Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with ...select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal ...data. Here, we introduce “weighted-nearest neighbor” analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.
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•“Weighted nearest neighbor” analysis integrates multimodal single-cell data•A multimodal reference “atlas” of the circulating human immune system•Identification and validation of novel sources of lymphoid heterogeneity•“Reference-based” mapping of query datasets onto a multimodal atlas
A framework that allows for the integration of multiple data types using single cells is applied to understand distinct immune cell states, previously unidentified immune populations, and to interpret immune responses to vaccinations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mapping single-cell sequencing profiles to comprehensive reference datasets provides a powerful alternative to unsupervised analysis. However, most reference datasets are constructed from single-cell ...RNA-sequencing data and cannot be used to annotate datasets that do not measure gene expression. Here we introduce 'bridge integration', a method to integrate single-cell datasets across modalities using a multiomic dataset as a molecular bridge. Each cell in the multiomic dataset constitutes an element in a 'dictionary', which is used to reconstruct unimodal datasets and transform them into a shared space. Our procedure accurately integrates transcriptomic data with independent single-cell measurements of chromatin accessibility, histone modifications, DNA methylation and protein levels. Moreover, we demonstrate how dictionary learning can be combined with sketching techniques to improve computational scalability and harmonize 8.6 million human immune cell profiles from sequencing and mass cytometry experiments. Our approach, implemented in version 5 of our Seurat toolkit ( http://www.satijalab.org/seurat ), broadens the utility of single-cell reference datasets and facilitates comparisons across diverse molecular modalities.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Recent studies suggest that alterations in the gut phagobiota may contribute to pathophysiological processes in mammals; however, the association of bacteriophage community structure with Parkinson's ...disease (PD) has not been yet characterized. Towards this end, we used a published dataset to analyse bacteriophage composition and determine the phage/bacteria ratio in faecal samples from drug-naive PD patients and healthy participants. Our analyses revealed significant alterations in the representation of certain bacteriophages in the phagobiota of PD patients. We identified shifts of the phage/bacteria ratio in lactic acid bacteria known to produce dopamine and regulate intestinal permeability, which are major factors implicated in PD pathogenesis. Furthermore, we observed the depletion of Lactococcus spp. in the PD group, which was most likely due to the increase of lytic c2-like and 936-like lactococcal phages frequently present in dairy products. Our findings add bacteriophages to the list of possible factors associated with the development of PD, suggesting that gut phagobiota composition may serve as a diagnostic tool as well as a target for therapeutic intervention, which should be confirmed in further studies. Our results open a discussion on the role of environmental phages and phagobiota composition in health and disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
As the number of single-cell datasets continues to grow rapidly, workflows that map new data to well-curated reference atlases offer enormous promise for the biological community. In this ...perspective, we discuss key computational challenges and opportunities for single-cell reference-mapping algorithms. We discuss how mapping algorithms will enable the integration of diverse datasets across disease states, molecular modalities, genetic perturbations, and diverse species and will eventually replace manual and laborious unsupervised clustering pipelines.
Single-cell datasets are increasing in number and size. To leverage this rich resource, new workflows can reveal novel insights and discoveries. This perspective discusses the computational challenges and opportunities for single-cell reference-mapping algorithms that may eventually replace manual and laborious unsupervised clustering pipelines.
Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The ...Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been ...fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
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•PPP and OXPHOS compensate for Gpi1 loss in Th17 cells in normoxic environment•Gpi1 is essential in hypoxic inflamed tissue due to reduced OXPHOS compensation•Metabolic redundancy can vary according to the microenvironment•Targeting Gpi1 could be a tolerable approach for therapeutic glycolysis inhibition
Metabolic redundancy differs according to microenvironments, making the glycolysis gene Gpi1 dispensable for homeostatic Th17 cells in normal tissue but essential for pathogenic Th17 cells in hypoxic inflamed tissue.
ATR （ataxia telangiectasia-mutated and Rad3-related） protein kinase and ATRIP （ATR-interacting protein） form a complex and play a critical role in response to replication stress and DNA damage. Here, ...we determined the cryo-electron microscopy （EM） structure of the human ATR-ATRIP complex at 4.7 ~ resolution and built an atomic model of the C-terminal catalytic core of ATR （residues 1 521-2 644） at 3.9 ~ resolution. The complex adopts a hollow ＂heart＂ shape, consisting of two ATR monomers in distinct conformations. The EM map for ATRIP reveals 14 HEAT repeats in an extended ＂S＂ shape. The conformational flexibility of ATR allows ATRIP to properly lock the N-termini of the two ATR monomers to favor ATR-ATRIP complex formation and functional diversity. The isolated ＂head-head＂ and ＂taft-tail＂ each adopts a pseudo 2-fold symmetry. The catalytic pockets face outward and substrate access is not restricted by inhibitory dements. Our studies provide a structural basis for understanding the assembly of the ATR- ATRIP complex and a framework for characterizing ATR-mediated DNA repair pathways.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The aim of this study was to carry out a case control study comparing the HPV genome in patients with oral cavity squamous cell carcinoma (OC-SCC) to normal patients using metagenomic shotgun ...sequencing. We recruited 50 OC-SCC cases which were then matched with a control patient by age, gender, race, smoking status and alcohol status. DNA was extracted from oral wash samples from all patients and whole genome shotgun sequencing performed. The raw sequence data was cleaned, reads aligned with the human genome (GRCH38), nonhuman reads identified and then HPV genotypes identified using HPViewer. In the 50 patients with OC-SCC, the most common subsite was tongue in 26 (52%). All patients were treated with primary resection and neck dissection. All but 2 tumors were negative on p16 immunohistochemistry. There were no statistically significant differences between the cases and controls in terms of gender, age, race/ethnicity, alcohol drinking, and cigarette smoking. There was no statistically significant difference between the cancer samples and control samples in the nonhuman DNA reads (medians 4,228,072 vs. 5,719,715, P value = 0.324). HPV was detected in 5 cases (10%) of OC-SCC (genotypes 10, 16, 98) but only 1 tumor sample (genotype 16) yielded a high number of reads to suggest a role in the etiology of OC-SCC. HPV was detected in 4 control patients (genotypes 16, 22, 76, 200) but all had only 1-2 HPV reads per human genome. Genotypes of HPV are rarely found in patients with oral cancer.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Members of the HSP70 family function as molecular chaperones to maintain cellular homeostasis and help plants cope with environmental stimuli. However, due to functional redundancy and lack of ...effective chemical inhibitors, our knowledge of functions of individual HSP70s has remained limited. Here, we confirmed a subclass of HSP70s, including HSP70-1, -2, -3, -4, and -5, localized to the cytosol and nucleus in
Arabidopsis thaliana
. Histochemical analyses of promoter:GUS reporter lines showed that
HSP70-1, -2, -3
, and -
4
genes were widely expressed, but
HSP70-5
was not. In addition, individual
HSP70
showed not only similar but also distinct transcriptions when treated by different abiotic stresses and phytohormones. No apparent phenotype was observed when individual
HSP70
genes were overexpressed or knocked-out/down, but the double mutant
hsp70-1 hsp70-4
and triple mutant
hsp70-2 hsp70-4 hsp70-5
plants exhibited developmental phenotypes with shortened specific growth periods, curly and round leaves, twisted petioles, thin stems, and short siliques. Moreover, both mutants were hypersensitive to heat, cold, high glucose, salt and osmotic stress, but hyposensitive to abscisic acid. Genes related to flowering, and the cytokinin, brassinosteroid, and abscisic acid signaling pathways were differentially expressed in both mutants. Our studies suggest that, the individual HSP70 possibly performs both redundant and specific functions with the other members in the cytosolic/nuclear HSP70 subclass, and apart from enabling plants to cope with abiotic stresses, this subclass of cytosolic/nuclear HSP70 proteins also participates in diverse developmental processes and signaling pathways.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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