CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

Ferroptosis is an iron-dependent cell death mechanism characterized by the accumulation of toxic lipid peroxides and cell membrane rupture. GPX4 (glutathione peroxidase 4) prevents ferroptosis by reducing these lipid peroxides into lipid alcohols. Ferroptosis induction by GPX4 inhibition has emerged as a vulnerability of cancer cells, highlighting the need to identify ferroptosis regulators that may be exploited therapeutically. Through genome-wide CRISPR activation screens, we identify the SWI/SNF (switch/sucrose non-fermentable) ATPases BRM (SMARCA2) and BRG1 (SMARCA4) as ferroptosis suppressors. Mechanistically, they bind to and increase chromatin accessibility at NRF2 target loci, thus boosting NRF2 transcriptional output to counter lipid peroxidation and confer resistance to GPX4 inhibition. We further demonstrate that the BRM/BRG1 ferroptosis connection can be leveraged to enhance the paralog dependency of BRG1 mutant cancer cells on BRM. Our data reveal ferroptosis induction as a potential avenue for broadening the efficacy of BRM degraders/inhibitors and define a specific genetic context for exploiting GPX4 dependency.

Systemic inflammatory response syndrome triggered by blood-borne pathogens induces prolonged dendritic cell paralysis and immunosuppression.

Blood-borne pathogens can cause systemic inflammatory response syndrome (SIRS) followed by protracted, potentially lethal immunosuppression. The mechanisms responsible for impaired immunity post-SIRS remain unclear. We show that SIRS triggered by pathogen mimics or malaria infection leads to functional paralysis of conventional dendritic cells (cDCs). Paralysis affects several generations of cDCs and impairs immunity for 3-4 weeks. Paralyzed cDCs display distinct transcriptomic and phenotypic signatures and show impaired capacity to capture and present antigens in vivo. They also display altered cytokine production patterns upon stimulation. The paralysis program is not initiated in the bone marrow but during final cDC differentiation in peripheral tissues under the influence of local secondary signals that persist after resolution of SIRS. Vaccination with monoclonal antibodies that target cDC receptors or blockade of transforming growth factor ß partially overcomes paralysis and immunosuppression. This work provides insights into the mechanisms of paralysis and describes strategies to restore immunocompetence post-SIRS.

A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies.

The success of CRISPR-mediated gene perturbation studies is highly dependent on the quality of gRNAs, and several tools have been developed to enable optimal gRNA design. However, these tools are not all adaptable to the latest CRISPR modalities or nucleases, nor do they offer comprehensive annotation methods for advanced CRISPR applications. Here, we present a new ecosystem of R packages, called crisprVerse, that enables efficient gRNA design and annotation for a multitude of CRISPR technologies. This includes CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR base editing (CRISPRbe) and CRISPR knockdown (CRISPRkd). The core package, crisprDesign, offers a user-friendly and unified interface to add off-target annotations, rich gene and SNP annotations, and on- and off-target activity scores. These functionalities are enabled for any RNA- or DNA-targeting nucleases, including Cas9, Cas12, and Cas13. The crisprVerse ecosystem is open-source and deployed through the Bioconductor project ( https://github.com/crisprVerse ).

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

SpatialExperiment: infrastructure for spatially-resolved transcriptomics data in R using Bioconductor.

SUMMARY: SpatialExperiment is a new data infrastructure for storing and accessing spatially-resolved transcriptomics data, implemented within the R/Bioconductor framework, which provides advantages of modularity, interoperability, standardized operations and comprehensive documentation. Here, we demonstrate the structure and user interface with examples from the 10x Genomics Visium and seqFISH platforms, and provide access to example datasets and visualization tools in the STexampleData, TENxVisiumData and ggspavis packages. AVAILABILITY AND IMPLEMENTATION: The SpatialExperiment, STexampleData, TENxVisiumData and ggspavis packages are available from Bioconductor. The package versions described in this manuscript are available in Bioconductor version 3.15 onwards. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Locus-specific expression of transposable elements in single cells with CELLO-seq.

Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.

Doublet identification in single-cell sequencing data using scDblFinder.

Doublets are prevalent in single-cell sequencing data and can lead to artifactual findings. A number of strategies have therefore been proposed to detect them. Building on the strengths of existing approaches, we developed scDblFinder, a fast, flexible and accurate Bioconductor-based doublet detection method. Here we present the method, justify its design choices, demonstrate its performance on both single-cell RNA and accessibility sequencing data, and provide some observations on doublet formation, detection, and enrichment analysis. Even in complex datasets, scDblFinder can accurately identify most heterotypic doublets, and was already found by an independent benchmark to outcompete alternatives.

Homeopathic Clinical Features of 18 Patients in COVID-19 Outbreaks in Hong Kong.

BACKGROUND: Hong Kong is geographically located in the province of Guangdong which, after Hubei, has been the region of China second-most affected by the COVID-19 pandemic. Compared to the pathognomonic symptoms of the named disease, homeopathic symptoms are always more helpful for homeopathic prescriptions. AIM: This study reports and summarizes the homeopathic symptoms observed in 18 confirmed/suspected epidemiologically related cases in cluster outbreaks of COVID-19 in Hong Kong in early 2020. METHODS: Homeopathic symptoms from this case series were collected from 18 consecutive patients who, in addition to their concurrent conventional treatment or traditional Chinese medicine, actively sought help from homeopathy as an adjunctive measure for symptomatic relief from COVID-19. Cases were categorized according to outbreak clusters, focusing mainly on the homeopathic symptoms. In the analysis, frequency of all homeopathic medicines, common rubrics in all the cases, common rubrics in each of the top-ranked remedies, and differentiating symptoms for each top-ranked remedy were determined. RESULTS: Homeopathic symptoms of 18 cases, each identified as mild and belonging to one of six separate clusters, are reported. Eighteen common symptoms screened out of 79 selected rubrics constituted two sets of homeopathic symptom pictures: Bryonia alba (n = 4) and Gelsemium sempervirens (n = 12). Eight and seven differentiating features, respectively, were identified for Bryonia alba and Gelsemium sempervirens. CONCLUSION: The common symptoms of 18 mild COVID-19 cases constituted two sets of homeopathic symptom pictures, indicating Bryonia alba or Gelsemium sempervirens; they were indicated in 4 and 12 cases, respectively, out of the 18 in total.

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division.

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.

Orchestrating single-cell analysis with Bioconductor.

Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.

Publisher Correction: Orchestrating single-cell analysis with Bioconductor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data.

Droplet-based single-cell RNA sequencing protocols have dramatically increased the throughput of single-cell transcriptomics studies. A key computational challenge when processing these data is to distinguish libraries for real cells from empty droplets. Here, we describe a new statistical method for calling cells from droplet-based data, based on detecting significant deviations from the expression profile of the ambient solution. Using simulations, we demonstrate that EmptyDrops has greater power than existing approaches while controlling the false discovery rate among detected cells. Our method also retains distinct cell types that would have been discarded by existing methods in several real data sets.

Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution.

Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood. Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific. We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution.

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT.

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.

Transcription-factor-mediated supervision of global genome architecture maintains B cell identity.

Recent studies have elucidated cell-lineage-specific three-dimensional genome organization; however, how such specific architecture is established or maintained is unclear. We hypothesized that lineage-defining transcription factors maintain cell identity via global control of genome organization. These factors bind many genomic sites outside of the genes that they directly regulate and thus are potentially implicated in three-dimensional genome organization. Using chromosome-conformation-capture techniques, we show that the transcription factor Paired box 5 (Pax5) is critical for the establishment and maintenance of the global lineage-specific architecture of B cells. Pax5 was found to supervise genome architecture throughout B cell differentiation, until the plasmablast stage, in which Pax5 is naturally silenced and B cell-specific genome structure is lost. Crucially, Pax5 did not rely on ongoing transcription to organize the genome. These results implicate sequence-specific DNA-binding proteins in global genome organization to establish and maintain lineage fidelity.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

iSEE: Interactive SummarizedExperiment Explorer.

Data exploration is critical to the comprehension of large biological data sets generated by high-throughput assays such as sequencing. However, most existing tools for interactive visualisation are limited to specific assays or analyses. Here, we present the iSEE (Interactive SummarizedExperiment Explorer) software package, which provides a general visual interface for exploring data in a SummarizedExperiment object. iSEE is directly compatible with many existing R/Bioconductor packages for analysing high-throughput biological data, and provides useful features such as simultaneous examination of (meta)data and analysis results, dynamic linking between plots and code tracking for reproducibility. We demonstrate the utility and flexibility of iSEE by applying it to explore a range of real transcriptomics and proteomics data sets.

Detection and removal of barcode swapping in single-cell RNA-seq data.

Barcode swapping results in the mislabelling of sequencing reads between multiplexed samples on patterned flow-cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays; however, the severity and consequences of barcode swapping remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in two plate-based single-cell RNA-sequencing datasets. We found that approximately 2.5% of reads were mislabelled between samples on the HiSeq 4000, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Furthermore, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA-sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10x Genomics experiments, allowing the continued use of cutting-edge sequencing machines for these assays.

T cell cytolytic capacity is independent of initial stimulation strength.

How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8+ T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells. Our data revealed that strong ligands drove more efficient and uniform activation than did weak ligands, but all activated cells were fully cytolytic. Notably, activation followed the same transcriptional pathways regardless of ligand potency. Thus, stimulation strength did not intrinsically dictate the T cell-activation route or phenotype; instead, it controlled how rapidly and simultaneously the cells initiated activation, allowing limited machinery to elicit wide-ranging responses.