Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with poorly understood clinical heterogeneity, underscored by significant differences in patient age at onset, symptom progression, therapeutic response, disease duration, and comorbidity presentation. We perform a patient stratification analysis to better understand the variability in ALS pathology, utilizing postmortem frontal and motor cortex transcriptomes derived from 208 patients. Building on the emerging role of transposable element (TE) expression in ALS, we consider locus-specific TEs as distinct molecular features during stratification. Here, we identify three unique molecular subtypes in this ALS cohort, with significant differences in patient survival. These results suggest independent disease mechanisms drive some of the clinical heterogeneity in ALS.

Abstract. This paper defines the best practices for documenting ocean acidification (OA) data and presents a framework for an OA metadata template. Metadata is structured information that describes and locates an information resource. It is the key to ensuring that a data set will be accessible into the future. With the rapid expansion of studies on biological responses to OA, the lack of a common metadata template to document the resulting data poses a significant hindrance to effective OA data management efforts. In this paper, we present a metadata template that can be applied to a broad spectrum of OA studies, including those studying the biological responses to OA. The "variable metadata section", which includes the variable name, observation type, whether the variable is a manipulation condition or response variable, and the biological subject on which the variable is studied, forms the core of this metadata template. Additional metadata elements, such as investigators, temporal and spatial coverage, and data citation, are essential components to complete the template. We explain the structure of the template, and define many metadata elements that may be unfamiliar to researchers. Template availability-Available at

The perivascular niche (PVN) is a glioblastoma tumor microenvironment (TME) that serves as a safe haven for glioma stem cells (GSCs), and acts as a reservoir that inevitably leads to tumor recurrence. Understanding cellular interactions in the PVN that drive GSC treatment resistance and stemness is crucial to develop lasting therapies for glioblastoma. The limitations of in vivo models and in vitro assays have led to critical knowledge gaps regarding the influence of various cell types in the PVN on GSCs behavior. This study developed an organotypic triculture microfluidic model as a means to recapitulate the PVN and study its impact on GSCs. This triculture platform, comprised of endothelial cells (ECs), astrocytes, and GSCs, is used to investigate GSC invasion, proliferation and stemness. Both ECs and astrocytes significantly increased invasiveness of GSCs. This study futher identified 15 ligand‐receptor pairs using single‐cell RNAseq with putative chemotactic mechanisms of GSCs, where the receptor is up‐regulated in GSCs and the diffusible ligand is expressed in either astrocytes or ECs. Notably, the ligand–receptor pair SAA1‐FPR1 is demonstrated to be involved in chemotactic invasion of GSCs toward PVN. The novel triculture platform presented herein can be used for therapeutic development and discovery of molecular mechanisms driving GSC biology.

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Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood.Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence.Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2 h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury.Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Protein adhesins are an important type of surface structure, and are components of some Gramnegative bacterial outer membranes, such as Pseudomonas fluorescens. The LapA adhesin is a major surface protein on this bacterium, which is required for irreversible adhesion and the initiation of biofilm formation. Atomic force microscopy (AFM) was used to characterize surface structures of P. fluorescens Pf0-1. The wild-type and three genetically modified strains were studied, namely a strain consisting of a single cross-over knockout mutation disrupting the lapA gene, a strain consisting of a single cross-over knockout mutation disrupting the lapB gene, (LapA is maintained in the cytoplasm and not transported to the cell surface), and a lapG deletion mutant (LapA is unable to be cleaved from the cell surface). AFM data was modeled using the Alexander-de Gennes (A-dG) relation for steric repulsion. We calculated the equilibrium layer thickness of the surface structures as well as spacing between adhesins. The wild-type strain and the lapA and lapB mutants all showed similar spacing for surface proteins. The strain lacking LapG had the smallest spacing between molecules. This suggests that the absence of the LapG protease allowed the LapA protein to accumulate, thus decreasing the overall molecular spacing of the protein on the bacterial surface compared to the wild-type strain. We found that the lapG mutant strain of P. fluorescens behaved like a classical polymer brush, in which the spacing between molecules was very small (3.3 nm), which would allow intermolecular interactions between protein units. Recent work has shown that the lapG mutant has greater adhesion and biofilm formation than the wild-type, lapA, and lapB strains, and exhibits stiffer conformation of LapA due to higher protein density and aggregation. Taken together, our results and these recent studies support the finding that LapA adhesin conformation is related to irreversible bacterial adhesion.

Research in understanding biofilm formation is dependent on accurate and representative measurements of the steric forces related to brush on bacterial surfaces. A MATLAB program to analyze force curves from an AFM efficiently, accurately, and with minimal user bias has been developed. The analysis is based on a modified version of the Alexander and de Gennes (AdG) polymer model, which is a function of equilibrium polymer brush length, probe radius, temperature, separation distance, and a density variable. Automating the analysis reduces the amount of time required to process 100 force curves from several days to less than 2min. The use of this program to crop and fit force curves to the AdG model will allow researchers to ensure proper processing of large amounts of experimental data and reduce the time required for analysis and comparison of data, thereby enabling higher quality results in a shorter period of time.