CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Resident memory T cells: Possible players in periodontal disease recurrence.

BACKGROUND/OBJECTIVES: Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in barrier tissues after previous bacterial or viral infection to support early/immediate defense mechanisms, providing site-specific protection from pathogen challenge. As data on Trm cells in human gingiva are just emerging, the aim of the present study was to explore their presence and distribution in epithelial and connective periodontal tissues in relation to microbial exposure and periodontal damage. MATERIAL AND METHODS: Periodontitis tissue specimens were collected from 20 generalized chronic periodontitis patients at the time of osseous resective surgery. As a control, 18 healthy tissue specimens were harvested each from both the primary flap and the palatal graft in 18 periodontally healthy patients during mucogingival surgeries. As CD69 and CD103 are phenotypic markers associated with tissue residence, intraepithelial and stromal CD103+ and CD69+ cells per high-power field were counted in areas with highest expression. Double immunohistochemistry for CD3 and CD69 was performed to identify T cells. RESULTS: CD69 +and CD103+ cells showed a lymphocytic morphology, and double CD69 and CD3 staining confirmed the T cell phenotype of these cells. CD103 and CD69 expression was significantly enhanced in epithelial and connective tissues from patients with periodontitis compared with healthy controls (P < .001). Significant positive correlation between PD and both CD103 and CD69 epithelial expression was observed in tissue specimens from periodontitis patients (P < .001). CONCLUSION: Within the limits of the present study, these results indicate that Trm cells are higher in periodontitis lesions. They could orchestrate the host response to microbial challenge, leading to a faster reactivation of periodontal disease.

Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

MaREA4Galaxy: Metabolic reaction enrichment analysis and visualization of RNA-seq data within Galaxy.

We present MaREA4Galaxy, a user-friendly tool that allows a user to characterize and to graphically compare groups of samples with different transcriptional regulation of metabolism, as estimated from cross-sectional RNA-seq data. The tool is available as plug-in for the widely-used Galaxy platform for comparative genomics and bioinformatics analyses. MaREA4Galaxy combines three modules. The Expression2RAS module, which, for each reaction of a specified set, computes a Reaction Activity Score (RAS) as a function of the expression level of genes encoding for the associated enzyme. The MaREA (Metabolic Reaction Enrichment Analysis) module that allows to highlight significant differences in reaction activities between specified groups of samples. The Clustering module which employs the RAS computed before as a metric for unsupervised clustering of samples into distinct metabolic subgroups; the Clustering tool provides different clustering techniques and implements standard methods to evaluate the goodness of the results.