Single-cell Approaches Research Articles

Elevated CDC45 Expression Predicts Poorer Overall Survival Prognoses and Worse Immune Responses for Kidney Renal Clear Cell Carcinoma via Single-Cell and Bulk RNA-Sequencing.

The main objective of this paper is to analyze the prognostic and immunological value of CDC45 in kidney renal clear cell carcinoma (KIRC) using single-cell and bulk RNA-sequencing approaches. The expression of CDC45 in KIRC was evaluated by the HPA database, the TCGA-KIRC dataset and verified by PCR analysis and single-cell RNA-sequencing. The ability of CDC45 to independently predict prognosis in KIRC was confirmed by univariate/multivariate regression analysis. Gene set enrichment analysis (GSEA) was employed to explore CDC45-related pathways in KIRC. In addition, Relationships between CDC45 and immunity were also examined. Elevated CDC45 expression in KIRC was demonstrated at mRNA and protein levels. The results of the correlation analysis showed that as CDC45 expression increased, so did the histological grade, clinical stage, and TNM stage of the patients (p < 0.05). Univariate/multivariate regression analysis suggested CDC45 as an independent prognostic factor for KIRC. Seven pathways related to CDC45 were screened through GSEA. Meanwhile, we found that CDC45 was correlated with tumor mutational burden (TMB) and microsatellite instability (MSI) but not tumor neoantigen burden (TNB). Regarding immunity, CDC45 exhibited correlations with the tumor microenvironment, immune cell infiltration, and immune checkpoints. Besides, low CDC45 expression was shown to be associated with a better response to immunotherapy. Single-cell RNA-sequencing revealed that CDC45 was differently expressed in T cells (p < 0.05). CDC45 showed potential as a prognostic biomarker and therapeutic target for KIRC. Meanwhile, the CDC45 low expression group was more sensitive to immunotherapy.

Magnetron-Sputtered Thin-Film Catalyst with Low-Ir-Ru Content for Water Electrolysis: Long-Term Stability and Degradation Analysis

Proton Exchange Membrane Water Electrolyzers (PEM-WEs) are entering the phase of commercial mass production. However, the issue of an iridium catalyst for the anode remains. This work presents an iridium-ruthenium-based catalyst (25% Ir = 158 µg cm-2, 75% Ru) prepared as a thin film on the surface-enhanced-anode of PEM-WE via magnetron sputtering. Using a strictly practical single-cell approach, without any iR corrections, we show its excellent activity – 1 A cm-2 at 1.606 V, 80 °C, and stability – 1.3 µV h-1 at 1 A cm-2 for 500 hours. Together with a purely Ir-based catalyst (158 µg cm-2), we subject it to a massive electrochemical and material analysis, showing that the thickness and interconnectivity are essential for the catalyst’s stability. This conclusion is based on results from single-cell galvanostatic measurements, Potential Electrochemical Impedance Spectroscopy (PEIS), Scanning Electron Micrsocopy (SEM), X-Ray Photoelectron Spectroscopy (XPS), and Energy Dispersive X-ray spectroscopy (EDX). Consequently, we believe that magnetron sputtering is currently the most perspective method for preparing low-Ir-loading catalysts. Compared to the nanoparticle approach, it allows the creation of very thin films with unprecedented interconnectivity.

Opportunities in optical and electrical single-cell technologies to study microbial ecosystems.

New techniques are revolutionizing single-cell research, allowing us to study microbes at unprecedented scales and in unparalleled depth. This review highlights the state-of-the-art technologies in single-cell analysis in microbial ecology applications, with particular attention to both optical tools, i.e., specialized use of flow cytometry and Raman spectroscopy and emerging electrical techniques. The objectives of this review include showcasing the diversity of single-cell optical approaches for studying microbiological phenomena, highlighting successful applications in understanding microbial systems, discussing emerging techniques, and encouraging the combination of established and novel approaches to address research questions. The review aims to answer key questions such as how single-cell approaches have advanced our understanding of individual and interacting cells, how they have been used to study uncultured microbes, which new analysis tools will become widespread, and how they contribute to our knowledge of ecological interactions.

Dynamic single-cell regulomes characterize human peripheral blood innate lymphoid cell subpopulations

Innate lymphoid cells (ILCs) are plastic immune cells divided into 3 main subsets, characterized by distinct phenotypic and functional profiles. Using single cell approaches, heightened heterogeneity of mouse ILCs has been appreciated, imprinted by tissue signals that shape their transcriptome and epigenome. Intra-subset diversity has also been observed in human ILCs. However, combined transcriptomic and epigenetic analyses of single ILCs in humans are lacking. Here, we show high transcriptional and epigenetic heterogeneity among human circulating ILCs in healthy individuals. We describe phenotypically distinct subclusters and diverse chromatin accessibility within main ILC populations, compatible with differentially poised states. We validate the use of this healthy donor-based analysis as resource dataset to help inferring ILC changes occurring in disease conditions. Overall, our work provides insights in the complex human ILC biology. We anticipate it to facilitate hypothesis-driven studies in patients, without the need to perform single cell OMICs using precious patients' material.

Lineage commitment of dermal fibroblast progenitors is controlled by Kdm6b-mediated chromatin demethylation.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the epigenetic mechanisms that regulate DFP differentiation are not known. Our objective was to use multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanism that governs its differentiation potential. Our initial results indicated that the overall transcription profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage-specific genes. Surprisingly, the repressive chromatin profile of DFPs renders them unable to reform the skin in allograft assays despite their multipotent potential. We hypothesized that chromatin derepression was modulated by the H3K27me3 demethylase, Kdm6b/Jmjd3. Dermal fibroblast-specific deletion of Kdm6b/Jmjd3 in mice resulted in adipocyte compartment ablation and inhibition of mature dermal papilla functions, confirmed by additional single-cell RNA-seq, ChIP-seq, and allografting assays. We conclude that DFPs are functionally derepressed during murine skin development by Kdm6b/Jmjd3. Our studies therefore reveal a multimodal understanding of how DFPs differentiate into distinct fibroblast lineages and provide a novel publicly available multiomics search tool.

Toward the functional interpretation of somatic structural variations: bulk- and single-cell approaches.

Structural variants (SVs) are genomic rearrangements that can take many different forms such as copy number alterations, inversions and translocations. During cell development and aging, somatic SVs accumulate in the genome with potentially neutral, deleterious or pathological effects. Generation of somatic SVs is a key mutational process in cancer development and progression. Despite their importance, the detection of somatic SVs is challenging, making them less studied than somatic single-nucleotide variants. In this review, we summarize recent advances in whole-genome sequencing (WGS)-based approaches for detecting somatic SVs at the tissue and single-cell levels and discuss their advantages and limitations. First, we describe the state-of-the-art computational algorithms for somatic SV calling using bulk WGS data and compare the performance of somatic SV detectors in the presence or absence of a matched-normal control. We then discuss the unique features of cutting-edge single-cell-based techniques for analyzing somatic SVs. The advantages and disadvantages of bulk and single-cell approaches are highlighted, along with a discussion of their sensitivity to copy-neutral SVs, usefulness for functional inferences and experimental and computational costs. Finally, computational approaches for linking somatic SVs to their functional readouts, such as those obtained from single-cell transcriptome and epigenome analyses, are illustrated, with a discussion of the promise of these approaches in health and diseases.

Does Chronic Obstructive Pulmonary Disease Originate from Different Cell Types?

The onset of chronic obstructive pulmonary disease (COPD) is heterogeneous, and current approaches to define distinct disease phenotypes are lacking. In addition to clinical methodologies, subtyping COPD has also been challenged by the reliance on human lung samples from late-stage diseases. Different COPD phenotypes may be initiated from the susceptibility of different cell types to cigarette smoke, environmental pollution, and infections at early stages that ultimately converge at later stages in airway remodeling and destruction of the alveoli when the disease is diagnosed. This perspective provides discussion points on how studies to date define different cell types of the lung that can initiate COPD pathogenesis, focusing on the susceptibility of macrophages, T and B cells, mast cells, dendritic cells, endothelial cells, and airway epithelial cells. Additional cell types, including fibroblasts, smooth muscle cells, neuronal cells, and other rare cell types not covered here, may also play a role in orchestrating COPD. Here, we discuss current knowledge gaps, such as which cell types drive distinct disease phenotypes and/or stages of the disease and which cells are primarily affected by the genetic variants identified by whole genome-wide association studies. Applying new technologies that interrogate the functional role of a specific cell type or a combination of cell types as well as single-cell transcriptomics and proteomic approaches are creating new opportunities to understand and clarify the pathophysiology and thereby the clinical heterogeneity of COPD.

Single-cell analysis of spinal cord injury reveals functional heterogeneity of oligodendrocyte lineage cells

Spinal cord injury (SCI) is a traumatic condition that causes myelin destruction and neuronal death, making it challenging to reverse. In spinal cord tissue, oligodendrocyte progenitor cells and oligodendrocytes are essential for maintaining myelin morphology and axon regeneration. The decrease in oligodendrocyte lineage cells after SCI is a major factor contributing to the difficulty in restoring spinal cord function. However, there is still a lack of research on the status and intercellular communication between oligodendrocyte lineage cells after injury. The development of single-cell sequencing technology has enabled researchers to obtain highly accurate cellular transcriptional information, facilitating detailed studies of cellular subpopulations. This study delved into the cellular heterogeneity of oligodendrocyte lineage cells using a single-cell transcriptomic approach to uncover functional changes and cellular interactions during different time points after SCI. Our findings highlighted the critical roles of Psap (Prosaposin)/Gpr37l1 and Psap/Gpr37 ligand-receptor pairs among oligodendrocyte lineage cells. Furthermore, we predicted the transcription factors that may play a key regulatory role. We demonstrated for the first time that Junb acts almost exclusively in mature oligodendrocytes, which provides a potential target for the study of oligodendrocyte transcriptional mechanisms.

The σB alternative sigma factor circuit modulates noise to generate different types of pulsing dynamics.

Single-cell approaches are revealing a high degree of heterogeneity, or noise, in gene expression in isogenic bacteria. How gene circuits modulate this noise in gene expression to generate robust output dynamics is unclear. Here we use the Bacillus subtilis alternative sigma factor σB as a model system for understanding the role of noise in generating circuit output dynamics. σB controls the general stress response in B. subtilis and is activated by a range of energy and environmental stresses. Recent single-cell studies have revealed that the circuit can generate two distinct outputs, stochastic pulsing and a single pulse response, but the conditions under which each response is generated are under debate. We implement a stochastic mathematical model of the σB circuit to investigate this and find that the system's core circuit can generate both response types. This is despite one response (stochastic pulsing) being stochastic in nature, and the other (single response pulse) being deterministic. We demonstrate that the main determinant for whichever response is generated is the degree with which the input pathway activates the core circuit, although the noise properties of the input pathway also biases the system towards one or the other type of output. Thus, our work shows how stochastic modelling can reveal the mechanisms behind non-intuitive gene circuit output dynamics.

BSBM-07 HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Rheumatoid arthritis specific autoimmunity in the lung before and at the onset of disease.

The lung is implicated as a site for breaching tolerance prior to onset of seropositive RA. To substantiate this, we have investigated lung-resident B cells in bronchoalveolar lavage (BAL) samples from early untreated RA patients (n=9) and anti-citrullinated protein antibody (ACPA)-positive individuals at risk of developing RA (n=3). Single B cells (n=7680) were phenotyped and isolated from BAL of individuals during the risk-RA phase and at RA diagnosis. The Immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n=141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils. By our single cell approach, we found significantly increased proportions of B lymphocytes in autoantibody-positive as compared to negative individuals. Memory and double negative (DN) B cells were prominent in all subgroups. Upon antibody re-expression, seven highly mutated citrulline autoreactive clones originating from different memory B cell subsets where identified, both in at-risk and early RA patients. Lung IgG variable gene transcripts from ACPA-positive individuals carry frequent mutation-induced N-linked Fab glycosylation sites (p<0.001), often in the framework-3 of the variable region. Two of the lungs ACPAs bound to activated neutrophils, one from an at-risk individual and one from early RA. We conclude that T cell driven B cell differentiation, resulting in local class switching and somatic hypermutation are evident in lungs before, as well as in, early stages of ACPA-positive RA. Our findings add to the notion of lung mucosa being a site for initiation of the citrulline autoimmunity preceding seropositive RA. This article is protected by copyright. All rights reserved.

Physiological response of Symbiodiniaceae to thermal stress: Reactive oxygen species, photosynthesis, and relative cell size.

This study investigates the physiological response to heat stress of three genetically different Symbiodiniaceae strains isolated from the scleractinian coral Mussismilia braziliensis, endemic of the Abrolhos Bank, Brazil. Cultures of two Symbiodinium sp. and one Cladocopium sp. were exposed to a stepwise increase in temperature (2°C every second day) ranging from 26°C (modal temperature in Abrolhos) to 32°C (just above the maximum temperature registered in Abrolhos during the third global bleaching event-TGBE). After the cultures reached their final testing temperature, reactive oxygen species (ROS) production, single cell attributes (relative cell size and chlorophyll fluorescence), and photosynthetic efficiency (effective (Y(II)) and maximum (Fv/Fm) quantum yields) were measured within 4 h and 72 h. Non-photochemical coefficient (NPQ) was estimated based on fluorescence values. Population average ROS production was variable across strains and exposure times, reaching up a 2-fold increase at 32°C in one of the Symbiodinium sp. strains. A marked intrapopulation difference was observed in ROS production, with 5 to 25% of the cells producing up to 10 times more than the population average, highlighting the importance of single cell approaches to assess population physiology. Average cell size increases at higher temperatures, likely resulting from cell cycle arrest, whereas chlorophyll fluorescence decreased, especially in 4 h, indicating a photoacclimation response. The conditions tested do not seem to have elicited loss of photosynthetic efficiency nor the activation of non-photochemical mechanisms in the cells. Our results unveiled a generalized thermotolerance in three Symbiodiniaceae strains originated from Abrolhos' corals. Inter and intra-specific variability could be detected, likely reflecting the genetic differences among the strains.

16:40-16-50 Immune cells at the maternal fetal interface during placental development: a single cell approach in mice

16:40-16-50 Immune cells at the maternal fetal interface during placental development: a single cell approach in mice

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

Single-Cell Proliferation Microfluidic Device for High Throughput Investigation of Replicative Potential and Drug Resistance of Cancer Cells.

Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01µM, 0.001µM, 0.0001µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells. The online version contains supplementary material available at 10.1007/s12195-023-00773-z.

Immune-related adverse events after immune check point inhibitors: Understanding the intersection with autoimmunity.

Immune checkpoint inhibitor therapies act through blockade of inhibitory molecules involved in the regulation of T cells, thus releasing tumor specific T cells to destroy their tumor targets. However, immune checkpoint inhibitors (ICI) can also lead to a breach in self-tolerance resulting in immune-related adverse events (irAEs) that include tissue-specific autoimmunity. This review addresses the question of whether the mechanisms that drive ICI-induced irAEs are shared or distinct with those driving spontaneous autoimmunity, focusing on ICI-induced diabetes, ICI-induced arthritis, and ICI-induced thyroiditis due to the wealth of knowledge about the development of autoimmunity in type 1 diabetes, rheumatoid arthritis, and Hashimoto's thyroiditis. It reviews current knowledge about role of genetics and autoantibodies in the development of ICI-induced irAEs and presents new studies utilizing single-cell omics approaches to identify T-cell signatures associated with ICI-induced irAEs. Collectively, these studies indicate that there are similarities and differences between ICI-induced irAEs and autoimmune disease and that studying them in parallel will provide important insight into the mechanisms critical for maintaining immune tolerance.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks

Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA sequencing (scRNA-seq) is increasingly used to study the host factors underlying diverse cellular phenotypes but has limited capacity to analyze the role of bacterial factors. Here, we developed scPAIR-seq, a single-cell approach to analyze infection with a pooled library of multiplex-tagged, barcoded bacterial mutants. Infected host cells and barcodes of intracellular bacterial mutants are both captured by scRNA-seq to functionally analyze mutant-dependent changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We analyzed redundancy between effectors and mutant-specific unique fingerprints and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

Peripheral Blood Omics and Other Multiplex-based Systems in Pulmonary and Critical Care Medicine.

Over the last years, the use of peripheral blood-derived big datasets in combination with machine learning technology has accelerated the understanding, prediction, and management of pulmonary and critical care conditions. The goal of this article is to provide the readers with an introduction to the methods and applications of blood omics- and other multiplex-based technologies in the pulmonary and critical care medicine setting to better appreciate the current literature in the field. To accomplish that, we provide essential concepts needed to rationalize this approach and introduce the readers to the types of molecules that can be obtained from the circulating blood to generate big datasets, elaborate on the differences between bulk, sorted and single cell approaches, along with the basic analytical pipelines required for clinical interpretation. Examples of peripheral blood-derived big datasets used in recent literature are presented, and limitations of that technology are highlighted to qualify both the present and future value of these methodologies.