bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mass spectrometric method for the unambiguous profiling of cellular dynamic glycosylation
Authors: Shajahan Asif , Supekar Nitin T. , Wu Han , Wands Amberlyn M. , Bhat Ganapati , Kalimurthy Aravind , Matsubara Masaaki , Ranzinger Rene , Kohler Jennifer J. , Azadi Parastoo 
Published: May 2020

ABSTRACTVarious biological processes at the cellular level are regulated by glycosylation which is a highly micro-heterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through bio-orthogonal tagging of metabolically engineered non-natural sugars into glycan epitopes. However, this approach possesses a significant drawback due to non-specific background reactions and ambiguity of non-natural sugar metabolism. Here we report a tag-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry. The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, GalNAc, and GlcNAc biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this we could deduce some interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.
A consistent evaluation of miRNA-disease association prediction models
Authors: Dong Ngan Thi , Khosla Megha 
Published: May 2020

AbstractMotivationA variety of machine learning based approaches have been applied to predicting miRNA-disease association. Although promising, the evaluation set up to measure prediction performance is inconsistent making it difficult to assess the actual progress. A more acute problem is that most of the models overlook the problem of data leakage due to the use of precomputed miRNA and disease similarity features.ResultsWe unearth a crucial problem of data leakage in evaluation of machine learning models for miRNA-disease association prediction. In particular, information from test set, in the form of precomputed input features for miRNA and disease, is used during training of the model. Moreover, we point out problems in the widely used performance metrics used in model evaluation. While resolving the issues of data leakage and model evaluation, we perform an indepth study of 3 recent models along with our proposed 9 variants of these models. Our proposed variants have resulted in improvements in Average Precision scores (as compared to original models) by approximately 287.7% and 36.7% on HMDDv2.0 (AP:0.504) and HMDDv3.0 (AP: 0.216) datasets respectively.Availability and ImplementationWe release a unified evaluation framework including all models and datasets athttps://git.l3s.uni-hannover.de/dong/simplifying_mirna_disease.
Inference of Population Admixture Network from Local Gene Genealogies: a Coalescent-based Maximum Likelihood Approach
Authors: Wu Yufeng 
Published: May 2020

AbstractPopulation admixture is an important subject in population genetics. Inferring population demographic history with admixture under the so-called admixture network model from population genetic data is an established problem in genetics. Existing admixture network inference approaches work with single genetic variation sites. While these methods are usually very fast, they don’t fully utilize the information (e.g., linkage disequilibrium or LD) contained in population genetic data. In this paper, we develop a new admixture network inference method called GTmix. Different from existing methods, GTmix works with local gene genealogies that can be inferred from population haplotypes. Local gene genealogies represent the evolutionary history of sampled alleles and contain the LD information. GTmix performs coalescent-based maximum likelihood inference of admixture networks with the inferred genealogies based on the well-known multispecies coalescent (MSC) model. GTmix utilizes various techniques to speed up likelihood computation on the MSC model and optimal network search. Our simulations show that GTmix can infer more accurate admixture networks with much smaller data than existing methods, even when these existing methods are run with much larger data. GTmix is reasonably efficient and can analyze genetic datasets of current interests.
Multistep substrate binding and engagement by the AAA+ ClpXP protease
Authors: Saunders Reuben A. , Stinson Benjamin M. , Baker Tania A. , Sauer Robert T. 
Published: May 2020

AbstractE. coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.SignificanceAAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show thatE. coliClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.
Identification and selection of optimal reference genes for qPCR-based gene expression analysis inFucus distichusunder various abiotic stresses
Authors: Linardić Marina , Braybrook Siobhan A 
Published: May 2020

AbstractQuantitative gene expression analysis is an important tool in the scientist’s belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown algaFucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter’s unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.
Mycobacterium marinumphthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage
Authors: Osman Morwan M. , Pagán Antonio J. , Shanahan Jonathan K. , Ramakrishnan Lalita 
Published: May 2020

AbstractPhthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence inMycobacterium tuberculosisandMycobacterium marinum. It has recently been shown that PDIMs work in concert with theM. tuberculosisType VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinummodel of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization inM. marinum. Using anΔmmpL7mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in anM. marinummacrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using anin vitrohemolysis assay— a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization inM. marinuminfection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.
Low Intensity Vibration Restores Nuclear YAP Levels and Acute YAP Nuclear Shuttling in Mesenchymal Stem Cells Subjected to Simulated Microgravity
Authors: Thompson M , Woods K , Newberg J , Oxford JT , Uzer G 
Published: May 2020

AbstractReducing the bone deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of rigorous and time-expensive exercise regimens under microgravity. The ability of low intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC) which maintain bone-making osteoblasts is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein) which is shuttled into the nucleus in response cyto-mechanical forces. Here, using YAP nuclear shuttling as a measure of MSC mechanoresponse, we tested the effect of 72 hours of simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied at the end of the 72h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry will be alleviated by daily application of LIVDT. Results showed that while both acute LIVATand LPA treatments increased nuclear YAP entry by 50% and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. Daily dosing of LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over the non-LIVDTtreated, SMG only, counterparts. These cell level observations suggest that utilizing daily LIV treatments is a feasible countermeasure for increasing the YAP-mediated anabolic responsiveness of MSCs to subsequent mechanical challenge under SMG.
The Proline-rich Domain Promotes Tau Liquid Liquid Phase Separation in Cells
Authors: Zhang Xuemei , Vigers Michael , McCarty James , Rauch Jennifer N. , Fredrickson Glenn H. , Wilson Maxwell Z. , Shea Joan-Emma , Han Songi , Kosik Kenneth S. 
Published: May 2020

AbstractTau proteinin vitrocan undergo liquid liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells we found that Cry2 can optogentically increase the association of full lengh tau with microtubules. To probe this mechanism, we identified tau domains that drive tau clustering on microtubules in living cells. The polyproline rich domain (PRD) drives LLPS and does so under the control of phosphorylation. These readily observable cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the ability of the PRD to undergo LLPSin vitro. In absence of the MTBD, the tau PRD co-condensed with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that utilize LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding alpha-beta tubulin heterodimers to the larger proportions of microtubules.
ESAT-6 ofMycobacterium tuberculosisdownregulates cofilin1 and reduces the phagosome acidification in infected macrophages
Authors: Mahesh P P , Retnakumar R J , Sivakumar K C , Mundayoor Sathish 
Published: May 2020

AbstractMycobacterium tuberculosiswhen phagocytosed by macrophages is not cleared completely and many of the bacteria remain in phagosomes indefinitely. In this study we considered abnormal retention of filamentous actin on early phagosomes contributing to defective phagosome acidification. Phosphocofilin1, the inactive form of actin depolymerizing protein cofilin1, which leads to retention of filamentous actin, and the total filamentous actin itself were found upregulated in macrophages infected with virulentM. tuberculosis. Over expression of constitutively active cofilin1 in macrophages was found to increase phagosome acidification when infected with virulentM. tuberculosis. The anticancer drug sorafenib which activates cofilin1 in PI3K dependent manner was also found to increase phagosome acidification. Cofilin1, known to be positively regulated by superoxide was found to be downregulated by ESAT-6 ofM. tuberculosiswhere the latter is known to reduce ROS in macrophages. Ectopic expression of ESAT-6 in macrophages was found to increase filamentous actin and to transform the macrophages more spindle shaped. ESAT-6 was also found to decrease phagosome acidification in macrophages infected with an avirulentM. tuberculosisstrain. Finally, this study proposes a role for the amino acid methionine in resisting ROS by creating M93 mutants of ESAT-6.
Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice
Authors: Yermanos Alexander , Neumeier Daniel , Sandu Ioana , Borsa Mariana , Waindok Ann Cathrin , Merkler Doron , Oxenius Annette , Reddy Sai T. 
Published: May 2020

AbstractNeuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer’s disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system (CNS). Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor (BCR) and T cell receptor (TCR) repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system (CNS) of aged mice. Furthermore, many of these B cells were of the IgM and IgD isotype and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.
E prostanoid receptor 4 expressing macrophages promote the regeneration of the intestinal epithelial barrier upon inflammation
Authors: Na Yi Rang , Jung Daun , Stakenborg Michelle , Gu Gyo Jeong , Jeong Mi Reu , Jang Hye Ri , Suh Soo Youn , Kim Hak Jae , Kwon Yoon Hey , Sung Tae Sik , Ryoo Seung Bum , Park Kyu Joo , Im Jong Pil , Park Ji Yong , Lee Yun Sang , Han Heon Jong , Park Bo Youn , Lee Sung Wook , Lee Ho Su , Cleynen Isabelle , Matteoli Gianluca , Seok Seung Hyeok 
Published: May 2020

AbstractDysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of resolution of inflammation but the mechanisms underlying their mucosal healing capacity remains elusive. Here, we describe a subset of E prostanoid receptor 4 (EP4) expressing intestinal macrophages with mucosal healing properties both in human and mice. Notably, Csf1r-iCre EP4-fl/fl mice showed defective mucosal healing and intestinal epithelial barrier regeneration in a dextran sodium sulfate-induced colitis model. Mechanistically, an increased mucosal level of prostaglandin E2 (PGE2) triggers the secretion of chemokine (C-X-C motif) ligand 1 (CXCL1) in monocyte-derived EP4+macrophages via MAPKs. Subsequently, CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during the resolution phase of colitis. Thus, EP4+intestinal macrophages are essential for the support of the intestinal stem cell niche and for the regeneration of the injured epithelium.One Sentence SummaryProstaglandin E2 licenses E-type prostanoid receptor 4 intestinal macrophage regenerative capacity promoting mucosal healing via the secretion of CXCL1
Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling
Authors: Amundsen Susan K. , Taylor Andrew F. , Smith Gerald R. 
Published: May 2020

AbstractRepair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5’ GCTGGTGG 3’) recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, >25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi-recognition-deficient mutant in the RecB helicase domain located >45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in the crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path ∼185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.
Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity
Authors: Tüting Christian , Iacobucci Claudio , Ihling Christian H. , Kastritis Panagiotis L. , Sinz Andrea 
Published: May 2020

AbstractThe ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of theE. coliribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143E. coliribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolutionE. coliribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimizedE. coliribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.
Reconstruction of natural images from responses of primate retinal ganglion cells
Authors: Brackbill Nora , Rhoades Colleen , Kling Alexandra , Shah Nishal P. , Sher Alexander , Litke Alan M. , Chichilnisky E.J. 
Published: May 2020

AbstractThe visual message conveyed by a retinal ganglion cell (RGC) is often summarized by its spatial receptive field, but in principle also depends on the responses of other RGCs and natural image statistics. This possibility was explored by linear reconstruction of natural images from responses of the four numerically-dominant macaque RGC types. Reconstructions were highly consistent across retinas. The optimal reconstruction filter for each RGC – its visual message – reflected natural image statistics, and resembled the receptive field only when nearby, same-type cells were included. ON and OFF cells conveyed largely independent, complementary representations, and parasol and midget cells conveyed distinct and expected features. Correlated activity and nonlinearities had statistically significant but minor effects on reconstruction. Simulated reconstructions, using linear-nonlinear cascade models of RGC light responses that incorporated measured spatial properties and nonlinearities, produced similar results. Spatiotemporal reconstructions exhibited similar spatial properties, suggesting that the results are relevant for natural vision.