Cardiac Pericytes Acquire a Fibrogenic Phenotype and Contribute to Vascular Maturation After Myocardial Infarction.

BACKGROUND: Pericytes have been implicated in tissue repair, remodeling, and fibrosis. Although the mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. METHODS: We used NG2Dsred;PDGFRαEGFP pericyte:fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in myocardial infarction. The transcriptomic profile of pericyte-derived cells was studied using polymerase chain reaction arrays and single-cell RNA sequencing. The role of transforming growth factor-ß (TGF-ß) signaling in regulation of pericyte phenotype was investigated in vivo using pericyte-specific TGF-ß receptor 2 knockout mice and in vitro using cultured human placental pericytes. RESULTS: In normal hearts, neuron/glial antigen 2 (NG2) and platelet-derived growth factor receptor α (PDGFRα) identified distinct nonoverlapping populations of pericytes and fibroblasts, respectively. After infarction, a population of cells expressing both pericyte and fibroblast markers emerged. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes exhibited transient expression of fibroblast markers. Pericyte-derived cells accounted for ~4% of PDGFRα+ infarct fibroblasts during the proliferative phase of repair. Pericyte-derived fibroblasts were overactive, expressing higher levels of extracellular matrix genes, integrins, matricellular proteins, and growth factors, when compared with fibroblasts from other cellular sources. Another subset of pericytes contributed to infarct angiogenesis by forming a mural cell coat, stabilizing infarct neovessels. Single-cell RNA sequencing showed that NG2 lineage cells diversify after infarction and exhibit increased expression of matrix genes, and a cluster with high expression of fibroblast identity markers emerges. Trajectory analysis suggested that diversification of infarct pericytes may be driven by proliferating cells. In vitro and in vivo studies identified TGF-ß as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific TGF-ß receptor 2 disruption had no significant effects on infarct myofibroblast infiltration and collagen deposition. Pericyte-specific TGF-ß signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels and protecting from dilative remodeling. CONCLUSIONS: In the healing infarct, cardiac pericytes upregulate expression of fibrosis-associated genes, exhibiting matrix-synthetic and matrix-remodeling profiles. A fraction of infarct pericytes exhibits expression of fibroblast identity markers. Pericyte-specific TGF-ß signaling plays a central role in maturation of the infarct vasculature and protects from adverse dilative remodeling, but it does not modulate fibrotic remodeling.

Polycomb repressive complex 2 in adult hair follicle stem cells is dispensable for hair regeneration.

Hair follicle stem cells (HFSCs) are multipotent cells that cycle through quiescence and activation to continuously fuel the production of hair follicles. Prior genome mapping studies had shown that tri-methylation of histone H3 at lysine 27 (H3K27me3), the chromatin mark mediated by Polycomb Repressive Complex 2 (PRC2), is dynamic between quiescent and activated HFSCs, suggesting that transcriptional changes associated with H3K27me3 might be critical for proper HFSC function. However, functional in vivo studies elucidating the role of PRC2 in adult HFSCs are lacking. In this study, by using in vivo loss-of-function studies we show that, surprisingly, PRC2 plays a non-instructive role in adult HFSCs and loss of PRC2 in HFSCs does not lead to loss of HFSC quiescence or changes in cell identity. Interestingly, RNA-seq and immunofluorescence analyses of PRC2-null quiescent HFSCs revealed upregulation of genes associated with activated state of HFSCs. Altogether, our findings show that transcriptional program under PRC2 regulation is dispensable for maintaining HFSC quiescence and hair regeneration.

Molecular and network disruptions in neurodevelopment uncovered by single cell transcriptomics analysis of CHD8 heterozygous cerebral organoids.

More than 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ∼1000 genes that may contribute. The new challenge is to investigate the molecular and cellular functions of these genes during neural and brain development, and then even more challenging, to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study, we used single-cell RNA-seq analysis to study one of the top risk genes, CHD8, in cerebral organoids, which models early neural development. We identified 21 cell clusters in the organoid samples, representing non-neuronal cells, neural progenitors, and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of a CHD8 knockout allele, generated by CRISPR/Cas9 editing, and their isogenic controls uncovered thousands of differentially expressed genes, which were enriched with functions related to neural and brain development, cilium organization, and extracellular matrix organization. The affected genes were also enriched with genes and pathways previously implicated in ASD, but surprisingly not for schizophrenia and intellectual disability risk genes. The comparisons also uncovered cell composition changes, indicating potentially altered neural differential trajectories upon CHD8 reduction. Moreover, we found that cell-cell communications were affected in the CHD8 knockout organoids, including the interactions between neural and glial cells. Taken together, our results provide new data and information for understanding CHD8 functions in the early stages of neural lineage development and interaction.

Selenocysteine tRNA methylation promotes oxidative stress resistance in melanoma metastasis.

Selenocysteine-containing proteins play a central role in redox homeostasis. Their translation is a highly regulated process and is dependent on two tRNASec isodecoders differing by a single 2'-O-ribose methylation called Um34. Here we characterized FTSJ1 as the Um34 methyltransferase and show that its activity is required for efficient selenocysteine insertion at the UGA stop codon during translation. Specifically, loss of Um34 leads to ribosomal stalling and decreased UGA recoding. FTSJ1-deficient cells are more sensitive to oxidative stress and show decreased metastatic colonization in xenograft models of melanoma metastasis. We found that FTSJ1 mediates efficient translation of selenoproteins essential for the cellular antioxidant response. Our findings uncover a role for tRNASec Um34 modification in oxidative stress resistance and highlight FTSJ1 as a potential therapeutic target specific for metastatic disease.

SMARCA4 controls state plasticity in small cell lung cancer through regulation of neuroendocrine transcription factors and REST splicing.

INTRODUCTION: Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. METHODS: ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. RESULTS: SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. CONCLUSIONS: This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.

COVID-19 immune signatures in Uganda persist in HIV co-infection and diverge by pandemic phase.

Little is known about the pathobiology of SARS-CoV-2 infection in sub-Saharan Africa, where severe COVID-19 fatality rates are among the highest in the world and the immunological landscape is unique. In a prospective cohort study of 306 adults encompassing the entire clinical spectrum of SARS-CoV-2 infection in Uganda, we profile the peripheral blood proteome and transcriptome to characterize the immunopathology of COVID-19 across multiple phases of the pandemic. Beyond the prognostic importance of myeloid cell-driven immune activation and lymphopenia, we show that multifaceted impairment of host protein synthesis and redox imbalance define core biological signatures of severe COVID-19, with central roles for IL-7, IL-15, and lymphotoxin-α in COVID-19 respiratory failure. While prognostic signatures are generally consistent in SARS-CoV-2/HIV-coinfection, type I interferon responses uniquely scale with COVID-19 severity in persons living with HIV. Throughout the pandemic, COVID-19 severity peaked during phases dominated by A.23/A.23.1 and Delta B.1.617.2/AY variants. Independent of clinical severity, Delta phase COVID-19 is distinguished by exaggerated pro-inflammatory myeloid cell and inflammasome activation, NK and CD8+ T cell depletion, and impaired host protein synthesis. Combining these analyses with a contemporary Ugandan cohort of adults hospitalized with influenza and other severe acute respiratory infections, we show that activation of epidermal and platelet-derived growth factor pathways are distinct features of COVID-19, deepening translational understanding of mechanisms potentially underlying SARS-CoV-2-associated pulmonary fibrosis. Collectively, our findings provide biological rationale for use of broad and targeted immunotherapies for severe COVID-19 in sub-Saharan Africa, illustrate the relevance of local viral and host factors to SARS-CoV-2 immunopathology, and highlight underemphasized yet therapeutically exploitable immune pathways driving COVID-19 severity.

Molecular and network disruptions in neurodevelopment uncovered by single cell transcriptomics analysis of CHD8 heterozygous cerebral organoids.

About 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ~1000 genes that may be involved. The new challenge now is to investigate the molecular and cellular functions of these genes during neural and brain development, and then even more challenging, to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study, we use single cell RNA-seq analysis to study one of the top risk gene, CHD8, in cerebral organoids, which models early neural development. We identify 21 cell clusters in the organoid samples, representing non-neuronal cells, neural progenitors, and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of the CHD8 knockout and their isogenic controls uncover thousands of differentially expressed genes, which are enriched with function related to neural and brain development, with genes and pathways previously implicated in ASD, but surprisingly not for Schizophrenia and intellectual disability risk genes. The comparisons also find cell composition changes, indicating potential altered neural differential trajectories upon CHD8 reduction. Moreover, we find that cell-cell communications are affected in the CHD8 knockout organoids, including the interactions between neural and glial cells. Taken together, our results provide new data for understanding CHD8 functions in the early stages of neural lineage development and interaction.

Characterization of cell-cell communication in autistic brains with single-cell transcriptomes.

BACKGROUND: Autism spectrum disorder is a neurodevelopmental disorder, affecting 1-2% of children. Studies have revealed genetic and cellular abnormalities in the brains of affected individuals, leading to both regional and distal cell communication deficits. METHODS: Recent application of single-cell technologies, especially single-cell transcriptomics, has significantly expanded our understanding of brain cell heterogeneity and further demonstrated that multiple cell types and brain layers or regions are perturbed in autism. The underlying high-dimensional single-cell data provides opportunities for multilevel computational analysis that collectively can better deconvolute the molecular and cellular events altered in autism. Here, we apply advanced computation and pattern recognition approaches on single-cell RNA-seq data to infer and compare inter-cell-type signaling communications in autism brains and controls. RESULTS: Our results indicate that at a global level, there are cell-cell communication differences in autism in comparison with controls, largely involving neurons as both signaling senders and receivers, but glia also contribute to the communication disruption. Although the magnitude of changes is moderate, we find that excitatory and inhibitor neurons are involved in multiple intercellular signaling that exhibits increased strengths in autism, such as NRXN and CNTN signaling. Not all genes in the intercellular signaling pathways show differential expression, but genes in the affected pathways are enriched for axon guidance, synapse organization, neuron migration, and other critical cellular functions. Furthermore, those genes are highly connected to and enriched for genes previously associated with autism risks. CONCLUSIONS: Overall, our proof-of-principle computational study using single-cell data uncovers key intercellular signaling pathways that are potentially disrupted in the autism brains, suggesting that more studies examining cross-cell type effects can be valuable for understanding autism pathogenesis.

Detection of significant SNP associated with production and oil quality traits in interspecific oil palm hybrids using RARSeq.

A RARSeq based Association mapping study was performed in a population of 104 Elaeis oleifera x E. guineensis hybrids of five origins with the aim of finding functional markers associated to six productive and 19 oil quality traits. For this purpose mRNA of each genotype was isolated and double stranded cDNA was synthesized. Following digestion with two restriction enzymes and adapter ligation, a size selected pool of barcoded amplicons was produced and sequenced using Illumina MiSeq. The obtained sequences were processed with a "snakemake" pipeline, filtered and missing values were imputed. For all traits except two significant effects of the origin was observed. Genetic diversity analyses revealed high variability within origins and an excess of heterozygosity in the population. Two GLM models with Q matrix or PCA matrix as covariates and two MLM models incorporating in addition a Kinship matrix were tested for genotype-phenotype associations using GAPIT software. Using unadjusted p values (< 0.01) 78 potential associations were detected involving 25 SNP and 20 traits. When applying FDR multiple testing with p < 0.05, 25 significant associations remained involving eight SNP and six quality traits. Four SNP were located in genes with a potential relevant biological meaning.

Association Mapping Between Candidate Gene SNP and Production and Oil Quality Traits in Interspecific Oil Palm Hybrids.

Oil palm production is gaining importance in Central and South America. However, the main species Elaeis guineensis (Eg) is suffering severely from bud rod disease, restricting the potential cultivation areas. Therefore, breeding companies have started to work with interspecific Elaeis oleifera × Eg (Eo × Eg) hybrids which are tolerant to this disease. We performed association studies between candidate gene (CG) single nucleotide polymorphisms (SNP) and six production and 19 oil quality traits in 198 accessions of interspecific oil palm hybrids from five different origins. For this purpose, barcoded amplicons of initially 167 CG were produced from each genotype and sequenced with Ion Torrent. After sequence cleaning 115 SNP remained targeting 62 CG. The influence of the origins on the different traits was analyzed and a genetic diversity study was performed. Two generalized linear models (GLM) with principle component analysis (PCA) or structure (Q) matrixes as covariates and two mixed linear models (MLM) which included in addition a Kinship (K) matrix were applied for association mapping using GAPIT. False discovery rate (FDR) multiple testing corrections were applied in order to avoid Type I errors. However, with FDR adjusted p values no significant associations between SNP and traits were detected. If using unadjusted p values below 0.05, seven of the studied CG showed potential associations with production traits, while 23 CG may influence different quality traits. Under these conditions the current approach and the detected candidate genes could be exploited for selecting genotypes with superior CG alleles in Marker Assisted Selection systems.

A novel strategy for Cr(III) and Cr(VI) analysis in dietary supplements by speciated isotope dilution mass spectrometry.

In recent years, Cr speciation in dietary supplements has become decisive in the evaluation of their health risks. Despite being an beneficial micronutrient, Cr(III) can be toxic at living organisms at high concentrations, while Cr(VI) is known to be highly toxic and carcinogenic. The main objective of this work was to optimize an analytical methodology for the extraction and accurate quantification of Cr(III) and Cr(VI) in dietary supplements. The extraction of Cr species was carried out with 50mM EDTA solution on a hotplate under optimized conditions. Special attention was paid to bidirectional species transformations. No noticeable oxidation of Cr(III) into Cr(VI) was observed and the reduction to Cr(III) only occurred at very high Cr(VI) concentrations. Cr(III) as Cr(EDTA)(-) complex was chromatographically separated from Cr(VI), retained as CrO4(2-), on an anion exchange column coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). The limit of quantification (0.08µgg(-1)) was below the limit established for Cr enriched yeasts by the European Union. Eleven dietary supplements were analyzed and Cr(III) and Cr(VI) quantification was carried out by external calibration monitoring (52)Cr isotope and by speciated isotope dilution mass spectrometry (SIDMS) adding (50)Cr(III) and (53)Cr(VI) spikes. Total Cr was also quantified by ICP-MS and mass balance between total Cr and the sum of Cr(III) and Cr(VI) was achieved. In eight of the eleven tested supplements Cr(III) calculated amounts were higher than those indicated by the manufacturer, but only one of them exceeded the 250µgday(-1) recommended by World Health Organization (WHO). In contrast, it is worth noting that Cr(VI) amounts beyond the recommendations of the European Union for Cr enriched yeasts were found in five supplements. These results revealed that more accurate and rigorous quality assurance protocols should be applied to the testing of the final products, including the analysis of both Cr(III) and Cr(VI).