Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles

Background: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles.Results: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFa signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust.Conclusions: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health

Expression of S561F CDKAL1 variant modifies the constitutive trafficking and affects insulin release in INS1E cells

Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), characterized by hypoglycaemia due to inappropriate insulin secretion. A Whole Exome Sequencing (WES) analysis performed on CHI patients lacking mutations in ABCC8/KCNJ11 identified a polimorfism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL is a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Interestingly, CDKAL is a susceptibility genes for type 2 diabetes and CDKAL knock-out (cdkal1 -/-) mice showed impaired glucose homeostasis, thus indicating the protein involvement in beta-cell function. Aim of this work was to understand the impact of the CDKAL1 variant S561F on the insulin content, trafficking and release in pancreatic beta cells. Materials and methods: Clonal INS1-E cells expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize S561F-CDKAL1 impact on beta cell function. The localization of the variant protein was monitored by immunofluorescence and insulin content and release were measured with ELISA test. An acridine orange assay was performed to evaluate the constitutive and regulated trafficking and possible alterations in vesicle protein expression were evaluated by western blotting. Results: Wild type CDKAL1 overexpressed in INS1-E cells localized in the reticular compartment. The S561F variant was similarly confined to the reticular compartment, although its localization was enriched in spot-like structures distributed in the perinuclear region. Insulin content was increased by overexpression of WT CDKAL1 (2 fold over INS1E, p<0.05) while it was decreased by S561F-CDKAL1 variant expression. Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation in normal glucose concentrations was increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p<0.05), thus suggesting a different insulin processing/secretion in the mutant CDKAL1. An acridina orange assay performed to measure the constitutive and regulated trafficking in INS1E cells revealed more basal exocytosis in S561F-CDKAL1 then WT clones. Interestingly, the basal release was not further increased by potassium chloride or high glucose. Western blotting experiments revealed up-regulation of proteins involved in the secretion machinery in mutant clones compared to WT. Conclusion: The S561F-CDKAL1 variant expression leads to an increased basal insulin release in INS1E cell line. Such an increase is associated to a defect in the vesicle trafficking and correlates with altered expression of proteins involved in the secretory machinery , further studies are needed to clarify molecular mechanisms linking CDKAL to insulin processing and membrane trafficking. Our findings confirm the importance of CDKAL1 in insulin release and suggest a possible mechanism by which this variant can participate to the development of congenital hyperinsulinism

Whole-exome sequencing identification of non-synonymous SNP variants of 10 CHI patients.

*<p>Mutation reported in Human Genome Mutation Database( HGMD).</p><p>PolyPhen/SIFT: (−) benign or tolerated; (+) possibly damaging/DAMAGING Low confidence;</p><p>(++) probably damaging/DAMAGING.</p><p>In bold known causative genes.</p

Functional annotation of novel variants.

<p>Functional annotation of novel variants.</p

Exome coverage and target coverage statistics of ten CHI probands.

<p>Exome coverage and target coverage statistics of ten CHI probands.</p