bluuebunny/crossref_metadata_2025_split huggingface dataset:
Coupling of spliceosome complexity to intron diversity
Authors: Sales-Lee Jade , Perry Daniela S. , Bowser Bradley A. , Diedrich Jolene K. , Rao Beiduo , Beusch Irene , Yates John R. , Roy Scott W. , Madhani Hiten D. 
Published: March 2021

SUMMARYWe determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution ofS. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeastCryptococcus neoformans.We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.
Asc-Seurat – Analytical single-cell Seurat-based web application
Authors: Pereira WJ , Almeida FM , Balmant KM , Rodriguez DC , Triozzi PM , Schmidt HW , Dervinis C , Pappas GJ , Kirst M 
Published: March 2021

AbstractSummarySingle-cell RNA sequencing (scRNA-seq) has become a popular approach for studying the transcriptome, providing a powerful tool for discovering and characterizing cell types and their developmental trajectories. However, scRNA-seq analysis is complex, requiring a continuous, iterative process to refine the data processing and uncover relevant biological information. We present Asc-Seurat, a feature rich workbench, providing a user-friendly and easy-to-install web application encapsulating the necessary tools for an all-encompassing and fluid scRNA-seq data analysis.Availability and implementationAsc-Seurat is available athttps://github.com/KirstLab/asc_seurat/and released under GNU 3 license.Contactmkirst@ufl.eduSupplementary informationSupplementary data are available atBioinformaticsonline.
ChromWave: Deciphering the DNA-encoded competition between transcription factors and nucleosomes with deep neural networks
Authors: Cakiroglu Sera Aylin , Steinhauser Sebastian , Smith Jon , Xing Wei , Luscombe Nicholas M. 
Published: March 2021

SummaryTranscription factors (TFs) regulate gene expression by recognising and binding specific DNA sequences. At times, these regulatory elements may be occluded by nucleosomes, making them inaccessible for TF-binding. The competition for DNA occupancy between TFs and nucleosomes, and associated gene regulatory outputs, are important consequences of the cis-regulatory information encoded in the genome. However, these sequence patterns are subtle and remain difficult to interpret. Here, we introduce ChromWave, a deep-learning model that, for the first time, predicts the competing profiles for TF and nucleosomes occupancies with remarkable accuracy. Models trained using short- and long-fragment MNase-Seq data successfully learn the sequence preferences underlying TF and nucleosome occupancies across the entire yeast genome. They recapitulate nucleosome evictions from regions containing “strong” TF binding sites and knock-out simulations show nucleosomes gaining occupancy in the absence of these TFs, accompanied by lateral rearrangement of adjacent nucleosomes. At a local level, models anticipate with high accuracy the outcomes of detailed experimental analysis of partially unwrapped nucleosomes at the GAL4 UAS locus. Finally, we trained a ChromWave model that successfully predicts nucleosome positions at promoters in the human genome. We find that human promoters generally contain few sites at which simple sequence changes can alter nucleosome occupancies and that these positions align well with causal variants linked to DNase hypersensitivity.
Correcting for Background Noise Improves Phenotype Prediction from Human Gut Microbiome Data
Authors: Briscoe Leah , Balliu Brunilda , Sankararaman Sriram , Halperin Eran , Garud Nandita R. 
Published: March 2021

AbstractThe ability to predict human phenotypes accurately from metagenomic data is crucial for developing biomarkers and therapeutics for diseases. However, metagenomic data is commonly affected by technical or biological variables, unrelated to the phenotype of interest, such as sequencing protocol or host sex, which can greatly reduce or, when correlated to the phenotype of interest, inflate prediction accuracy. We perform a comparative analysis of the ability of different data transformations and existing supervised and unsupervised methods to correct microbiome data for background noise. We find that supervised methods are limited because they cannot account for unmeasured sources of variation. In addition, we observe that unsupervised approaches are often superior in addressing these issues, but existing methods developed for other ‘omic data types, e.g., gene expression and methylation, are restricted by parametric assumptions unsuitable for microbiome data, which is typically compositional, highly skewed, and sparse. We show that application of the centered log-ratio transformation prior to correction with unsupervised approaches improves prediction accuracy for many phenotypes while simultaneously reducing variance due to unwanted sources of variation. As new and larger metagenomic datasets become increasingly available, background noise correction will become essential for generating reproducible microbiome analyses.
JASMONIC ACID PRIMING OF POTATO USES HYPERSENSITIVE RESPONSE-DEPENDENT DEFENSE AND DELAYS NECROTROPHIC PHASE CHANGE AGAINST PHYTOPHTHORA INFESTANS
Authors: Arévalo-Marín Diego F , Briceño-Robles Daniel M , Mosquera Teresa , Melgarejo Luz Marina , Sarmiento Felipe 
Published: March 2021

AbstractOne of the most important diseases affecting potato is late blight, caused by the oomycete Phtytophthora infestans. The use of jasmonic acid has been reported to reduce the progression of the disease in potato, but the defense mechanisms involved in this response are unknown. In this study we described the effect of jasmonic acid as a priming agent over time in the defense response of potato against the invasion of P. infestans. We observed that the initial stimulus generated by the exogenous application of jasmonic acid had an effect on the stomatal conductance of the treated tissue and activated StMYC2 expression. Results reveal a priming effect in plants inoculated 11 days after treatment with jasmonic acid, evidenced by an increased transcriptional induction of defense-associated genes, decrease in the number of necrotic lesions and an evident reduction of lesion area (72.23%). Furthermore, in this study, we show that the tested concentration of jasmonic acid does not have an adverse effect at the physiological level in plants, since variation in stomatal conductance was transient, no change in chlorophyll a fluorescence and no early senescence in leaves was observed.
A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads
Authors: Geithe Christiane , Zeng Bo , Schmidt Carsten , Dinter Franziska , Roggenbuck Dirk , Lehmann Werner , Dame Gregory , Schierack Peter , Hanack Katja , Rödiger Stefan 
Published: March 2021

AbstractBackgroundMicroRNAs (miRNAs) are small, conserved, noncoding RNAs regulating gene expression that functions in RNA silencing and post-transcriptional regulation of gene expression. Altered miRNA profiles have been implicated in many human diseases, and due to their circulating abilities, they have excited great interest in their use as clinical biomarkers. The development of innovative methods for miRNA detection has become of high scientific and clinical interest.MethodsWe developed a diffusion-driven microbead assay and combined it with an antibody-based miRNA detection. The diffusion process was carried out in two different approaches a) co-diffusion of miRNA and antibodies (termed diffusion approach I, DAI) and b) diffusion of miRNA in an antibody-saturated environment (DAII). In both approaches, neutravidin-coated microbeads were loaded with specific biotinylated DNA capture probes, which targets either miR-21-5p, miR-30a-3p or miR-93-5p. The miRNAs were time- and dose-dependently detected in a diffusion microchamber by primary anti-DNA:RNA hybrid and fluorescence-labeled secondary antibodies using our in-house developed inverse fluorescence microscope imaging platform VideoScan.ResultsOur assay offers the advantage that several target molecules can be detected simultaneously and in real-time in one reaction environment (multiplex), without any amplification steps. We recorded the diffusion process over a period of 24 h and found that the reaction was almost completed after 2 h. The specificity of the assay was 96.7 % for DAI and 92.3 % for DAII. The detection limits were in a concentration range of 0.03-0.43 nM for DAI and 0.14-1.09 nM for DAII, depending on the miRNA.ConclusionThe miRNAs are successively exposed to the capture probe-loaded randomly ordered microbeads (p value of CSR 0.23-0.96), which leads to microbeads that become saturated with the target molecules first in front rows. Non-bonded miRNAs continue to diffuse further and can therefore subsequently bind to the microbeads with free binding sites. Our detection principle differs from other microbead assays, in which all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind equally distributed to the microbeads, resulting in an averaged signal intensity.
HLA Type and Chronic Viral Infection Impact Peripheral T-cell Receptor Sharing Between Unrelated Individuals
Authors: Johnson Sarah A. , Seale Spencer L. , Gittelman Rachel M. , Rytlewski Julie A. , Robins Harlan S. , Fields Paul A. 
Published: March 2021

ABSTRACTThe human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, certain factors such as low junctional diversity, thymic selection, and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRβ variable region of 426 healthy individuals, we find that fewer than 1% of TCRβ clones are shared between individuals on average, consistent with largely private TCRβ repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRβ clones, with each additional shared HLA allele contributing to an increase in ∼0.01% of the total TCRβ clones being shared, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRβ clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRβ clone sharing, indicating that the pattern of private TCRβ clonal expansion is a general feature of the T-cell response to other infectious antigens. All of these factors contribute to shaping the TCRβ repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.
Generation ofDrosophilaattP containing cell lines using CRISPR-Cas9
Authors: Mariyappa Daniel , Luhur Arthur , Overton Danielle , Zelhof Andrew C. 
Published: March 2021

AbstractThe generation ofDrosophilastable cell lines have become invaluable for complementingin vivoexperiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation ofDrosophilacells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100Drosophilacell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the availableDrosophila melanogastercell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic thein vivoattP sites located at 25C6 and 99F8 of theDrosophilagenome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existingD. melanogastercell lines.
Systemic and mucosal mobilization of granulocyte subsets during lentivirus infection
Authors: Jones Rhianna , Manickam Cordelia , Ram Daniel R. , Kroll Kyle , Hueber Brady , Woolley Griffin , Shah Spandan V. , Smith Scott , Varner Valerie , Reeves R. Keith 
Published: March 2021

AbstractGranulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentivirus infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood from naïve, and chronically SHIVsf162p3-infected RM were analyzed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils – CD45+CD66+CD49d+; neutrophils – CD45+CD66+CD14+; and basophils – CD45+CD123+FcRε+. Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcRγII) but did not express CD16 (FcRγIII). Additionally, constitutive expression of CD64 (FcRγI) on neutrophils and FcRε on basophils, indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naïve whole blood ranged 25.4-81.5% neutrophils, 0.59-13.3% eosinophils and 0.059-1.8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils, and colorectal eosinophils were all observed in chronic lentivirus disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils, and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentivirus infection, most notably in the gastrointestinal mucosae where significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.
Proteus mirabilisemploys a contact-dependent killing system against competingEnterobacteriaceae
Authors: Kiani Dara , Santus William , Kiernan Kaitlyn , Behnsen Judith 
Published: March 2021

ABSTRACTMany bacterial species encode systems for interference competition with other microorganisms. Some systems are effective without contact (e.g. through secretion of toxins), while other systems (e.g. Type VI secretion system (T6SS)) require direct contact between cells. Here, we provide the initial characterization of a novel contact-dependent competition system forProteus mirabilis. In neonatal mice, a commensalP. mirabilisstrain apparently eliminated commensalEscherichia coli. We replicated the phenotypein vitroand showed thatP. mirabilisefficiently reduced viability of severalEnterobacteriaceaespecies, but not Gram-positive species or yeast cells. Importantly,P. mirabilisstrains isolated from humans also killedE. coli. Reduction of viability occurred from early stationary phase to 24h of culture and was observed in shaking liquid media as well as on solid media. Killing required contact, but was independent of T6SS, the only contact-dependent killing system described forP. mirabilis. Expression of the killing system was regulated by osmolarity and components secreted into the supernatant. Stationary phaseP. mirabilisculture supernatant itself did not kill but was sufficient to induce killing in an exponentially growing co-culture. In contrast, killing was largely prevented in media with low osmolarity. In summary, we provide the initial characterization of a potentially novel interbacterial competition system encoded inP. mirabilis.IMPORTANCEThe study of bacterial competition systems has received significant attention in recent years. These systems collectively shape the composition of complex ecosystems like the mammalian gut. They are also being explored as narrow-spectrum alternatives to specifically eliminate problematic pathogenic species. However, many competition systems that effectively workin vitrodo not show strong phenotypes in the gut. Our study was informed by an observation in infant mice. Furtherin vitrostudies confirmed thatP. mirabiliswas able to kill severalEnterobacteriaceaespecies. This killing system is novel forP. mirabilisand might represent a new function of a known system or even a novel system, as the observed characteristics do not fit with described contact-dependent competition systems. Competition systems are frequently present in multipleEnterobacteriaceaespecies. If present or transferred into a probiotic, it might be used in the future to reduce blooms of pathogenicEnterobacteriaceaeassociated with disease.
Evolutionary conserved protein motifs drive attachment of the plant nucleoskeleton at nuclear pores
Authors: Mermet Sarah , Voisin Maxime , Mordier Joris , Dubos Tristan , Tutois Sylvie , Tuffery Pierre , Baroux Célia , Tamura Kentaro , Probst Aline V. , Vanrobays Emmanuel , Tatout Christophe 
Published: March 2021

ABSTRACTThe nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear morphology and gene expression. To understand how the Arabidopsis nucleoskeleton physically connects to the nuclear periphery, we investigated the nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. Computational predictions identified three evolutionary conserved peptide motifs within the N-terminal region of KAKU4. Functional analysis revealed that these motifs are required for homomerization of KAKU4, interaction with the nucleoskeleton proteins CROWDED NUCLEI (CRWN) and localization at the nuclear periphery. We find that similar protein motifs are present in NUP82 and NUP136, two plant specific nucleoporins from the Nuclear Pore Complex (NPC) basket. These conserved motifs allow the two nucleoporins to bind CRWN proteins, thus revealing a physical link between the nucleoskeleton and nuclear pores in plants. Finally, whilst NUP82, NUP136 and KAKU4 have a common evolutionary history predating non-vascular land plants, KAKU4 mainly localizes outside the NPC suggesting neofunctionalization of an ancient nucleoporin into a new nucleoskeleton component.
Marchantia polymorphamodel reveals conserved infection mechanisms in the vascular wilt fungal pathogenFusarium oxysporum
Authors: Redkar Amey , Ibanez Selena Gimenez , Sabale Mugdha , Zechmann Bernd , Solano Roberto , Di Pietro Antonio 
Published: March 2021

AbstractThe non-vascular plantMarchantia polymorphahas emerged as a valuable model for studying evolutionarily conserved microbial infection strategies and plant immune responses. However, only a handful of fungal pathogens ofMarchantiahave been described so far. Here we establish a new pathosystem using the root-infecting vascular wilt fungusFusarium oxysporum. On angiosperms, this fungus exhibits exquisite adaptation to the plant vascular niche and host-specific pathogenicity, both of which are conferred by lineage-specific effectors secreted during growth in the xylem. We show thatF. oxysporumisolates with different lifestyles - pathogenic or endophytic - are able to infect this non-vascular liverwort causing tissue maceration and plant cell killing. Similar to bacterial pathogens,F. oxysporuminduces a PAMP-triggered immune response inM. polymorpha. Analysis of isogenic fungal mutants established that infection ofMarchantiarequires conserved fungal pathogenicity mechanisms such as mitogen activated protein kinases, transcriptional regulators and cell wall remodeling enzymes. Remarkably, lineage-specific virulence effectors are dispensable for infection, most likely due to the absence of xylem tissue in this non-vascular plant. TheF. oxysporum-M. polymorphasystem provides new insights into the mechanism and evolution of pathogenic and endophytic fungus-plant interactions.Significance statementRoot-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they infect non-vascular plants, which diverged from vascular plants over 450 million years ago. Here we show that two strains of the fungusFusarium oxysporumwith opposed lifestyles, causing either wilting and death or beneficial protection on tomato, produce similar disease symptoms on the non-vascular plantMarchantia polymorpha.We define a set of core fungal pathogenicity factors required on both vascular and non-vascular plants and show that host-specific effectors contributing to disease on tomato are dispensable onMarchantia. These findings suggest that systemic wilt disease evolved in fungal pathogens after the emergence of vascular land plants.
GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control
Authors: Yang Yi , Cao Weiqian , Yan Guoquan , Kong Siyuan , Wu Mengxi , Yang Pengyuan , Qiao Liang 
Published: March 2021

AbstractLarge-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further adapt a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data dependent acquisition (DDA) based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.