Single-cell RNA sequencing (scRNA-seq) identifies cell subpopulations within tissue but does not capture their spatial distribution nor reveal local networks of intercellular communication acting in ...situ. A suite of recently developed techniques that localize RNA within tissue, including multiplexed in situ hybridization and in situ sequencing (here defined as high-plex RNA imaging) and spatial barcoding, can help address this issue. However, no method currently provides as complete a scope of the transcriptome as does scRNA-seq, underscoring the need for approaches to integrate single-cell and spatial data. Here, we review efforts to integrate scRNA-seq with spatial transcriptomics, including emerging integrative computational methods, and propose ways to effectively combine current methodologies.
To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging ...from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
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•Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI•Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges•Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.•Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening
Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.
Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 ...SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Network biology is useful for modeling complex biological phenomena; it has attracted attention with the advent of novel graph-based machine learning methods. However, biological ...applications of network methods often suffer from inadequate follow-up. In this perspective, we discuss obstacles for contemporary network approaches—particularly focusing on challenges representing biological concepts, applying machine learning methods, and interpreting and validating computational findings about biology—in an effort to catalyze actionable biological discovery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only ...generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9).
In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression.
We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.
Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell–cell interactions, but conserved or divergent mechanisms governing ...this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell–type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.
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Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric ...disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.
Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and ...not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.
Agricultural production is hampered by disease, pests, and environmental stresses. To minimize yield loss, it is important to develop crop cultivars with resistance or tolerance to their respective ...biotic and abiotic constraints. Transformation techniques are not optimized for many species and desirable cultivars may not be amenable to genetic transformation, necessitating inferior cultivar usage and time-consuming introgression through backcrossing to the preferred variety. Overcoming these limitations will greatly facilitate the development of disease, insect, and abiotic stress tolerant crops. One such avenue for rapid crop improvement is the development of viral systems to deliver CRISPR/Cas-based genome editing technology to plants to generate targeted beneficial mutations. Viral delivery of genomic editing constructs can theoretically be applied to span the entire host range of the virus utilized, circumventing the challenges associated with traditional transformation and breeding techniques. Here we explore the types of viruses that have been optimized for CRISPR/Cas9 delivery, the phenotypic outcomes achieved in recent studies, and discuss the future potential of this rapidly advancing technology.
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