Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology. This approach utilized the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings identified three profiles of reactive astrocyte responses, categorized by their effects on cell size- and shape- related morphological parameters: \"moderate\", \"strong,\" and \"very strong\". We also examined the heterogeneity of astrocyte reactivity, focusing on spatial and clonal distribution. Our research revealed a notable enrichment of protoplasmic and fibrous astrocytes within the \"strong\" and \"very strong\" response subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By characterizing the diverse reactive responses among astrocyte subpopulations, we provide insights that could guide future research aimed at identifying novel therapeutic targets to mitigate brain damage and promote neural repair.

Inferring gene regulatory networks from single-cell RNA-sequencing trajectories has been an active area of research yet methods are still needed to identify regulators governing cell transitions. We developed DREAMIT (Dynamic Regulation of Expression Across Modules in Inferred Trajectories) to annotate transcription-factor activity along single-cell trajectory branches, using ensembles of relations to target genes. Using a benchmark representing several different tissues, as well as external validation with ATAC-Seq and Perturb-Seq data on hematopoietic cells, the method was found to have higher tissue-specific sensitivity and specificity over competing approaches.

Alzheimer\'s disease (AD) and Parkinson\'s disease (PD) cause significant neuronal loss and severely impair daily living. Despite different clinical manifestations, these disorders share common pathological molecular hallmarks, including mitochondrial dysfunction and synaptic degeneration. A detailed comparison of molecular changes at single-cell resolution in the cortex, as one of the main brain regions affected in both disorders, may reveal common susceptibility factors and disease mechanisms. We performed single-cell transcriptomic analyses of post-mortem cortical tissue from AD and PD subjects and controls to identify common and distinct disease-associated changes in individual genes, cellular pathways, molecular networks, and cell-cell communication events, and to investigate common mechanisms. The results revealed significant disease-specific, shared, and opposing gene expression changes, including cell type-specific signatures for both diseases. Hypoxia signaling and lipid metabolism emerged as significantly modulated cellular processes in both AD and PD, with contrasting expression alterations between the two diseases. Furthermore, both pathway and cell-cell communication analyses highlighted shared significant alterations involving the JAK-STAT signaling pathway, which has been implicated in the inflammatory response in several neurodegenerative disorders. Overall, the analyses revealed common and distinct alterations in gene signatures, pathway activities, and gene regulatory subnetworks in AD and PD. The results provide insights into coordinated changes in pathway activity and cell-cell communication that may guide future diagnostics and therapeutics.

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Central nervous system (CNS) tumors have complex causes and poor prognosis. Tumor heterogeneity is a major cause of treatment failure, and in-depth understanding of the biodiversity of CNS tumors is critical. Single-cell sequencing technology provides an opportunity to reveal the complex ecosystem of CNS tumors. In this study, we review the significance of single-cell sequencing in exploring the heterogeneity, diagnosis, treatment, and prognosis of CNS tumors from three aspects: tumor stem cells, tumor microenvironment, and cerebrospinal fluid. Although most of the findings have not been clinically applicable, they lay the foundation for the development of new guidelines for CNS tumors that help improve tumor prognosis and prevent tumor recurrence.
中枢神经系统（CNS）肿瘤起因复杂，预后极差，肿瘤异质性是治疗失败和复发的主要原因，深入了解CNS肿瘤的生物多样性至关重要。单细胞测序技术为揭示CNS肿瘤复杂的生态系统提供了契机。本研究从肿瘤干细胞、肿瘤微环境以及脑脊液三个方面阐述了单细胞测序对CNS肿瘤异质性的探索，以及临床诊断、治疗及预后的意义。尽管大多数研究成果尚未在临床上应用，但为CNS肿瘤新临床指南的制定打下基础，以改善患者预后，预防肿瘤复发。.

Spiders produce nature\'s toughest fiber using renewable components at ambient temperatures and with water as solvent, making it highly interesting to replicate for the materials industry. Despite this, much remains to be understood about the bioprocessing and composition of spider silk fibers. Here, we identify 18 proteins that make up the spiders\' strongest silk type, the major ampullate fiber. Single-cell RNA sequencing and spatial transcriptomics revealed that the secretory epithelium of the gland harbors six cell types. These cell types are confined to three distinct glandular zones that produce specific combinations of silk proteins. Image analysis of histological sections showed that the secretions from the three zones do not mix, and proteomics analysis revealed that these secretions form layers in the final fiber. Using a multi-omics approach, we provide substantial advancements in the understanding of the structure and function of the major ampullate silk gland as well as of the architecture and composition of the fiber it produces.

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

Histone post-translational modifications (PTMs) influence the overall structure of the chromatin and gene expression. Over the course of cell differentiation, the distribution of histone modifications is remodeled, resulting in cell type-specific patterns. In the past, their study was limited to abundant cell types that could be purified in necessary numbers. However, studying these cell type-specific dynamic changes in heterogeneous in vivo settings requires sensitive single-cell methods. Current advances in single-cell sequencing methods remove these limitations, allowing the study of nonpurifiable cell types. One complicating factor is that some of the most biologically interesting cell types, including stem and progenitor cells that undergo differentiation, only make up a small fraction of cells in a tissue. This makes whole-tissue analysis rather inefficient. In this chapter, we present a sort-assisted single-cell Chromatin ImmunoCleavage sequencing technique (sortChIC) to map histone PTMs in single cells. This technique combines the mapping of histone PTM location in combination with surface staining-based enrichment, to allow the integration of established strategies for rare cell type enrichment. In general terms, this will enable researchers to quantify local and global chromatin changes in dynamic complex biological systems and can provide additional information on their contribution to lineage and cell-type specification in physiological conditions and disease.

Protein disulfide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER) protein. It has different functions including glycoprotein folding in the ER. The unfavorable prognosis of cancer patients was related to the abnormal PDIA3 expression level. However, it is unclear how PDIA3 correlates with the malignant characteristics of different tumors and its impact on tumor immunity. Pan-cancer data were downloaded from several databases for large-scale bioinformatics analysis. The immunological functions of PDIA3 were systematically explored at the single-cell sequencing level, including cell communication, cell metabolism, cell evolution and epigenetic modification. We performed immunofluorescence staining to visualize PDIA3 expression and infiltration of macrophages in pan-cancer samples. Further, we performed a loss-of-function assay of PDIA3 in vitro. The CCK8 assay, clone formation assay, and transwell assay were performed. M2 macrophages were co-cultured with different cell lines before the transwell assay was performed. The immunofluorescence staining of pan-cancer samples presented a higher expression of PDIA3 than those of the paired normal tissues. According to single-cell sequencing analysis, expression of PDIA3 was closely associated with cell communication, cell metabolism, cell evolution and epigenetic modification. The knockdown of PDIA3 in tumor cells inhibited cell proliferation and invasion, and restrained cocultured M2 macrophage migration. Furthermore, PDIA3 displayed predictive value in immunotherapy response in human cancer cohorts, indicating a potential therapeutic target. Our study showed that PDIA3 was associated with tumor malignant characteristics and could mediate the migration of M2 macrophages in various tumor types. PDIA3 could be a promising target to achieve tumor control and improve the immune response on a pan-cancer scale.