bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal Transport improves cell-cell similarity inference in single-cell omics data
Authors: Huizing Geert-Jan , Peyré Gabriel , Cantini Laura
Published: March 2021

The recent advent of high-throughput single-cell molecular profiling is revolutionizing biology and medicine by unveiling the diversity of cell types and states contributing to development and disease. The identification and characterization of cellular heterogeneity is typically achieved through unsupervised clustering, which crucially relies on a similarity metric.

We here propose the use of Optimal Transport (OT) as a cell-cell similarity metric for single-cell omics data. OT defines distances to compare, in a geometrically faithful way, high-dimensional data represented as probability distributions. It is thus expected to better capture complex relationships between features and produce a performance improvement over state-of-the-art metrics. To speed up computations and cope with the high-dimensionality of single-cell data, we consider the entropic regularization of the classical OT distance. We then extensively benchmark OT against state-of-the-art metrics over thirteen independent datasets, including simulated, scRNA-seq, scATAC-seq and single-cell DNA methylation data. First, we test the ability of the metrics to detect the similarity between cells belonging to the same groups (e.g. cell types, cell lines of origin). Then, we apply unsupervised clustering and test the quality of the resulting clusters.

In our in-depth evaluation, OT is found to improve cell-cell similarity inference and cell clustering in all simulated and real scRNA-seq data, while its performances are comparable with Pearson correlation in scATAC-seq and single-cell DNA methylation data. All our analyses are reproducible through the OT-scOmics Jupyter notebook available athttps://github.com/ComputationalSystemsBiology/OT-scOmics.
Cellar: Interactive single cell data annotation tool
Authors: Hasanaj Euxhen , Wang Jingtao , Sarathi Arjun , Ding Jun , Bar-Joseph Ziv
Published: March 2021

Several recent technologies and platforms enable the profiling of various molecular signals at the single-cell level. A key question for all studies using such data is the assignment of cell types. To improve the ability to correctly assign cell types in single and multi-omics sequencing and imaging single-cell studies, we developed Cellar. This interactive software tool supports all steps in the analysis and assignment process. We demonstrate the advantages of Cellar by using it to annotate several HuBMAP datasets from multi-omics single-cell sequencing and spatial proteomics studies. Cellar is freely available and includes several annotated reference HuBMAP datasets.

Availability

https://data.test.hubmapconsortium.org/app/cellar
Isolation, Molecular identification, Molecular docking In Vitro and In Silico Evaluation study against Fusarium oxysporum sp. Lactucae
Authors: Shamkh Israa. M. , Pratiwi Dina , El Faki Ahmad S. , Reyad Nour El-Houda A.
Published: March 2021

bioRxiv has withdrawn this preprint as it was submitted and made public without the full consent of all the contributing authors.
Foxes engineer hotspots of wildlife activity on the nutrient-limited Arctic tundra
Authors: Zhao Shu-Ting , Johnson-Bice Sean M. , Roth James D.
Published: March 2021

Predators largely affect ecosystems through trophic interactions, but they also can have indirect effects by altering nutrient dynamics and acting as ecosystem engineers. Arctic foxes (Vulpes lagopus) are ecosystem engineers that concentrate nutrients on their dens, creating biogeochemical hotspots with lush vegetation on the nutrient-limited tundra. Red foxes (V. vulpes) similarly engineer subarctic environments through their denning behavior, and have recently expanded onto the tundra where they now often occupy historical Arctic fox dens. We evaluated the impact of fox denning activity on the spatial behavior of other tundra wildlife by comparing predator and herbivore visits to 12 natal dens and adjacent control sites over two years using camera traps in northeastern Manitoba, where both fox species are sympatric. Both the capture rates and species richness of wildlife were significantly greater at fox dens relative to control sites. Predators were detected almost exclusively on dens occupied by foxes, where they were observed investigating and scavenging prey remains (carrion, feathers), suggesting carcass presence or fox presence attracts predators to den sites. Caribou (Rangifer tarandus) also visited dens more often than control sites, likely attracted by the enhanced vegetation typically found on dens. Our results suggest fox ecosystem engineering affects the spatial distribution of herbivores by enriching vegetation at dens, and other predators by providing carrion. Understanding how predators affect other organisms via non-trophic interactions provides an enriched view of their functional roles within ecosystems.
Multi-substrate DNA stable isotope probing reveals guild structure in bacterially mediated soil carbon cycling
Authors: Barnett Samuel E. , Youngblut Nicholas D. , Koechli Chantal N. , Buckley Daniel H.
Published: March 2021

Soil microorganisms determine the fate of soil organic matter (SOM), and their activities comprise a major component of the global carbon (C) cycle. We employed a multi-substrate DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interact within soil. We identified and tracked 1,286 bacterial taxa that assimilated13C in an agricultural soil over a period of 48 days. Overall13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds comprised of taxa that had similar13C assimilation dynamics. We show that C source bioavailability explained significant variation in both C mineralization dynamics and guild structure. In addition, guild structure explained significant variation in bacterial growth dynamics. We demonstrate that the observed guild structure is consistent with predictions made by bacterial life history theory. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C-cycling.
Application of HPMCAS-coated Ctx(Ile21)-Ha peptide microparticles as a potential use to prevent systemic infection caused bySalmonellaEnteritidis in poultry
Authors: Roque Borda Cesar Augusto , de Mesquita Souza Saraiva Mauro , Monte Daniel F. M. , Rodrigues Alves Lucas Bocchini , de Almeida Adriana Maria , Ferreira Taísa Santiago , de Lima Túlio Spina , Benevides Valdinete Pereira , Cabrera Julia Memrava , Meneguin Andréia Bagliotti , Chorilli Marlus , Junior Angelo Berchieri , Vicente Eduardo Festozo
Published: March 2021

The transmission ofSalmonellaEnteritidis (SE) in poultry is most often by the fecal-oral route, which can be attributed to the population density. Consequently, the pathogen triggers stress response and virulence factors deploying it to survive in hosts. Therefore, this study proposed to evaluate HPMCAS-coated microparticles containing the Ctx(Ile21)-Ha antimicrobial peptide against SE in laying hens chicks’ infection model to determine whether Ctx(Ile21)-Ha-utilization confers a benefit in the intestinal lumen, as well as whether limits systemic infection. Importantly, while assessing whether AMP utilization confers reduction of SE in liver, it was noted that there was statistical significance between groups A (control, no Ctx(Ile21)-Ha peptide) and B (2.5 mg of Ctx(Ile21)-Ha/kg) at 2 dpi, potentially indicating the Ctx(Ile21)-Ha effectiveness in the first stage of infection by SE. Remarkably, it was also detected a statistical significance (p-value <0.0001) with lower counts of SE (∼ 0 CFU) in livers at 5, 7, and 14 dpi, regardless of Ctx(Ile21)-Ha dosage (2.5 mg or 5 mg/kg - group C). By using Chi-square test, the AMP effect on SE fecal excretion was evaluated. In this regard, it was noticed statistical significance (p< 0.05) among groups B and C in comparison with control group A, since those groups had lower bacterial excretion along 21 days. In summary, the role of HPMCAS-Ctx(Ile21)-Ha peptide microcapsules againstS. Enteritidis in laying hen chicks infection model was unraveled, providing a satisfactory results against this pathogen.
DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis
Authors: Chatterjee Purba , Goldenfeld Nigel , Kim Sangjin
Published: March 2021

Recent experiments showed that multiple copies of the molecular machine RNA polymerase (RNAP) can efficiently synthesize mRNA collectively in the active state of the promoter. However, environmentally-induced promoter repression results in long-distance antagonistic interactions that drastically reduce the speed of RNAPs and cause a quick arrest of mRNA synthesis. The mechanism underlying this transition between cooperative and antagonistic dynamics remains poorly understood. In this Letter, we introduce a continuum deterministic model for the translocation of RNAPs, where the speed of an RNAP is coupled to the local DNA supercoiling as well as the density of RNAPs on the gene. We assume that torsional stress experienced by individual RNAPs is exacerbated by high RNAP density on the gene and that transcription factors act as physical barriers to the diffusion of DNA supercoils. We show that this minimal model exhibits two transcription modes mediated by the torsional stress: a fluid mode when the promoter is active and a torsionally stressed mode when the promoter is repressed, in quantitative agreement with experimentally observed dynamics of co-transcribing RNAPs. Our work provides an important step towards understanding the collective dynamics of molecular machines involved in gene expression.
An atlas of inter- and intra-tumor heterogeneity of apoptosis competency in colorectal cancer tissue at single cell resolution
Authors: Lindner Andreas Ulrich , Salvucci Manuela , McDonough Elizabeth , Cho Sanghee , Stachtea Xanthi , O’Connell Emer Patsy , Corwin Alex D , Santamaria-Pang Alberto , Carberry Steven , Fichtner Michael , Van Schaeybroeck Sandra , Laurent-Puig Pierre , Burke John P , McNamara Deborah A , Lawler Mark , Soop Anup , Graf John F , Rehm Markus , Dunne Philip , Longley Daniel B , Ginty Fiona , Prehn Jochen HM
Published: March 2021

Cancer cells’ ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells and quantification of 20 cell lineage and apoptosis proteins. Ordinary differential equation-based modelling of apoptosis sensitivity at single cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. We identified an enrichment for BCL2 in immune, and BAK, SMAC and XIAP in cancer cells. ODE-based modelling at single cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, with significant inter- and intra-tumor heterogeneity. However, we did not find increased spatial heterogeneity of apoptosis signaling in cancer cells, suggesting that such heterogeneity is an intrinsic, non-genomic property not increased by the process of malignant transformation.
Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence thatRalstoniabiomass clogs xylem vessels
Authors: Ingel Brian , Caldwell Denise , Duong Fiona , Parkinson Dilworth Y. , McCulloh Katherine A. , Iyer-Pascuzzi Anjali S. , McElrone Andrew J. , Lowe-Power Tiffany M.
Published: March 2021

Plant pathogenicRalstoniacause wilt diseases by colonizing xylem vessels and disrupting water transport. Due to the abundance ofRalstoniacells in vessels, the dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap. However, the physiological mechanism of xylem disruption during bacterial wilt disease is untested. Using a tomato andRalstonia pseudosolanacearumGMI1000 model, we visualized and quantified the spatiotemporal dynamics of xylem disruption during bacterial wilt disease. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets located on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged stems on plants with intact roots and leaves to quantify embolized vessels. Embolized vessels were rare, but there was a slight trend of increased vessel embolisms in infected plants with low bacterial population sizes. To test the hypothesis that vessels are clogged during bacterial wilt, we imaged excised stems after allowing the sap to evaporate during a brief dehydration. Most xylem vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5×108cfu/g stem tissue, approximately half of the xylem vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in the X-ray microCT reconstructions or light microscopy on the preserved stems. Bacterial blockage of vessels appears to be the principal cause of vascular disruption duringRalstoniawilt.
Platinized graphene fiber electrodes revealed differential activity in terminal splenic neurovascular plexi supporting a direct spleen-vagus communication
Authors: González-González María A. , Bendale Geetanjali , Wang K , Wallace Gordon G. , Romero-Ortega Mario
Published: March 2021

Neural interfacing nerve fascicles along the splenic neurovascular plexi (SNVP) is needed to better understand the spleen physiology, and for selective neuromodulation of this major organ. However, the small size and anatomical location have proven to be a significant challenge. Here, we use a reduced liquid crystalline graphene oxide (rGO) fiber coated with platinum (Pt) as a super-flexible suture-like electrode to interface multiple SNVP. The Pt-rGO fibers were used as handover knot electrodes over the small plexi, allowing sensitive recording from the splenic nerve (SN) terminal branches. Asymmetric defasciculation of the SN branches was revealed by electron microscopy, and the functional compartmentalization in spleen innervation was evidenced in response to hypoxia and pharmacological modulation of mean arterial pressure. We demonstrate that electrical stimulation of cervical and subdiaphragmatic vagus nerve (VN), evoked direct activity in a subset of SN terminal branches, providing evidence for a direct VN control over the spleen. This notion was supported by tract-tracing of SN branches, revealing an unconventional direct spleen-VN projection. High-performance Pt-rGO fiber electrodes, may be used for the fine neural modulation of other small neurovascular plexi at the point of entry of the organs as a bioelectronic medical alternative.
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

We 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

Single-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 implementation

Asc-Seurat is available athttps://github.com/KirstLab/asc_seurat/and released under GNU 3 license.

Contact

mkirst@ufl.edu

Supplementary information

Supplementary 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

Transcription 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

The 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

One 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

Background

MicroRNAs (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.

Methods

We 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.

Results

Our 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.

Conclusion

The 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

The 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

The 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

Granulocytes 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

Many 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.

IMPORTANCE

The 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

The 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

The 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 statement

Root-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

Large-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.
The Aliment to Bodily Condition knowledgebase (ABCkb): A database connecting plants and human health
Authors: Trautman Aaron , Linchangco Richard , Walstead Rachel , Jay Jeremy J , Brouwer Cory
Published: March 2021

Objective

Overconsumption of processed foods has led to an increase in chronic diet-related diseases such obesity and type 2 diabetes. Although diets high in fresh fruits and vegetables are linked with healthier outcomes, the specific mechanisms for these relationships are poorly understood. Experiments examining plant phytochemical production and breeding programs, or separately on the health effects of nutritional supplements have yielded results that are sparse, siloed, and difficult to integrate between the domains of human health and agriculture. To connect plant products to health outcomes through their molecular mechanism an integrated computational resource is necessary.

Results

We created the Aliment to Bodily Condition Knowledgebase (ABCkb) to connect plants to human health by creating a stepwise path from plant → plant product → human gene → pathways → indication. ABCkb integrates 11 curated sources as well as relationships mined from Medline abstracts by loading into a graph database which is deployed via a Docker container. This new resource, provided in a queryable container with a user-friendly interface connects plant products with human health outcomes for generating nutritive hypotheses. All scripts used are available on github (https://github.com/atrautm1/ABCkb) along with basic directions for building the knowledgebase.
A non-canonical microRNA derived from the snaR-A non-coding RNA targets a metastasis inhibitor
Authors: Stribling Daniel , Lei Yi , Guardia Casey M. , Li Lu , Fields Christopher J. , Nowialis Pawel , Opavsky Rene , Renne Rolf , Xie Mingyi
Published: March 2021

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical post-transcriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small non-coding RNA. Here, we develop theTarget-OrientedmiRNADiscovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs fromArgonautecrosslinkingandsequencing ofhybrids (Ago-CLASH) datasets. Using this technique, we discovered a novel miRNA derived from a primate specific non-coding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independent of Drosha processing, but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently upregulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A’s role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor non-coding RNA.
A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo
Authors: Hanke Leo , Das Hrishikesh , Sheward Daniel J , Vidakovics Laura Perez , Urgard Egon , Moliner-Morro Ainhoa , Kim Changil , Karl Vivien , Pankow Alec , Smith Natalie L , Porebski Bartlomiej , Fernandez-Capetillo Oscar , Sezgin Erdinc , Pedersen Gabriel K , Coquet Jonathan M , Hällberg B Martin , Murrell Ben , McInerney Gerald M
Published: March 2021

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we isolated a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the B.1.351 variant, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the B.1.351 variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes a novel strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.
Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children
Authors: Han Alvin X. , Garza Zandra C. Felix , Welkers Matthijs R. A. , Vigeveno René M. , Duong Tran Nhu , Quynh Mai Le Thi , Thai Pham Quang , Thoang Dang Dinh , Ngoc Anh Tran Thi , Tuan Ha Manh , Hung Nguyen Thanh , Thinh Le Quoc , Hai Le Thanh , Bich Ngoc Hoang Thi , Chokephaibulkit Kulkanya , Puthavathana Pilaipan , Van Vinh Chau Nguyen , Ngoc Nghiem My , Van Kinh Nguyen , Trinh Dao Tuyet , Hien Tran Tinh , Wertheim Heiman F. L. , Horby Peter , Fox Annette , van Doorn H. Rogier , Eggink Dirk , de Jong Menno D. , Russell Colin A.
Published: March 2021

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on within-host virus diversity of infections in otherwise healthy adults based on single timepoint data. Here, we analysed the within-host evolution of 82 longitudinally-sampled individuals, mostly young children, infected with A/H3N2 or A/H1N1pdm09 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous changes were common early in infection but decreased or remained constant throughout infection. For A/H3N2 viruses, early infection was dominated by purifying selection. However, as infections progressed, nonsynonymous variants increased in frequencies even though within-host virus titres decreased, leading to the maintenance of virus diversity via mutation-selection balance. Our findings suggest that this maintenance of genetic diversity in these children combined with their longer duration of infection may provide important opportunities for within-host virus evolution.
Transcriptome-wide spatial RNA profiling maps the cellular architecture of the developing human neocortex
Authors: Roberts Kenny , Aivazidis Alexander , Kleshchevnikov Vitalii , Li Tong , Fropf Robin , Rhodes Michael , Beechem Joseph M. , Hemberg Martin , Bayraktar Omer Ali
Published: March 2021

Spatial genomic technologies can map gene expression in tissues, but provide limited potential for transcriptome-wide discovery approaches and application to fixed tissue samples. Here, we introduce the GeoMX Whole Transcriptome Atlas (WTA), a new technology for transcriptome-wide spatial profiling of tissues with cellular resolution. WTA significantly expands the Digital Spatial Profiling approach to enablein situhybridisation against 18,190 genes at high-throughput using a sequencing readout. We applied WTA to generate the first spatial transcriptomic map of the fetal human neocortex, validating transcriptome-wide spatial profiling on formalin-fixed tissue material and demonstrating the spatial enrichment of autism gene expression in deep cortical layers. To demonstrate the value of WTA for cell atlasing, we integrated single-cell RNA-sequencing (scRNA-seq) and WTA data to spatially map dozens of neural cell types and showed that WTA can be used to directly measure cell type specific transcriptomesin situ. Moreover, we developed computational tools for background correction of WTA data and accurate integration with scRNA-seq. Our results present WTA as a versatile transcriptome-wide discovery tool for cell atlasing and fixed tissue spatial transcriptomics.