bluuebunny/crossref_metadata_2025_split huggingface dataset:
Host-associated microbe PCR (hamPCR): accessing new biology through convenient measurement of both microbial load and community composition
Authors: Lundberg Derek S. , Ayutthaya Pratchaya Pramoj Na , Strauß Annett , Shirsekar Gautam , Lo Wen-Sui , Lahaye Thomas , Weigel Detlef 
Published: May 2020

AbstractThe ratio of microbial population size relative to the amount of host tissue, or “microbial load”, is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because conventional methods to determine load, such as serial dilution plating or quantitative PCR, add substantial experimental burden, they are only rarely paired with amplicon sequencing. Alternatively, whole metagenome sequencing of DNA contributed by host and microbes both reveals microbial community composition and enables determination of microbial load, but host DNA typically greatly outweighs microbial DNA, severely limiting the cost-effectiveness and scalability of this approach. We introduce host-associated microbe PCR (hamPCR), a robust amplicon sequencing strategy to quantify microbial load and describe interkingdom microbial community composition in a single, cost-effective library. We demonstrate its accuracy and flexibility across multiple host and microbe systems, including nematodes and major crops. We further present a technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without loss of information. Because of its simplicity, and the fact that it provides an experimental solution to the well-known statistical challenges provided by compositional data, hamPCR will become a transformative approach throughout culture-independent microbiology.
Gbx2 identifies two amacrine cell subtypes with distinct molecular, morphological, and physiological properties
Authors: Kerstein Patrick C. , Leffler Joseph , Sivyer Benjamin , Taylor W. Rowland , Wright Kevin M. 
Published: May 2020

ABSTRACTOur understanding of how the nervous sytem works is limited by our ability to identify the neuronal subtypes that comprise functional circuits. Using a genetic approach, we show that the transcription factorGbx2labels two amacrine cell (AC) subtypes in the mouse retina that have distinct morphological, physiological, and molecular properties. One subtype of Gbx2+ ACs are likely the previously characterized On-type GABAergic CRH-1 AC. The other Gbx2+ AC population is a previously uncharacterized non-GABAergic, non-Glycinergic (nGnG) AC subtype. Gbx2+ nGnG ACs are On-Off type cells with asymmetric dendritic arbors. Gbx2+ nGnG ACs also exhibit tracer coupling to bipolar cells (BCs) through gap junctions that are modulated by dopamine signaling. This study genetically identifies a previously uncharacterized AC subtype and reveals an unusual AC-BC connectivity through gap junctions that may provide a novel model of synaptic communication and visual circuit function.
Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment
Authors: Nicolas-Boluda Alba , Vaquero Javier , Barrin Sarah , Kantari-Mimoun Chahrazade , Ponzo Matteo , Renault Gilles , Deptuła Piotr , Pogoda Katarzyna , Bucki Robert , Cascone Ilaria , Courty Jose , Fouassier Laura , Gazeau Florence , Donnadieu Emmanuel 
Published: May 2020

ABSTRACTOnly a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing 5 preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase (LOX) was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.
A novel essential RNA-binding protein complex inTrypanosoma brucei
Authors: Bajak Kathrin , Leiss Kevin , Clayton Christine , Erben Esteban 
Published: May 2020

AbstractZC3H5 is an essential cytoplasmic trypanosome protein with a single Cx7Cx5Cx3H zinc finger domain. We here show that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. ZC3H5 interacts directly with Tb927.11.4900, which in turn interacts with Tb927.7.3040. Tb927.11.4900 has a circularly permuted GTPase domain, which is required for the Tb927.7.3040 interaction. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5’-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). Tethering of ZC3H5, or other complex components, to a reporter repressed its expression. However, depletion of ZC3H5in vivodid not increase the abundance of ZC3H5-bound mRNAs: instead, counter-intuitively, there were very minor decreases in a few targets, and marked increases in the abundances of very stable mRNAs encoding ribosomal proteins. Depletion also resulted in an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of sub-optimal open reading frames; complex assembly might be regulated by GTP hydrolysis and GTP-GDP exchange.
Insights into rumen microbial biosynthetic gene cluster diversity through genome-resolved metagenomics
Authors: Anderson Christopher L. , Fernando Samodha C. 
Published: May 2020

AbstractRuminants are critical to global food security as they transform lignocellulosic biomass into high-quality protein products. The rumen microbes ferment feed to provide necessary energy and nutrients for the ruminant host. However, we still lack insight into the metabolic processes encoded by most rumen microbial populations. In this study, we implemented metagenomic binning approaches to recover 2,809 microbial genomes from cattle, sheep, moose, deer, and bison. By clustering genomes based on average nucleotide identity, we demonstrate approximately one-third of the metagenome-assembled genomes (MAGs) to represent species not present in current reference databases and rumen microbial genome collections. Combining these MAGs with other rumen genomic datasets permitted a phylogenomic characterization of the biosynthetic gene clusters (BGCs) from 8,160 rumen microbial genomes, including the identification of 5,346 diverse gene clusters for nonribosomal peptide biosynthesis. A subset ofPrevotellaandSelenomonasBGCs had higher expression in steers with lower feed efficiency. Moreover, the microdiversity of BGCs was fairly constant across types of BGCs and cattle breeds. The reconstructed genomes expand the genomic representation of rumen microbial lineages, improve the annotation of multi-omics data, and link microbial populations to the production of secondary metabolites that may constitute a source of natural products for manipulating rumen fermentation.
In vivoCRISPR screening identifies Fli1 as a transcriptional safeguard that restrains effector CD8 T cell differentiation during infection and cancer
Authors: Chen Zeyu , Arai Eri , Khan Omar , Zhang Zhen , Ngiow Shin Foong , He Yuan , Huang Hua , Manne Sasikanth , Cao Zhendong , Baxter Amy E. , Cai Zhangying , Freilich Elizabeth , Ali Mohammed A. , Giles Josephine R. , Wu Jennifer E. , Greenplate Allison R. , Kurachi Makoto , Nzingha Kito , Ekshyyan Viktoriya , Wen Zhuoyu , Speck Nancy A. , Battle Alexis , Berger Shelley L. , Wherry E. John , Shi Junwei 
Published: May 2020

SummaryImproving effector activity of antigen specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TF), the transcriptional coordination of TEFFbiology remains poorly understood. We developed anin vivoT cell CRISPR screening platform and identified a novel mechanism restraining TEFFbiology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFFresponses without compromising memory or exhaustion precursors. Fli1 restrained TEFFlineage differentiation by binding tocis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs allowing more efficient Runx3-driven TEFFbiology. CD8 T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8 T cell transcriptional landscape from excessive ETS:RUNX-driven TEFFcell differentiation. Moreover, genetic deletion of Fli1 improves TEFFdifferentiation and protective immunity in infections and cancer.
RdRp mutations are associated with SARS-CoV-2 genome evolution
Authors: Eskier Doğa , Karakülah Gökhan , Suner Aslı , Oktay Yavuz 
Published: May 2020

AbstractCOVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic. With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of viral genome over time as it spread across the world. An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics. Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C>T mutation, on mutation rate and viral spread. By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure. Our results indicate that 14408C>T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C>T, has the opposite effect. It is possible that 14408C>T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.
A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition
Authors: Dieterle M. Eugenia , Haslwanter Denise , Bortz Robert H. , Wirchnianski Ariel S. , Lasso Gorka , Vergnolle Olivia , Abbasi Shawn A. , Fels J. Maximilian , Laudermilch Ethan , Florez Catalina , Mengotto Amanda , Kimmel Duncan , Malonis Ryan J. , Georgiev George , Quiroz Jose , Barnhill Jason , Pirofski Liise-anne , Daily Johanna P. , Dye John M. , Lai Jonathan R. , Herbert Andrew S. , Chandran Kartik , Jangra Rohit K. 
Published: May 2020

SummaryThere is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.
Glyphosate Inhibits Melanization and Increases Susceptibility to Infection in Insects
Authors: Smith Daniel F. Q. , Camacho Emma , Thakur Raviraj , Barron Alexander J. , Dong Yuemei , Dimopoulos George , Broderick Nichole A. , Casadevall Arturo 
Published: May 2020

ABSTRACTMelanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including: UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin’s broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate – the most widely used herbicide globally – inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in two evolutionary distant species,Galleria mellonella(Lepidoptera: Pyralidae) andAnopheles gambiae(Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival ofG. mellonellacaterpillars following infection with the fungusCryptococcus neoformansand decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria- causing parasitePlasmodium falciparuminA. gambiaemosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate’s mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance Overall, these findings suggest that glyphosate’s environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.
Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus
Authors: Nigg Jared C. , Kuo Yen-Wen , Falk Bryce W. 
Published: May 2020

AbstractPartial integrations of DNA and non-retroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce PIWI-interacting RNAs (piRNAs) speculated to target cognate viruses through the ping-pong cycle, a post-transcriptional RNA silencing mechanism. Here we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome ofDiaphorina citri. We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed amongD. citripopulations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent viral piRNAs (vpiRNAs) inD. citrilacking the DcDV-derived EVE, while four naturally infecting RNA viruses ofD. citriare not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection inD. citriharboring the EVE. These results represent the first report of ping-pong-dependent vpiRNAs outside of mosquitoes.
Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites
Authors: Crow Jacob J. , Albig Allan R. 
Published: May 2020

ABSTRACTNotch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that each family member is capable of dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.
Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2-complex
Authors: Lind Simon , Dahlgren Claes , Holmdahl Rikard , Olofsson Peter , Forsman Huamei 
Published: May 2020

AbstractTwo formyl peptide receptors (FPR1 and FPR2), abundantly expressed by neutrophils, regulate both pro-inflammatory tissue recruitment of neutrophils and resolution of inflammatory reactions. This dual functionality of the FPRs, opens for a possibility to develop receptor selective therapeutics as mechanism for novel anti-inflammatory treatments. In line with this, high throughput screening studies have identified numerous FPR ligands belonging to different structural classes, but a potent FPR1 agonist with defined biased signaling and functional selectivity has not yet been reported. In this study, we used an FPR1 selective small compound agonist (RE) that represents a chemical entity developed from NOX2 activators identified from our earlier screening studies (WO2012127214). This FPR1 agonist potently activates neutrophils to produce reactive oxygen species (ROS, EC50~1 nM), whereas it is a weaker chemoattractant than the prototype FPR1 agonist fMLF. At the signaling level, RE has a strong bias towards the PLC-PIP2-Ca2+pathway and ERK1/2 activation but away from β-arrestin recruitment and the ability to recruit neutrophils chemotactically. In addition, FPR1 when activated by RE could cross-regulate other receptor-mediated neutrophil functions. In comparison to the peptide agonist fMLF, RE is more resistant to oxidization-induced inactivation by the MPO-H2O2-halide system. In summary, this study describes as a novel FPR1 agonist displaying a biased signaling and functional selectivity when activating FPR1 in human blood neutrophils. RE could possibly be a useful tool compound not only for further mechanistic studies of the regulatory role of FPR1 in inflammationin vitroandin vivo, but also for developing FPR1specific drug therapeutics.
CONTAIN: An open-source shipping container laboratory optimised for automated COVID-19 diagnostics
Authors: Walker Kenneth T. , Donora Matthew , Thomas Anthony , Phillips Alexander James , Ramgoolam Krishma , Pilch Kjara S , Oberacker Phil , Jurkowski Tomasz Piotr , Gosman Rares Marius , Fleiss Aubin , Perkins Alex , MacKenzie Neil , Zuckerman Mark , Danovi Davide , Steiner Helene , Meany Thomas 
Published: May 2020

AbstractThe COVID-19 pandemic has challenged diagnostic systems globally. Expanding testing capabilities to conduct population-wide screening for COVID-19 requires innovation in diagnostic services at both the molecular and industrial scale. No report to-date has considered the complexity of laboratory infrastructure in conjunction with the available molecular assays to offer a standardised solution to testing. Here we present CONTAIN. A modular biosafety level 2+ laboratory optimised for automated RT-qPCR COVID-19 testing based on a standard 40ft shipping container. Using open-source liquid-handling robots and RNA extraction reagents we demonstrate a reproducible workflow for RT-qPCR COVID-19 testing. With five OT2 liquid handlers, a single CONTAIN unit reaches a maximum daily testing capacity of 2400 tests/day. We validate this workflow for automated RT-qPCR testing, using both synthetic SARS-CoV-2 samples and patient samples from a local NHS hospital. Finally, we discuss the suitability of CONTAIN and its flexibility in a range of diagnostic testing scenarios including high-density urban environments and mobile response units.Visual abstract
Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time
Authors: Narayanasamy Kaarjel K. , Price Joshua C. , Mesquita-Riberio Raquel , Mather Melissa L. , Jayasinghe Izzy 
Published: May 2020

AbstractStochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the ‘off’-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV-is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at ∼ 3-times the speed and ∼ 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.
Discovery of potent inhibitors of PLproCoV2 by screening a library of selenium-containing compounds
Authors: Węglarz-Tomczak Ewelina , Tomczak Jakub M. , Giurg Mirosław , Burda-Grabowska Małgorzata , Brul Stanley 
Published: May 2020

AbstractA collection of twelve organoselenium compounds, structural analogues of antioxidant drug ebselen were screened for inhibition of the papain-like protease (PLpro) from the acute respiratory syndrome coronavirus 2 (SARS-CoV-2, CoV2). This cysteine protease, being responsible for the hydrolysis of peptide bonds between specific amino acids, plays a critical role in CoV2 replication and in assembly of new viral particles within human cells. The activity of the PLproCoV2 is essential for the progression of coronavirus disease 2019 (COVID-19) and it constitutes a key target for the development of anti-COVID-19 drugs. Here, we identified four strong inhibitors that bind favorably to the PLproCoV2 with theIC50in the nanomolar range.