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Massively parallel single-cell and single-nucleus RNA sequencing has opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so is the need for computational pipelines for scaled analysis. Here we developed Cumulus—a cloud-based framework for analyzing large-scale single-cell and single-nucleus RNA sequencing datasets. Cumulus combines the power of cloud computing with improvements in algorithm and implementation to achieve high scalability, low cost, user-friendliness and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies. Cumulus is a cloud-based framework enabling large-scale single-cell and single-nucleus RNA sequencing data analysis.

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost. Droplet microfluidics enables high-throughput single-cell sequencing, but often with increased noise. Here the authors report spinDrop (sorting picoinjection inDrop) to increase gene detection and reduce noise; they use this to generate a high-quality molecular atlas of mouse brain development.

Bimodality of gene expression, as a mechanism contributing to phenotypic diversity, enhances the survival of cells in a fluctuating environment. To date, the bimodal response of a gene regulatory system has been attributed to the cooperativity of transcription factor binding or to feedback loops. It has remained unclear whether noncooperative binding of transcription factors can give rise to bimodality in an open-loop system. We study a theoretical model of gene expression in a two-step cascade (a deterministically monostable system) in which the regulatory gene produces transcription factors that have a nonlinear effect on the activity of the target gene. We show that a unimodal distribution of transcription factors over the cell population can generate a bimodal steadystate output without cooperative transcription factor binding. We introduce a simple method of geometric construction that allows one to predict the onset of bimodality. The construction only involves the parameters of bursting of the regulatory gene and the dose—response curve of the target gene. Using this method, we show that the gene expression may switch between unimodal and bimodal as the concentration of inducers or corepressors is varied. These findings may explain the experimentally observed bimodal response of cascades consisting of a fluorescent protein reporter controlled by the tetracycline repressor. The geometric construction provides a useful tool for designing experiments and for interpretation of their results. Our findings may have important implications for understanding the strategies adopted by cell populations to survive in changing environments.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6βrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration. This preclinical study demonstrates the potential of a drug repurposing strategy using the coadministration of tamsulosin, metoprolol, and bromocriptine to provide a mutation-agnostic therapy for inherited retinal degeneration.

Tumor-associated macrophages (TAMs) of classic Hodgkin Lymphoma (cHL) contribute to the development of immunosuppressive tumor microenvironment (TME) and are associated with worse treatment outcomes. However, detailed features, functions and therapeutic vulnerabilities of cHL TAMs remain largely unknown. To address this, we analyzed cHL diagnostic biopsies by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and assessed transcriptional, proteomic and metabolic profiles of in vitro TAM models. We show that Reed–Sternberg (RS) cells induce a disease-specific TAM phenotype, characterized by elevated expression of factors involved in chemotaxis, angiogenesis, extracellular matrix remodeling and tumor immune escape. RS cell-conditioned TAMs expressed TGFβ, CCL17 and tryptophan catabolizing enzymes, IDO1 and IL4I1, promoting regulatory T cell recruitment and activation. In addition, we identified the expression of PIM1/2/3 kinases in cHL TAMs and characterized PIMs as critical hubs orchestrating RS-macrophage interactions. Pharmacological PIM blockade attenuated the RS-induced TAM transcriptional program. In established TAMs, PIM inhibition or PROTAC-mediated degradation decreased the expression of multiple factors associated with pro-tumoral TAM functions, including IL8, MMP9, CHI3L1/2, CD206, CD209, PD-L1, CCL17, TGFβ, IL4I1 and IDO1. PIM blockade attenuated TAM-dependent eosinophil chemoattraction, extracellular matrix remodeling, angiogenesis and regulatory T-cell development. Taken together, our study highlights the role of PIMs in the regulation of pathogenic TAM functions in cHL, further supporting the rationale of PIM targeting in this disease.

Embryonic stem cells co-express Oct4 and Oct1, a related protein with similar DNA-binding specificity. To study the role of Oct1 in ESC pluripotency and transcriptional control, we constructed germline and inducible-conditional Oct1-deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal and growth but manifest defects upon differentiation. They fail to form beating cardiomyocytes, generate neurons poorly, form small, poorly differentiated teratomas, and cannot generate chimeric mice. Upon RA-mediated differentiation, Oct1-deficient cells induce lineage-appropriate developmentally poised genes poorly while lineage-inappropriate genes, including extra-embryonic genes, are aberrantly expressed. In ESCs, Oct1 co-occupies a specific set of targets with Oct4, but does not occupy differentially expressed developmental targets. Instead, Oct1 occupies these targets as cells differentiate and Oct4 declines. These results identify a dynamic interplay between Oct1 and Oct4, in particular during the critical window immediately after loss of pluripotency when cells make the earliest developmental fate decisions. Humans and most other animals are composed of hundreds of different types of cell, including nerve cells, muscle cells and blood cells. Despite performing many different roles, these cells all develop from a single fertilized egg, which divides to make a particular group of cells that when studied in the laboratory are called embryonic stem cells (or ESCs for short). The ability of a cell to become a different cell type is defined as “potency”. ESCs are unique because they can specialize into any type of cell present in the adult organism, and they are therefore called “pluripotent”. However, as the embryo develops, its ESCs gradually lose their potency, and become more and more specialized. The activity of a great number of genes must be regulated during the transition from pluripotent to specialized cells, and some of the mechanisms involved in this transition are still unclear. ESCs are known to need a gene-regulating protein called Oct4 to remain pluripotent and Shen, Kang, Shakya et al. now show that a similar protein named Oct1 is essential for their transition to becoming more specialized. When the gene for Oct1 was deleted from mouse ECSs, they behaved largely like “normal” ESCs, but could not properly mature into certain cell types such as heart and nerve cells. Molecular analyses revealed that Oct4 and Oct1 compete to regulate the activity of many common genes with opposing outcomes: Oct4 keeps ESCs pluripotent while Oct1 leads them to specialize. The Oct4 protein is abundant in ESCs and prevails over Oct1, but as the cells mature, the levels of Oct4 drop, and Oct1 takes over in the regulation of their common target genes. Going forward, a better understanding of how ESCs become specialized will help basic research in the laboratory and allow scientists to tackle new questions about how the human body develops and how our organs work. In the longer-term, these findings might also have applications in the field of regenerative medicine, which aims to repair or replace a person’s cells, tissues or organs to improve their health.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1 . We discovered an accumulation of ceramides in the AdipoR1 –/– retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1 –/– mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1- related retinopathy.

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.

Abstract The recent expansion of single-cell technologies has enabled simultaneous genome-wide measurements of multiple modalities in the same single cell. The potential to jointly profile such modalities as gene expression, chromatin accessibility, protein epitopes, or multiple histone modifications at single-cell resolution represents a compelling opportunity to study developmental processes at multiple layers of gene regulation. Here, we present Ocelli, a lightweight Python package implemented in Ray for scalable visualization and analysis of developmental multimodal single-cell data. The core functionality of Ocelli focuses on diffusion-based modeling of biological processes involving cell state transitions. Ocelli addresses common tasks in single-cell data analysis, such as visualization of cells on a low-dimensional embedding that preserves the continuity of the developmental progression of cells, identification of rare and transient cell states, integration with trajectory inference algorithms, and imputation of undetected feature counts. Extensive benchmarking shows that Ocelli outperforms existing methods regarding computational time and quality of the reconstructed low-dimensional representation of developmental data. Graphical Abstract Graphical Abstract

T cell immunoglobulin and mucin-containing molecule 3 (TIM-3), first identified as a molecule expressed on interferon-γ producing T cells 1 , is emerging as an important immune-checkpoint molecule, with therapeutic blockade of TIM-3 being investigated in multiple human malignancies. Expression of TIM-3 on CD8 + T cells in the tumour microenvironment is considered a cardinal sign of T cell dysfunction; however, TIM-3 is also expressed on several other types of immune cell, confounding interpretation of results following blockade using anti-TIM-3 monoclonal antibodies. Here, using conditional knockouts of TIM-3 together with single-cell RNA sequencing, we demonstrate the singular importance of TIM-3 on dendritic cells (DCs), whereby loss of TIM-3 on DCs—but not on CD4 + or CD8 + T cells—promotes strong anti-tumour immunity. Loss of TIM-3 prevented DCs from expressing a regulatory program and facilitated the maintenance of CD8 + effector and stem-like T cells. Conditional deletion of TIM-3 in DCs led to increased accumulation of reactive oxygen species resulting in NLRP3 inflammasome activation. Inhibition of inflammasome activation, or downstream effector cytokines interleukin-1β (IL-1β) and IL-18, completely abrogated the protective anti-tumour immunity observed with TIM-3 deletion in DCs. Together, our findings reveal an important role for TIM-3 in regulating DC function and underscore the potential of TIM-3 blockade in promoting anti-tumour immunity by regulating inflammasome activation. Mouse genetic studies and single-cell transcriptome analysis demonstrate that TIM-3 on dendritic cells has a key role in regulating antitumour immunity via inflammasome activation and is a potential target for anticancer therapy.