Showing papers by "Howard Hughes Medical Institute published in 2019"
Northern Arizona University1, National Institutes of Health2, University of Minnesota3, University of California, Davis4, Woods Hole Oceanographic Institution5, Massachusetts Institute of Technology6, University of Copenhagen7, University of Trento8, Chinese Academy of Sciences9, University of California, San Francisco10, University of Pennsylvania11, Pacific Northwest National Laboratory12, North Carolina State University13, University of California, San Diego14, Institute for Systems Biology15, Dalhousie University16, University of British Columbia17, Statens Serum Institut18, Anschutz Medical Campus19, University of Washington20, Michigan State University21, Stanford University22, Broad Institute23, Harvard University24, Australian National University25, University of Düsseldorf26, University of New South Wales27, Sookmyung Women's University28, San Diego State University29, Howard Hughes Medical Institute30, Cornell University31, Max Planck Society32, Colorado State University33, Google34, Syracuse University35, Webster University36, United States Department of Agriculture37, University of Arkansas for Medical Sciences38, Colorado School of Mines39, University of Southern Mississippi40, National Oceanic and Atmospheric Administration41, University of California, Merced42, Wageningen University and Research Centre43, University of Arizona44, Environment Agency45, University of Florida46, Merck & Co.47
TL;DR: QIIME 2 development was primarily funded by NSF Awards 1565100 to J.G.C. and R.K.P. and partial support was also provided by the following: grants NIH U54CA143925 and U54MD012388.
Abstract: QIIME 2 development was primarily funded by NSF Awards 1565100 to J.G.C. and 1565057 to R.K. Partial support was also provided by the following: grants NIH U54CA143925 (J.G.C. and T.P.) and U54MD012388 (J.G.C. and T.P.); grants from the Alfred P. Sloan Foundation (J.G.C. and R.K.); ERCSTG project MetaPG (N.S.); the Strategic Priority Research Program of the Chinese Academy of Sciences QYZDB-SSW-SMC021 (Y.B.); the Australian National Health and Medical Research Council APP1085372 (G.A.H., J.G.C., Von Bing Yap and R.K.); the Natural Sciences and Engineering Research Council (NSERC) to D.L.G.; and the State of Arizona Technology and Research Initiative Fund (TRIF), administered by the Arizona Board of Regents, through Northern Arizona University. All NCI coauthors were supported by the Intramural Research Program of the National Cancer Institute. S.M.G. and C. Diener were supported by the Washington Research Foundation Distinguished Investigator Award.
8,821 citations
TL;DR: A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.
Abstract: Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
2,260 citations
TL;DR: The original Cancer Cell Line Encyclopedia is expanded with deeper characterization of over 1,000 cell lines, including genomic, transcriptomic, and proteomic data, and integration with drug-sensitivity and gene-dependency data, which reveals potential targets for cancer drugs and associated biomarkers.
Abstract: Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
1,801 citations
TL;DR: Investigation of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8 and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.
Abstract: Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.
1,222 citations
TL;DR: It is found that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity.
1,189 citations
TL;DR: This review highlights recent progress in understanding the function of N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors, and emphasizes the importance of context for RNA modification regulation and function.
909 citations
TL;DR: The ‘jGCaMP7’ sensors are four genetically encoded calcium indicators with better sensitivity than state-of-the-art GCaMP6 and specifically improved for applications such as neuropil or wide-field imaging.
Abstract: Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
723 citations
TL;DR: In this article, the authors present CRISPResso2 to analyze base editors, perform allele-specific quantification or incorporate biologically-informed and scalable alignment approaches, and demonstrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.
Abstract: Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.
696 citations
TL;DR: An atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals is generated, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which are associated with resistance to anti-TNF treatment.
680 citations
TL;DR: It is shown that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15, which suppresses the clearance of tumours by enhancing the translation of lysosomal proteases in dendritic cells and thereby suppressing tumour antigen presentation.
Abstract: There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.
568 citations
TL;DR: This Review focuses on how quorums are detected and how quorum sensing controls group behaviours in complex and dynamically changing environments such as multi-species bacterial communities, in the presence of flow, in 3D non-uniform biofilms and in hosts during infection.
Abstract: Quorum sensing is a process of bacterial cell-to-cell chemical communication that relies on the production, detection and response to extracellular signalling molecules called autoinducers. Quorum sensing allows groups of bacteria to synchronously alter behaviour in response to changes in the population density and species composition of the vicinal community. Quorum-sensing-mediated communication is now understood to be the norm in the bacterial world. Elegant research has defined quorum-sensing components and their interactions, for the most part, under ideal and highly controlled conditions. Indeed, these seminal studies laid the foundations for the field. In this Review, we highlight new findings concerning how bacteria deploy quorum sensing in realistic scenarios that mimic nature. We focus on how quorums are detected and how quorum sensing controls group behaviours in complex and dynamically changing environments such as multi-species bacterial communities, in the presence of flow, in 3D non-uniform biofilms and in hosts during infection.
TL;DR: It is shown that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron–glioma synapses, which indicates that synaptic and electrical integration into neural circuits promotesglioma progression.
Abstract: High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth, but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron–glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections, forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation, whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth, human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together, these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression. Neurons form synapses onto glioma cells, and depolarization of glioma membranes promotes glioma growth in vivo, whereas blocking electrochemical signalling blocks tumour growth.
TL;DR: Two derivatives of lithocholic acid are revealed that act as regulators of T helper cells that express IL-17a and regulatory T cells, thus influencing host immune responses.
Abstract: Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells. Screening of a library of bile acid metabolites revealed two derivatives of lithocholic acid that act as regulators of T helper cells that express IL-17a and regulatory T cells, thus influencing host immune responses.
TL;DR: This process of “clonal hematopoiesis,” including the mechanisms by which it arises and the current state of knowledge regarding its effects on human health is reviewed, including the prevalence and clinical associations of somatic, clonal mutations in blood cells of individuals without hematologic malignancies.
Abstract: As people age, their tissues accumulate an increasing number of somatic mutations. Although most of these mutations are of little or no functional consequence, a mutation may arise that confers a fitness advantage on a cell. When this process happens in the hematopoietic system, a substantial proportion of circulating blood cells may derive from a single mutated stem cell. This outgrowth, called "clonal hematopoiesis," is highly prevalent in the elderly population. Here we discuss recent advances in our knowledge of clonal hematopoiesis, its relationship to malignancies, its link to nonmalignant diseases of aging, and its potential impact on immune function. Clonal hematopoiesis provides a glimpse into the process of mutation and selection that likely occurs in all somatic tissues.
TL;DR: Quantitative connectivity matrices (or connectomes) for both adult sexes of the nematode Caenorhabditis elegans are presented that encompass all connections from sensory input to end-organ output across the entire animal.
Abstract: Knowledge of connectivity in the nervous system is essential to understanding its function. Here we describe connectomes for both adult sexes of the nematode Caenorhabditis elegans, an important model organism for neuroscience research. We present quantitative connectivity matrices that encompass all connections from sensory input to end-organ output across the entire animal, information that is necessary to model behaviour. Serial electron microscopy reconstructions that are based on the analysis of both new and previously published electron micrographs update previous results and include data on the male head. The nervous system differs between sexes at multiple levels. Several sex-shared neurons that function in circuits for sexual behaviour are sexually dimorphic in structure and connectivity. Inputs from sex-specific circuitry to central circuitry reveal points at which sexual and non-sexual pathways converge. In sex-shared central pathways, a substantial number of connections differ in strength between the sexes. Quantitative connectomes that include all connections serve as the basis for understanding how complex, adaptive behavior is generated.
TL;DR: It is demonstrated that radiotherapy induces tumor cell ferroptosis, an unappreciated mechanism and focus for the development of effective combinatorial cancer therapy.
Abstract: A challenge in oncology is to rationally and effectively integrate immunotherapy with traditional modalities, including radiotherapy. Here, we demonstrate that radiotherapy induces tumor-cell ferroptosis. Ferroptosis agonists augment and ferroptosis antagonists limit radiotherapy efficacy in tumor models. Immunotherapy sensitizes tumors to radiotherapy by promoting tumor-cell ferroptosis. Mechanistically, IFNγ derived from immunotherapy-activated CD8+ T cells and radiotherapy-activated ATM independently, yet synergistically, suppresses SLC7A11, a unit of the glutamate-cystine antiporter xc-, resulting in reduced cystine uptake, enhanced tumor lipid oxidation and ferroptosis, and improved tumor control. Thus, ferroptosis is an unappreciated mechanism and focus for the development of effective combinatorial cancer therapy. SIGNIFICANCE: This article describes ferroptosis as a previously unappreciated mechanism of action for radiotherapy. Further, it shows that ferroptosis is a novel point of synergy between immunotherapy and radiotherapy. Finally, it nominates SLC7A11, a critical regulator of ferroptosis, as a mechanistic determinant of synergy between radiotherapy and immunotherapy.This article is highlighted in the In This Issue feature, p. 1631.
TL;DR: Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components, is described and demonstrated by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.
Abstract: Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.
VU University Amsterdam1, Leibniz Institute for Neurobiology2, Broad Institute3, Tufts University4, University of Southern California5, Cornell University6, Johns Hopkins University School of Medicine7, Johns Hopkins University8, Max Planck Society9, Harvard University10, KAIST11, Utrecht University12, University of Lausanne13, University of Rome Tor Vergata14, European Bioinformatics Institute15, University of Milan16, Autonomous University of Barcelona17, Yale University18, Swiss Institute of Bioinformatics19, Stanford University20, University of California, San Francisco21, Genentech22, Howard Hughes Medical Institute23, National University of Singapore24, Montreal Neurological Institute and Hospital25, University of Southampton26
TL;DR: It is shown that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes, and among de novo variants associated with neurodevelopmental disorders, including schizophrenia.
TL;DR: Waddington-OT is introduced, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them that sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.
TL;DR: It is concluded that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c- Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
Abstract: Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
TL;DR: It is shown that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy, and it is suggested that the combination of FTO inhibition with anti- PD1 blockade may reduce the resistance to immunotherapy in melanoma.
Abstract: Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.
TL;DR: The ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.
Abstract: The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.
TL;DR: A multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq).
TL;DR: This work presents RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes and demonstrates the scalability and utility of the tool.
Abstract: We present RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes. After the pseudomolecules are constructed, RaGOO identifies structural variants, including those spanning sequencing gaps. We show that RaGOO accurately orders and orients 3 de novo tomato genome assemblies, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open source at https://github.com/malonge/RaGOO .
TL;DR: It is shown that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo, and these findings have implications for targeting the intact provirus that are a barrier to curing HIV infection.
Abstract: A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.
TL;DR: The dynamics of the effector response of CD8+ tumor‐infiltrating lymphocytes (TILs) after checkpoint blockade therapy was examined, showing a shift from naive‐like to memory‐precursor‐ and effector‐like subsets within PD‐1−CD8+ T cells in tumors.
Max Delbrück Center for Molecular Medicine1, National University of Singapore2, The Heart Research Institute3, Humboldt University of Berlin4, National Institutes of Health5, Hammersmith Hospital6, Charité7, University of Hamburg8, Utrecht University9, University College London10, Ruhr University Bochum11, University of Sydney12, Technical University of Berlin13, Max Planck Society14, University of Münster15, Harvard University16, Howard Hughes Medical Institute17, Brigham and Women's Hospital18
TL;DR: This work analyzes the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation, and shows extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner.
Harvard University1, Broad Institute2, Radcliffe Institute for Advanced Study3, University of California, Berkeley4, Howard Hughes Medical Institute5, Massachusetts Institute of Technology6, Sapienza University of Rome7, University of Padua8, Queen's University Belfast9, Russian Academy of Sciences10, Al-Farabi University11, University of Pennsylvania12, University College Dublin13, University of Vienna14, Pennsylvania State University15, Max Planck Society16, Centre for Cellular and Molecular Biology17, Birbal Sahni Institute of Palaeobotany18, Emory University19, Centre national de la recherche scientifique20, Kyrgyz National University21, Altai State University22, Academy of Sciences of the Czech Republic23, University of Oxford24, South Ural State University25, Kemerovo State University26, University College London27, Northwest University (China)28, University of Pittsburgh29, Samara State University30, Chelyabinsk State University31, University of Bologna32, Academy of Sciences of Uzbekistan33, University of Winnipeg34, Simon Fraser University35, National Museum of Natural History36, Tomsk State University37, Naturhistorisches Museum38, Národní muzeum39, Hazara University40, Deccan College Post-Graduate and Research Institute41, Pompeu Fabra University42, Hartwick College43, University of California, Santa Barbara44, Washington University in St. Louis45
TL;DR: It is shown that Steppe ancestry then integrated further south in the first half of the second millennium BCE, contributing up to 30% of the ancestry of modern groups in South Asia, supporting the idea that the archaeologically documented dispersal of domesticates was accompanied by the spread of people from multiple centers of domestication.
Abstract: By sequencing 523 ancient humans, we show that the primary source of ancestry in modern South Asians is a prehistoric genetic gradient between people related to early hunter-gatherers of Iran and Southeast Asia. After the Indus Valley Civilization's decline, its people mixed with individuals in the southeast to form one of the two main ancestral populations of South Asia, whose direct descendants live in southern India. Simultaneously, they mixed with descendants of Steppe pastoralists who, starting around 4000 years ago, spread via Central Asia to form the other main ancestral population. The Steppe ancestry in South Asia has the same profile as that in Bronze Age Eastern Europe, tracking a movement of people that affected both regions and that likely spread the distinctive features shared between Indo-Iranian and Balto-Slavic languages.
TL;DR: This work leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee to identify human-specific features of cortical development and identifies 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex.
TL;DR: Tagging live single cells and nuclei with lipid- or cholesterol-modified oligonucleotides enables massive scRNA-seq sample multiplexing, identifies doublets and recovers cells with low RNA content.
Abstract: Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.