High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we ...introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.
Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this ...approach to Arabidopsis (
) root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.
Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible ...pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of “chromatin hubs”—they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
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•Cicero connects regulatory DNA elements to target genes•Co-accessible elements form chromatin hubs•Chromatin hubs are co-regulated during skeletal muscle development•Cicero can help reveal the mechanisms of cis-regulation on a genome-wide scale
Pliner et al. introduce Cicero, a software program to connect distal regulatory elements with target genes using single-cell ATAC-seq data. They find evidence that groups of co-accessible elements form chromatin hubs and undergo coordinated changes in histone marks that are predictive of changes in gene expression in skeletal muscle development.
Latino males are effectively vanishing from the American higher education pipeline. Even as the number of Latinas/os attending college has actually increased steadily over the last few decades, the ...proportional representation of Latino males continues to slide relative to their Latina female counterparts. The question of why Latino males are losing ground in accessing higher education-relative to their peers-is an important and complex one, and it lies at the heart of this book. There are several broad themes highlighted, catalogued along with the four dimensions of policy, theory, research, and practice. The contributors to this book present new research on factors that inhibit or promote Latino success in both four-year institutions and community colleges in order to inform both policy and practice. They explore the social-cultural factors, peer dynamics, and labor force demands that may be perpetuating the growing gender gap, and consider what lessons can be learned from research on the success of Latinas. This book also closely examines key practices that enable first generation Latino male undergraduates to succeed which may seem counterintuitive to institutional expectations and preconceived notions of student behavior. Using narrative data, the book also explores the role of family in persistence; outlines how Latino men conceptualize fulfilling expectations, negotiate the emasculization of the educational process, and how they confront racialization in the pursuit of a higher education; uncovers attitudes to help-seeking that are detrimental to their success: and analyzes how those who succeed and progress in college apply their social capital - whether aspirational, navigational, social, linguistic, familial, or resistant. While uncovering the lack of awareness at all levels of our colleges and universities about the depth and severity of the challenges facing Latino males, this book provides the foundation for rethinking policy; challenges leaders to ins
Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to ...probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-β. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.
In the last 10 years, a great number of publications (both regular papers and reviews) have been published on the interesting molecules-mycosporine-like amino acids (MAAs). Despite significant ...advances in the research of MAAs, current overviews in the recent publications involving MAA research still need reporting. The aim of this Special Issue is to join, as an interdisciplinary approach, the photochemical and photobiological aspects, with emphasis on new natural resources to obtain both algae and zooplankton MAAs, advances in methodology of extraction and chemical identification of new MAAs. Finally, this Special Issue reviews the bioactivities of MAAs including UVR screen, antioxidant, immunostimulant, growth factor, DNA protection, inhibition of collagenase, elastase and hyaluronidase, and anti-photoaging, among others, and their potential use as nutracosmeceutic molecules (i.e., oral and topic photoprotector).
Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are ...limited in scope to common variants and in resolution by linkage disequilibrium. We present 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). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
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•Perturbed 5,920 human candidate enhancers for impact on gene expression•Multiplexed ∼28 CRISPRi perturbations per single-cell transcriptome•Adapted the eQTL analytical framework to identify 664 cis human enhancer-gene pairs•Characterized genomic features associated with these enhancer-gene pairs
A highly multiplexed CRISPRi screen uncovers gene-enhancer relationships at scale.
Summary Background Emotional stress is associated with increased risk of cardiovascular disease. We imaged the amygdala, a brain region involved in stress, to determine whether its resting metabolic ...activity predicts risk of subsequent cardiovascular events. Methods Individuals aged 30 years or older without known cardiovascular disease or active cancer disorders, who underwent18 F-fluorodexoyglucose PET/CT at Massachusetts General Hospital (Boston, MA, USA) between Jan 1, 2005, and Dec 31, 2008, were studied longitudinally. Amygdalar activity, bone-marrow activity, and arterial inflammation were assessed with validated methods. In a separate cross-sectional study we analysed the relation between perceived stress, amygdalar activity, arterial inflammation, and C-reactive protein. Image analyses and cardiovascular disease event adjudication were done by mutually blinded researchers. Relations between amygdalar activity and cardiovascular disease events were assessed with Cox models, log-rank tests, and mediation (path) analyses. Findings 293 patients (median age 55 years IQR 45·0–65·5) were included in the longitudinal study, 22 of whom had a cardiovascular disease event during median follow-up of 3·7 years (IQR 2·7–4·8). Amygdalar activity was associated with increased bone-marrow activity ( r =0·47; p<0·0001), arterial inflammation ( r =0·49; p<0·0001), and risk of cardiovascular disease events (standardised hazard ratio 1·59, 95% CI 1·27–1·98; p<0·0001), a finding that remained significant after multivariate adjustments. The association between amygdalar activity and cardiovascular disease events seemed to be mediated by increased bone-marrow activity and arterial inflammation in series. In the separate cross-sectional study of patients who underwent psychometric analysis (n=13), amygdalar activity was significantly associated with arterial inflammation ( r =0·70; p=0·0083). Perceived stress was associated with amygdalar activity ( r =0·56; p=0·0485), arterial inflammation ( r =0·59; p=0·0345), and C-reactive protein ( r =0·83; p=0·0210). Interpretation In this first study to link regional brain activity to subsequent cardiovascular disease, amygdalar activity independently and robustly predicted cardiovascular disease events. Amygdalar activity is involved partly via a path that includes increased bone-marrow activity and arterial inflammation. These findings provide novel insights into the mechanism through which emotional stressors can lead to cardiovascular disease in human beings. Funding None.
Decomposition is an essential ecosystem service driven by interacting biotic and abiotic factors. Increasing temperatures due to climate change can affect soil moisture, soil fauna, and subsequently, ...decomposition. Understanding how projected climate change scenarios will affect decomposition is of vital importance for predicting nutrient cycling and ecosystem health. In this study, we experimentally addressed the question of how the early stages of decomposition would vary along a gradient of projected climate change scenarios. Given the importance of biodiversity for ecosystem service provisioning, we measured the effect of invertebrate exclusion on red maple (Acer rubrum) leaf litter breakdown along a temperature gradient using litterbags in warming chambers over a period of five weeks. Leaf litter decomposed more slowly in the warmer chambers and in the litterbag treatment that minimized invertebrate access. Moreover, increasing air temperature reduced invertebrate abundance and richness, and altered the community composition, independent of exclusion treatment. Using structural equation models, we were able to disentangle the effects of average air temperature on leaf litter loss, finding a direct negative effect of warming on the early stages of decomposition, independent of invertebrate abundance. This result indicates that not only can climate change affect the invertebrate community, but may also directly influence how the remaining organisms interact with their environment and their effectiveness at provisioning ecosystem services. Overall, our study highlights the role of biodiversity in maintaining ecosystem services and contributes to our understanding of how climate change could disrupt nutrient cycling.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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