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.

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.

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).