wound and distances by 33.3% between the edges of the panniculus carnosus in SMF group at day 7 postoperatively. SMF promoted M2 macrophage recruitment by 87.5% and reduced M1 macrophages accumulation by 24.3%, which was observed by immunofluorescence staining in wound areas of db/db mice. Flow cytometry analysis also revealed that M2 macrophages (F4/80+CD206+) were increased and M1 macrophages (F4/80+CD206−) were significantly decreased, indicating SMF facilitates M2 polarization in inflamed tissues. The expression levels of pro‐inflammatory markers (iNOS, IL‐6, IL‐1β, CCR7) in injured tissues were paradoxically downregulated in the SMF group. Meanwhile, SMF significantly raised the expression levels of reparative genes (CD206, Fizz1, Arg‐1, IL‐10). The pro‐inflammatory iNOS expression was decreased and anti‐inflammatory Arg‐1 was increased in SMF/LPS/IL‐4‐treated macrophages. Furthermore, SMF inhibited LPS‐induced STAT1 phosphorylation and augmented IL‐4‐induced STAT6 phosphorylation in the macrophages.In summary, our study showed for the first time the effect of 0.6 T SMF on wound healing in diabetic mice. These results indicate that SMF accelerates diabetic wound healing by promoting macrophage polarization and resolution of inflammation through modulation of the JAK‐STAT pathway. Therefore, focusing on SMF in therapeutic interventions might be useful for treating diabetic wound by re‐normalizing the healing process.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (grants 81703591).
Fuzzy Interactions Between 14‐3‐3ζ And Tau Regulate Tau Aggregation
Authors: Huang Fan , Shu Qianhui , Qin Zhaojie , Tian Jianglin , Gao Meng , Huang Yongqi 
Published: April 2020

The microtubule associated protein tau is vital for elongation and stability of microtubules in the central nervous system. However, aberrant accumulation of tau amyloid fibrils in neurons is a character of a neurodegenerative pathology called Alzheimer’s disease. Extensive studies have revealed that several factors promote aggregation of tau, including mutations, polyanions, phosphorylation, and interactions with other proteins. Previous studies have shown that the conserved scaffolding protein family 14‐3‐3 proteins interact with tau in a phosphorylation dependent manner and 14‐3‐3 proteins promote tau aggregation. Crystal structure of 14‐3‐3/tau complex revealed that phosphorylated tau segments bind to the ligand binding grooves of 14‐3‐3 dimer. Consequently, it is promising that 14‐3‐3 specific binding ligands will reduce/inhibit tau aggregation. Indeed, rational designed inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. Although thermodynamics characterization showed that non‐phosphorylated tau segments bind to 14‐3‐3 much weaker than phosphorylated tau segments, 14‐3‐3ζ also promotes non‐phosphorylated tau aggregation. In this work, we investigated the interactions between tau K18 construct and 14‐3‐3ζ. We found that non‐phosphorylated and PKA‐phosphorylated tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Replacing Ser324 with Ala had little effect of tau aggregation mediated by 14‐3‐3ζ protein. We further saturated the 14‐3‐3ζ ligand binding grooves by a high affinity peptide R18 and found that the presence of R18 could not reduce tau aggregation. Molecular dynamics simulations showed that tau K18 interact with 14‐3‐3ζ via extensive fuzzy interactions. Our results suggest that besides binding to the canonical ligand binding groove, tau also interacts with 14‐3‐3ζ via nonspecific interactions which are responsible for tau aggregation and cannot be inhibited by 14‐3‐3ζ specific inhibitors.Support or Funding InformationThis work was supported by Hubei University of Technology
Mechanistic Roles of Electrostatic Interactions in the Molecular Recognition of Intrinsically Disordered Proteins
Authors: Gao Meng , Huang Yongqi 
Published: April 2020

Electrostatic interactions could play important roles in the molecular recognition process of intrinsically disordered proteins (IDPs) as many IDPs and the recognition elements contain charged residues. Previous studies have demonstrated that electrostatic forces could accelerate the encounter process via electrostatic steering effect and increase the efficiency of binding upon encounter for several IDPs. To further investigate the universal role of electrostatic interactions in IDPs molecular recognition, we collected the experimentally determined binding kinetics of IDPs. For many studied IDPs, the association rate constants are reduced for about ten folds when the salt concentration increases from low (~50 mM) to high (~500 mM), indicating the presence of favorable electrostatic interactions. We further performed molecular dynamics simulations using structure‐based coarse‐grained models. Simulations predicted that long‐range electrostatic interactions accelerate the binding rate in a range consistent with experimental results. Simulations also revealed that electrostatic forces generally enhance the binding kinetics by increasing the encounter rate and by enhancing the evolving efficiency. Correlating the charge density with binding kinetics revealed that the overall charge density is relate with enhancement of the capture rate constant and the charge distribution affects both the evolution rate constant and escape rate constant. Analysis of the transition path times showed that electrostatic interactions stabilize the intermediate state along the binding process. Besides affecting the binding kinetics, electrostatic interactions also redistributed the flux among multiple parallel binding transition paths. As a summary, our results suggest that favorable electrostatic interactions are present in many IDPs recognition processes. Under physiological salt concentrations, electrostatic interactions are expected to enhance the binding rate constant by several folds.Support or Funding InformationThis work was supported by Hubei University of Technology
Effect of knock out of ASIC3 on cardiovascular reflexes arising from hindlimb muscle in ligated rats
Authors: Kim Joyce S. , Ducrocq Guillaume P. , Kaufman Marc P. 
Published: April 2020

The exercise pressor reflex (EPR) arises from contracting the skeletal muscle and is manifested by increases in arterial pressure, heart rate and blood flow to working muscles. Group III and IV afferents comprise the sensory arm of the exercise pressor reflex. The exercise pressor reflex is exaggerated in patients with peripheral artery disease, which is an atherosclerotic process that occludes blood flow to working muscles. The exaggeration of the exercise pressor reflex is believed to be caused by a metabolite whose concentration is increased when the working muscles are inadequately perfused. Previous work has shown that pharmacological blockade of acid‐sensing ion channel 3, which is found on the endings of group III and IV afferents, prevents the exaggeration of the exercise pressor reflex in rats with simulated peripheral artery disease. In the present experiments, we compared the magnitude of the exercise pressor reflex in decerebrated rats with a functional knockout of the ASIC3 (KO) with the magnitude of the reflex in their decerebrated wild type counterparts (WT). We found that the exercise pressor reflex in ASIC3 KO rats was significantly smaller than the exercise pressor reflex in their WT counterparts (p<0.05). In addition, ASIC 3 KO rats demonstrated significantly smaller pressor responses to injection of diprotonated phosphate (86mM; pH 6.0), lactic acid (12mM; pH 2.85), and capsaicin (0.2μg; pH 7.2) into the arterial supply of the hindlimb (p< 0.05). In contrast, both ligated WT and ASIC3 KO rats displayed similar pressor responses to calcaneal tendon stretch (p>0.05). We conclude that ASIC3 plays an important role in evoking the exaggerated exercise pressor reflex seen in rats with peripheral artery disease.Support or Funding InformationNIH Grants R01‐AR‐059397 and P01‐HL‐134609
Structural Disorder and Induced Folding of the WWC Family Proteins
Authors: Rodriguez Diego Javier , Kwok Ethiene , Chatterjee Tilottama , Baker Kasie , Nyarko Afua 
Published: April 2020

The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. Each protein contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains are named for two conserved tryptophans spaced 23 residues apart and adopt a three‐stranded antiparallel beta sheet fold. The WWC proteins are “atypical” in that, the second WW domain (WW2) contains an isoleucine rather than a conserved tryptophan. Recent studies of the KIBRA WW domains shows that this substitution leads to unfolding of the WW2 domains. However, the domain undergoes binding‐induced folding when bound to some partners but remains largely unfolded when bound to others. High sequence similarity of the WW domains in the WWC family suggests that unfolding and binding induced folding of the WW2 domain may be conserved in this family, but it is unclear why some partners induce folding of the WW2 domain while others do not. Our working hypothesis is that partners which contain PPxY motifs in close proximity will bind the WWC proteins with relatively high affinity and induce folding of the WW2 domain. In this study, we test this hypothesis with two binding partners, Large Tumor Suppressor 1 (LATS1), a kinase and tumor suppressor, and Synaptopodin (SYNPO), a synaptic and podocyte protein. We demonstrate by isothermal titration calorimetry that wildtype LATS1 and SYNPO which have two PPxY motifs each, separated by 194 and 15 residues respectively, bound KIBRA and WWC3 with modest micromolar binding affinities. However, mutant LATS1 (LATS1‐VA) and SYNPO (SYNPO_AA) constructs in which the PPxY motifs are separated by two amino acids, bound KIBRA and WWC3 with 20 to 180‐fold enhanced affinity. Using solution magnetic resonance spectroscopy, we demonstrated that the WW2 domain of KIBRA and WWC3 remain unfolded when bound to wildtype LATS1 and SYNPO, while the enhanced binding of the mutants LATS1_VA and SYNPO_AA is accompanied by folding of the WW2 domain. The structural heterogeneity of the WW2 domain may be an important for modulating binding to diverse PPxY motif partners.Support or Funding InformationThis work is supported by start‐up funds from Oregon State University (OSU), OSU College of Science Disease Mechanism and Prevention Fund, and a New Investigator Award from the Medical Research Foundation of Oregon. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162
Functional Studies of the Non‐Ribosomal Peptide Synthetase Cyclization Domain in Epothilone Biosynthesis Using Site‐Directed Mutagenesis
Authors: Kim Christopher , Gonzalez Reyaz , Soule Jessica , Dieu Viet , MacRae Mark , Dowling Daniel Peter 
Published: April 2020

Epothilones are anticancer natural products from Sorangium cellulosum that are comprised of a 16‐membered macrolactone ring with a thiazole side chain. This family of products is synthesized by a hybrid assembly line of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins. PKS modules insert small acyl groups into natural products through C‐C bonds, whereas NRPS modules incorporate amino acids. Two proteins, EpoA and EpoB, form the hybrid PKS‐NRPS biosynthetic starter unit that generates 2‐methylthiazole, a chemical moiety that contributes to epothilone bioactivity. EpoB harbors a cyclization (Cy) domain that is responsible for the generating 2‐methylthiazoline, which is subsequently oxidized to the thiazole. Initial crystallographic studies of the Cy domain revealed a primary tunnel entrance for the upstream carrier protein to deliver its phosphopantetheine‐tethered substrate to the active site. Interestingly, Cy domain structures from other systems contain a different upstream tunnel location. To uncover the roles of the two possible upstream tunnels in EpoB Cy, site‐directed mutations were incorporated that contain larger, bulkier residues predicted to interfere with phosphopantheine arm access to the active site. A previously developed LC/MS assay for determining the 2‐methylthiazole product was adapted in order to monitor product formation among the mutants in comparison to the wild type. These results contribute to an understanding of the function of each tunnel and provide insight to how the protein modules of the PKS‐NRPS biosynthetic starter units work together to catalyze the formation of 2‐methylthiazole.Support or Funding InformationFunding provided by NIGMS (Project number: 1R15GM123425‐01)
The Role of TIMP3 in Microvascular Endothelial Cell‐Extracellular Matrix Interaction and Regulation of Microvascular Barrier Function
Authors: Kulkarni Nidhi , Mehta Sanjay , Wang Lefeng , Pape Cynthia , Gill Sean 
Published: April 2020

BackgroundDuring sepsis, disruption of the pulmonary microvascular endothelial cell (PMVEC) barrier is associated with lung dysfunction and death. PMVEC‐PMVEC interactions through adherens and tight junctions are crucial for maintaining barrier function, as disruption of these junctions causes protein leak. Multiple mechanisms support PMVEC junctional and barrier function, including PMVEC binding to the extracellular matrix (ECM). Data suggest that decreased PMVEC‐ECM interactions under pro‐inflammatory conditions, indicated by altered focal adhesion kinase (FAK) phosphorylation, may be associated with increased leak. Tissue inhibitor of metalloproteinases 3 (TIMP3) is shown to regulate barrier function as PMVEC from Timp3−/− mice have increased leak; however, the specific mechanisms are unknown. Interestingly, previous studies in the developing lung found that TIMP3 can mediate PMVEC‐ECM interactions. Based on this, I hypothesized that TIMP3 maintains PMVEC barrier function by promoting PMVEC‐ECM interactions.MethodPMVEC from wild type (WT) and Timp3−/− mice were cultured on different matrices and stimulated with pro‐inflammatory cytokines (sepsis) vs control PBS. PMVEC‐ECM interactions were assessed using adhesion assays, immunocytochemistry, and the XPERT‐permeability assay.ResultsExamination of PMVEC adhesion to fibronectin, laminin, vitronectin, and collagen I indicated preferential adhesion of WT PMVEC to fibronectin with lowest adhesion to collagen. While Timp3−/− PMVEC appeared to also preferentially adhere to fibronectin, overall adhesion to the ECM vs. WT PMVEC was reduced. Assessment of phosphorylated FAK (pFAK) through immunocytochemistry revealed reduced abundance of pFAK in both cell types under cytomix‐treatment vs. PBS. Additionally, pFAK organisation and localised distribution around the periphery of cell borders appeared to be reduced in Timp3−/− vs. WT PMVEC. The XPERT‐permeability assay revealed differences in total inter‐PMVEC avidin fluorescence and specifically avidin leak diffusion distance between PMVEC and the ECM in WT vs. Timp3−/− cells, especially under cytomix‐treatment.SignificanceThese findings demonstrate that sepsis disrupts the interaction of WT PMVEC with ECM, which is further enhanced in Timp3−/− cells. The results suggest that TIMP3 may play an important role in regulating PMVEC‐ECM interactions, and could help elucidate therapeutic targets to maintain or rescue PMVEC barrier integrity in sepsis.Support or Funding InformationHeart and Stroke Foundation of Ontario, Ontario Thoracic Society Grant‐in‐Aid, NSERC Discovery Grant
Membrane Proteins — the Lipid Connection
Authors: Robinson Carol 
Published: April 2020

We have used native MS of membrane proteins liberated form micelles and native membranes to study protein lipid interactions. Using different Orbitrap MS platforms developed for membrane proteins we show that it is possible to distinguish lipids, cofactors and drugs while in direct contact with membrane protein targets.We also showed how drug binding mediates phosphorylation in a class A GPCR (P2Y1R) and that purification of three further class A GPCRs results in binding of specific lipids that mediate downstream coupling.The ability to detect binding which has both structural and functional effects for example on lipid flippase activity or G‐protein coupling, is a powerful and important attribute of the study of membrane proteins by mass spectrometry.Very recently we have added yet further insights by ejecting membrane protein directly from their native membrane environments. Recent results will be presented.Support or Funding InformationWellcome Trust Investigator AwardERC Advanced Grant MRC Programme Grant