bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in theCaenorhabditis elegansgerm line
Authors: Li Qianyan , Kaur Arshdeep , Okada Kyoko , McKenney Richard J. , Engebrecht JoAnne 
Published: October 2022

AbstractThe tumor suppressor BRCA1-BARD1 complex functions in many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, thein vivorequirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activityin vivousingC. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity reveal both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is essential for BRCA1-BARD1 to concentrate at both DNA damage and recombination sites in meiotic germ cells, but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRC-1 serves a structural role for DNA damage signaling and repair while BRD-1 plays an accessory role to enhance BRC-1 function.Author SummaryBRCA1-BARD1 is a E3 ubiquitin ligase, which modifies proteins by the addition of the small protein ubiquitin. While mutations that disrupt E3 ligase activity and stability of the BRCA1-BARD1 complex lead to a predisposition for breast and ovarian cancer, the specific requirement for E3 ligase activity in tumor suppression is not known. Here we probe the function of E3 ligase activity and BARD1 in the maintenance of genome integrity by engineering point mutations that disrupt E3 ligase activity inC. elegansBRCA1 as well as a null mutation in BARD1. We find that while E3 ligase activity is important for genome integrity, the complex plays additional roles besides ubiquitylating proteins. Further, our data suggest that BRCA1 is the key functional unit of the complex while BARD1 is an accessory partner that enhances BRCA1’s function. These findings may help explain why there is a higher prevalence of cancer-causing mutations in BRCA1 compared to BARD1.
Observed Kinetics of Enterovirus Inactivation by Free Chlorine Is Host Cell-Dependent
Authors: Torii Shotaro , David Shannon Christa , Larivé Odile , Cariti Federica , Kohn Tamar 
Published: October 2022

AbstractThe virucidal efficacy of disinfectants is typically assessed by infectivity assay utilizing a single type of host cell. Enteroviruses infect multiple host cells via different entry routes, and each entry route may be impaired to a varying extent by a given disinfectant. Yet, it is not known how the choice of host cells for titration affects the observed inactivation kinetics. Here, we evaluated the inactivation kinetics of echovirus 11 (E11) by free chlorine, ultraviolet (UV) irradiation, and heat, using three different host cells (BGMK, RD, and A549). E11 inactivation by free chlorine occurred at a two-fold greater rate when enumerated on BGMK cells compared to RD and A549 cells. Conversely, a comparable inactivation rate was observed for UV and heat independent of the host cell used. Host cell-dependent inactivation kinetics by free chlorine were also observed for echovirus 7, 9 and 13, and coxsackievirus A9, confirming that this phenomenon is not serotype-specific. Inactivation of E11 was partly caused by a loss in host cell attachment, which was most pronounced for BGMK cells, and which may be promoted by a lack of CD55 attachment receptors on this cell type. Additionally, BGMK cells lack a key subunit of the uncoating receptor, β2M, which may further contribute to the differential inactivation kinetic for this cell type. Consequently, inactivation kinetics of enteroviruses should be assessed using host cells with different receptor profiles. This will yield a more complete understanding of the inactivating power of disinfectants targeting the viral attachment and/or uncoating.Graphic for Table of Contents (TOC)
RNA-sensing pattern-recognition receptors synergize with TLR2 or dectin-1 to trigger high production of IL-12p70 by human dendritic cells
Authors: Gilmour Brian C. , Corthay Alexandre , Øynebråten Inger 
Published: October 2022

AbstractIL-12p70 is crucial for T helper 1 polarization and the generation of type 1 immunity that is required to fight cancer and intracellular pathogens. Therefore, strategies to optimize the production of IL-12p70 by dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies for cancer. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing all five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by human DCs. We used ten well-characterized agonists and stimulated human monocyte-derived DCsin vitrowith either single agonists or 26 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and/or MDA5, was the only agonist that could elicit IL-12p70 production when used alone. Combinations of agonists of cell surface and intracellular PRRs were found to synergize to induce high IL-12p70 production, given that the combination included poly(I:C) or resiquimod, which both are agonists of intracellular, RNA sensing PRRs (TLR3/MDA5 and TLR7/8, respectively). Our data show that production of high IL-12p70 is strictly controlled, which is different from what we observed for IFNβ, whose production could be elicited by several intracellular PRRs. In conclusion, we identified six different combinations of PRR ligands able to induce high IL-12p70 production by human DCs. The identified synergistic PRR ligand combinations may represent strong adjuvant candidates in particular for therapeutic cancer vaccines.
More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases
Authors: Gaballa Ahmed , Wiedmann Martin , Carroll Laura M. 
Published: October 2022

AbstractMobilized colistin resistance genes (mcr) may confer resistance to colistin, a last-resort, critically important antimicrobial for human health. mcr can often be transmitted horizontally (e.g., via mobile genetic elements); however, mcr encode phosphoethanolamine transferases (PET) closely related to chromosomally encoded, intrinsic lipid modification enzymes (e.g., EptA, EptB, CptA). To explore the genetic diversity of mcr within the context of intrinsic lipid modification PET, we identified 9,836 non-redundant protein accession numbers associated with mcr-like genes, representing a total of 69,814 mcr-like genes present across 256 bacterial genera. We subsequently identified 125 unique, putative novel mcr-like genes encoded on the same contig as a plasmid replicon and other antimicrobial resistance genes. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like genes, and intrinsic lipid modification PET-encoding genes indicated that sequence similarity is insufficient to discriminate between genes involved in colistin resistance and genes encoding intrinsic lipid modification PET. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific diversifying positive selection might have played a role in the evolution of subvariants within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. These residues were found to be differentially conserved in different mcr families and thus may play a role in mcr subvariant phenotypic diversity. Moreover, we found that eptA and mcr are localized within different genomic contexts. Canonical eptA are typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. In contrast, mcr are encoded as single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to “colistin resistance genes” through various mechanisms, including selection and diversification of the genomic context, regulatory pathways, and mobilization. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.
The cellular states and fates of shed intestinal cells
Authors: Halpern Keren Bahar , Kohanim Yael Korem , Biram Adi , Egozi Adi , Shulman Ziv , Itzkovitz Shalev 
Published: October 2022

AbstractThe intestinal epithelium is replaced every few days1. Enterocytes are shed into the gut lumen predominantly from the tips of villi3,4, and are believed to rapidly die upon their dissociation from the tissue. However, technical limitations prohibited studying the cellular states and fates of shed intestinal cells. Here, we used bulk and single cell RNA sequencing of mouse intestinal fecal washes to demonstrate that shed epithelial cells remain viable and up-regulate distinct anti-microbial programs upon shedding. We further identify abundant shedding of immune cells, a process that is elevated in DSS-induced colitis. We find that fecal host transcriptomics mirrors changes in the intestinal tissue following perturbations. Our study suggests potential functions of shed cells in the intestinal lumen and demonstrates that host cell transcriptomes in intestinal washes can be used to probe tissue states.
Midbody proteins display distinct temporal dynamics during cytokinesis
Authors: Halcrow Ella F.J. , Mazza Riccardo , Diversi Anna , Enright Anton , D’Avino Pier Paolo 
Published: October 2022

AbstractThe midbody is an organelle that forms between the two daughter cells during cytokinesis. It co-ordinates the abscission of the nascent daughter cells and is composed of a multitude of proteins that are meticulously arranged into distinct temporal and spatial localization patterns. However, very little is known about the mechanisms that regulate the localization and function of midbody proteins. Here, we analyzed the temporal and spatial profiles of key midbody proteins during mitotic exit under normal conditions and after treatment with drugs that affect phosphorylation and proteasome-mediated degradation to decipher the impacts of post-translational modifications on midbody protein dynamics. Our results highlighted that midbody proteins show distinct spatio-temporal dynamics during mitotic exit and cytokinesis that depend on both ubiquitin-mediated proteasome degradation and phosphorylation/de-phosphorylation. They also identified two discrete classes of midbody proteins: ‘transient’ midbody proteins - including Anillin, Aurora B and PRC1- which rapidly accumulate at the midbody after anaphase onset and then slowly disappear, and ‘stable’ midbody proteins - including CIT-K, KIF14 and KIF23- which instead persist at the midbody throughout cytokinesis and also post abscission. These two classes of midbody proteins display distinct interaction networks with ubiquitylation factors, which could potentially explain their different dynamics and stability during cytokinesis.
Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast
Authors: Schindler Daniel , Walker Roy S.K. , Jiang Shuangying , Brooks Aaron N. , Wang Yun , Müller Carolin A. , Cockram Charlotte , Luo Yisha , García Alicia , Schraivogel Daniel , Mozziconacci Julien , Blount Benjamin A. , Cai Jitong , Ogunlana Lois , Liu Wei , Jönsson Katarina , Abramczyk Dariusz , Garcia-Ruiz Eva , Turowski Tomasz W. , Swidah Reem , Ellis Tom , Antequera Francisco , Shen Yue , Nieduszynski Conrad A. , Koszul Romain , Dai Junbiao , Steinmetz Lars M. , Boeke Jef D. , Cai Yizhi 
Published: October 2022

AbstractHere we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.HighlightsDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae.Increasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy.The tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain.Comprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology.
EngineeringEscherichia colito utilize erythritol as sole carbon source
Authors: Ba Fang , Ji Xiangyang , Huang Shuhui , Zhang Yufei , Liu Wan-Qiu , Liu Yifan , Ling Shengjie , Li Jian 
Published: October 2022

AbstractErythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Human themselves are not able to catabolize erythritol and their gut microbes lack related catabolic pathways either to metabolize erythritol. Here, we engineerEscherichia colito utilize erythritol as sole carbon source aiming for defined applications. First, we isolate the erythritol metabolic gene cluster and experimentally characterize the erythritol-binding transcriptional repressor and its DNA-binding site. Transcriptome analysis suggests that carbohydrate metabolism-related genes in the engineeredE. coliare overall upregulated, which then guides the selection of four genes for overexpression that notably enhances cell growth. Finally, engineeredE. colistrains can be used as a living detector to distinguish erythritol-containing soda soft drinks and can grow in the simulated intestinal fluid supplemented with erythritol. We expect our work will inspire the engineering of more hosts to respond and utilize erythritol for broad applications in metabolic engineering, synthetic biology, and biomedical engineering.
A membrane integral methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases
Authors: Batth Tanveer S. , Simonsen Jonas L. , Hernández-Rollán Cristina , Brander Søren , Morth Jens Preben , Johansen Katja S. , Nørholm Morten H. H. , Hoof Jakob B. , Olsen Jesper V. 
Published: October 2022

ABSTRACTLytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identified the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirmed that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies showed endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs resulted in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this unique post-translational modification.
The effect of morphine on rat microglial phagocytic activity: an in vitro study of brain region-, plating density-, sex-, morphine concentration-, and receptor-dependency
Authors: King’uyu David N. , Nti-Kyemereh Lily , Bonin Jesse L. , Feustel Paul J. , Tram Michelle , MacNamara Katherine C. , Kopec Ashley M. 
Published: October 2022

ABSTRACTOpioids have long been used for clinical pain management, but also have addictive properties that have contributed to the ongoing opioid epidemic. While opioid activation of opioid receptors is well known to contribute to reward and reinforcement, data now also suggest that opioid activation of immune signaling via toll-like receptor 4 (TLR4) may also play a role in addiction-like processes. TLR4 expression is enriched in immune cells, and in the nervous system is primarily expressed in microglia. Microglial phagocytosis is important for developmental, homeostatic, and pathological processes. To examine how morphine impacts microglial phagocytosis, we isolated microglia from adult male and female rat cortex and striatum and plated themin vitroat 10,000 (10K) or 50,000 cells/well densities. Microglia were incubated with neutral fluorescent microbeads to stimulate phagocytosis in the presence of one of four morphine concentrations. We found that the brain region from which microglia are isolated and plating density, but not morphine concentration, impact cell survivalin vitro. We found that 10-12M morphine, but not higher concentrations, increases phagocytosis in striatal microgliain vitroindependent of sex and plating density, while 10-12M morphine increased phagocytosis in cortical microgliain vitroindependent of sex, but contingent on plating density. Finally, we demonstrate that the effect of 10-12M morphine in striatal microglia plated at 10K density is mediated via TLR4, and not µORs. Overall, our data suggest that in rats, a morphine-TLR4 signaling pathway increases phagocytic activity in microglia independent of sex. This may be useful information for better understanding the possible neural outcomes associated with morphine exposures.
Microbial communities from weathered outcrops of a sulfide-rich ultramafic intrusion, and implications for mine waste management
Authors: Hobart Kathryn K. , Greensky ZhaaZhaawaanong , Hernandez Kimberly , Feinberg Joshua M. , Bailey Jake V. , Jones Daniel S. 
Published: October 2022

ABSTRACTThe Duluth Complex, Northeastern Minnesota, contains sulfide-rich magmatic intrusions that, collectively, represent one of the world’s largest known economic deposits of copper, nickel, and platinum group elements (Cu-Ni-PGEs). Previous work showed that microbial communities associated with experimentally-weathered Duluth Complex waste rock and tailings were dominated by uncultivated taxa and other populations not typically associated with mine waste. However, those experiments were designed for kinetic testing and do not necessarily represent the conditions expected for reclaimed mine waste or long-term weathering in the environment. We therefore used 16S rRNA gene methods to characterize the microbial communities present on the surfaces of naturally-weathered and historically disturbed outcrops of Duluth Complex material, as well as a circumneutral seep draining a reclaimed blast pit. Rock surfaces were dominated by diverse uncultured Ktedonobacteria, Acetobacteria, and Actinobacteria while seeps were dominated by Proteobacteria, including Leptothrix spp. and Methylovulum spp. All samples had abundant algae and other phototrophs. These communities were distinct from previously-described microbial assemblages from experimentally-weathered Duluth Complex rocks, suggested different energy and nutrient resources in the reclaimed rocks, outcrops, and seeps. Sulfide mineral incubations performed with and without algae showed that photosynthetic microorganisms could have an inhibitory effect on some of the autotrophic populations from the site, resulting in slightly lower sulfate release and differences in the dominant microorganisms. The microbial assemblages from these weathered outcrops show how communities are expected to develop during natural weathering of sulfide-rich Duluth Complex rocks, and represent baseline data that could be used to evaluate the effectiveness of future reclamation of tailings and waste rock produced by large scale mining operations.
Robust and easy-to-use one pot workflow for label free single cell proteomics
Authors: Matzinger Manuel , Müller Elisabeth , Dürnberger Gerhard , Pichler Peter , Mechtler Karl 
Published: October 2022

ABSTRACTThe analysis of ultra-low input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a comprehensive workflow that includes optimized strategies for all steps from cell lysis to data analysis. Thanks to convenient to handle 1 μL sample volume and standardized 384 well plates the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using the CellenONE®, which allows for highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced μ-pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA) and data-independent acquisition (DIA), and commonly used advanced data-analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single cell level input in a 20-min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.