Recent Advances In Single-cell Analysis Research Articles

Strategies for senolytic drug discovery.

Senolytics are a category of drugs that reduce the impact of cellular senescence, an effect associated with a range of chronic and age-related diseases. Since the discovery of the first senolytics in 2015, the number of known senolytic agents has grown dramatically. This review discusses the broad categories of known senolytics-kinase inhibitors, Bcl-2 family protein inhibitors, naturally occurring polyphenols, heat shock protein inhibitors, BET family protein inhibitors, P53 stabilizers, repurposed anti-cancer drugs, cardiac steroids, PPAR-alpha agonists, and antibiotics. The approaches used to screen for new senolytics are articulated including a range of methods to induce senescence, different target cell types, various senolytic assays, and markers. The choice of methods can greatly influence the outcomes of a screen, with high-quality screens featuring robust systems, adequate controls, and extensive validation in alternate assays. Recent advances in single-cell analysis and computational methods for senolytic identification are also discussed. There is significant potential for further drug discovery, but this will require additional research into drug targets and mechanisms of actions and their subsequent rigorous evaluation in pre-clinical models and human trials.

Mass spectrometry-based techniques for single-cell analysis.

The cell is the most basic structural unit and plays a vital role in the function of an organism. Studying the heterogeneity of cells, especially the qualitative and quantitative analyses of proteins and lipids at the cellular level and even at the subcellular level, is of great significance for the study of some important pathological or physiological processes. Due to the small size of a single cell, low content of analytes and large interference from the biological matrix within the single cell, analytical methods at the single cell level must be highly sensitive and selective. Mass spectrometry is a powerful technology for single-cell analysis, because it has high sensitivity, high selectivity and the ability to monitor multiple chemicals at the same time. In this review, four mass spectrometry-based methods applied to single-cell analysis are introduced and discussed in detail; these are electrospray ionization mass spectrometry (ESI-MS), laser desorption ionization mass spectrometry (LDI-MS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS). The recent advances in single-cell analysis with these mass spectrometry-based techniques are summarized. We believe that this review can provide some help and reference for single-cell analysis by mass spectrometry.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Transcriptomics Reveals Compartment-Specific Differences in Immune Responses and Contributions for Complement Factor 3 in Hemorrhagic Shock Plus Tissue Trauma.

Hemorrhagic shock with tissue trauma (HS/T) leads to the activation of a system-wide immune-inflammatory response that involves all organs and body compartments. Recent advances in single-cell analysis permit the simultaneous assessment of transcriptomic patterns in a large number of cells making it feasible to survey the landscape of immune cell responses across numerous anatomic sites. Here, we used single-cell RNA sequencing of leukocytes from the blood, liver, and spleen to identify the major shifts in gene expression by cell type and compartment in a mouse HS/T model. At 6 h, dramatic changes in gene expression were observed across multiple-cell types and in all compartments in wild-type mice. Monocytes from circulation and liver exhibited a significant upregulation of genes associated with chemotaxis and migration and a simultaneous suppression of genes associated with interferon signaling and antigen presentation. In contrast, liver conventional DC exhibited a unique pattern compared with other myeloid cells that included a pronounced increase in major histocompatibility complex class II (MHCII) gene expression. The dominant pattern across all compartments for B and T cells was a suppression of genes associated with cell activation and signaling after HS/T. Using complement factor 3 (C3) knockout mice we unveiled a role for C3 in the suppression of monocyte Major Histocompatibility Complex class II expression and activation of gene expression associated with migration, phagocytosis and cytokine upregulation, and an unexpected role in promoting interferon-signaling in a subset of B and T cells across all three compartments after HS/T. This transcriptomic landscape study of immune cells provides new insights into the host immune response to trauma, as well as a rich resource for further investigation of trauma-induced immune responses and complement in driving interferon signaling.

Tendon Stem/Progenitor Cell Subpopulations and Their Implications in Tendon Biology.

Tendon harbors a cell population that possesses stem cell characteristics such as clonogenicity, multipotency and self-renewal capacity, commonly referred to as tendon stem/progenitor cells (TSPCs). Various techniques have been employed to study how TSPCs are implicated in tendon development, homeostasis and healing. Recent advances in single-cell analysis have enabled much progress in identifying and characterizing distinct subpopulations of TSPCs, which provides a more comprehensive view of TSPCs function in tendon biology. Understanding the mechanisms of physiological and pathological processes regulated by TSPCs, especially a particular subpopulation, would greatly benefit treatment of diseased tendons. Here, we summarize the current scientific literature on the various subpopulations of TSPCs, and discuss how TSPCs can contribute to tissue homeostasis and pathogenesis, as well as examine the key modulatory signaling pathways that determine stem/progenitor cell state. A better understanding of the roles that TSPCs play in tendon biology may facilitate the development of novel treatment strategies for tendon diseases.

Recent advances in single-cell analysis by mass spectrometry.

Cells are the most basic structural units that play vital roles in the functioning of living organisms. Analysis of the chemical composition and content of a single cell plays a vital role in ensuring precise investigations of cellular metabolism, and is a crucial aspect of lipidomic and proteomic studies. In addition, structural knowledge provides a better understanding of cell behavior as well as the cellular and subcellular mechanisms. However, single-cell analysis can be very challenging due to the very small size of each cell as well as the large variety and extremely low concentrations of substances found in individual cells. On account of its high sensitivity and selectivity, mass spectrometry holds great promise as an effective technique for single-cell analysis. Numerous mass spectrometric techniques have been developed to elucidate the molecular profiles at the cellular level, including electrospray ionization mass spectrometry (ESI-MS), secondary ion mass spectrometry (SIMS), laser-based mass spectrometry and inductively coupled plasma mass spectrometry (ICP-MS). In this review, the recent advances in single-cell analysis by mass spectrometry are summarized. The strategies of different ionization modes to achieve single-cell analysis are classified and discussed in detail.

Advancing single-cell proteomics and metabolomics with microfluidic technologies.

Recent advances in single-cell analysis have unraveled substantial heterogeneity among seemingly identical cells at genomic and transcriptomic levels. These discoveries have urged scientists to develop new tools that are capable of investigating single cells from a broader set of "omics". Proteomics and metabolomics, for instance, are of particular interest as they are closely correlated with a dynamic picture of cellular behaviors and phenotypic identities. The development of such tools requires highly efficient isolation and processing of a large number of individual cells, where techniques such as microfluidics are extremely useful. Here, we review the recent advances in single-cell proteomics and metabolomics, with a focus on microfluidics-based platforms. We highlight a vast array of emerging microfluidic formats for single-cell isolation and manipulation, and how the state-of-the-art analytical tools are coupled with such platforms for proteomic and metabolomic profiling.

Advances in atomic force microscopy for single-cell analysis

Single-cell analysis has been considered as a promising way to uncover the underlying mechanisms guiding the mysteries of life activities, which considerably complements traditional ensemble assays and yields novel insights into cell biology. The advent of atomic force microscopy (AFM) provides a potent tool for investigating the structures and properties of native biological samples at the micro/nanoscale under near-physiological conditions, which promotes the studies of single-cell behaviors. In the past decades, AFM has achieved great success in single-cell observation and manipulation for biomedical applications, demonstrating the excellent capabilities of AFM in addressing biological issues at the single-cell level with unprecedented spatiotemporal resolution. In this article, we review the recent advances in single-cell analysis that has been made with the utilization of AFM, and provide perspectives for future progression.

Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles.

Recent advances in single-cell analysis have revealed the stochasticity and nongenetic heterogeneity inherent to cellular processes. However, our knowledge of the actual cellular behaviors in a living multicellular organism is still limited. By using a single-cell bioluminescence imaging technique on duckweed, Lemna gibba, we demonstrate that, under constant conditions, cells in the intact plant work as individual circadian clocks that oscillate with their own frequencies and respond independently to external stimuli. Quantitative analysis uncovered the heterogeneity and instability of cellular clocks and partial synchronization between neighboring cells. Furthermore, we found that cellular clocks in the plant body under light-dark cycles showed a centrifugal phase pattern in which the effect of cell-to-cell heterogeneity in period lengths was almost masked. The inherent heterogeneity in the properties of cellular clocks observed under constant conditions is corrected under light-dark cycles to coordinate the daily rhythms of the plant body. These findings provide a novel perspective of spatiotemporal architectures in the plant circadian system.