Project description:Atmospheric particulate matter (PM) is a recognized risk factor for the global burden of disease in human populations. We are presenting here the application of toxicogenomics in the evaluation of the toxic effects of organic content of atmospheric particle matter (PM), from urban and rural environments (city of Barcelona and village of La Pobla, NE Spain), using the developing zebrafish embryo. The main goal is to identify the metabolic pathways involved in the adverse effects observed in zebrafish embryos exposed to PM organic content from urban and rural environments, also allowing the selection of genes of interest that are differentially expressed. The relevance of particle size to the PM toxicity is also addressed. Indeed, the zebrafish embryos were exposed to PM of aerodynamic diameter larger than 7.2 μm and smaller than 0.5 μm (PM10 and PM0.5, respectively). PM0.5 concentrated biological and toxic activities linked to organic substances. Transcriptomic analyses showed strong induction of the AhR signalling pathway (a.k.a. dioxin-like activity) for embryos exposed to both rural and urban extracts, correlating with the concentrations of PAHs. Urban extracts, with strong contribution of traffic emissions, specifically de-regulated oxidative stress-related genes, as well as pancreatic and eye-lens specific genes, two organs known to be affected by air pollution in humans. Exposure to rural extracts, with high contribution of wood burning emissions, affected genes implicated in basic cellular functions, in agreement with their strong embryotoxicity. Extracts from rural and urban samples elicited both common and specific transcriptome responses, suggesting different potentially adverse outcomes depending on PM source and composition. The authors thank the financial support of the Spanish Ministry (project TEA-PARTICLE, grant number CGL2011-29621) and the Portuguese Foundation for Science and Technology for the doctoral grant of Sofia R. Mesquita (SFRH/BD/80710/2011) funded by the Program POPH-QREN through the Portuguese Ministry of Education and Science and the European Social Fund, and support through project PEst-C/MAR/LA0015/2013.

Project description:Atmospheric particulate matter (PM) is a recognized risk factor for the global burden of disease in human populations. We are presenting here the application of toxicogenomics in the evaluation of the toxic effects of organic content of atmospheric particle matter (PM), from urban and rural environments (city of Barcelona and village of La Pobla, NE Spain), using the developing zebrafish embryo. The main goal is to identify the metabolic pathways involved in the adverse effects observed in zebrafish embryos exposed to PM organic content from urban and rural environments, also allowing the selection of genes of interest that are differentially expressed. The relevance of particle size to the PM toxicity is also addressed. Indeed, the zebrafish embryos were exposed to PM of aerodynamic diameter larger than 7.2 Î¼m and smaller than 0.5 Î¼m (PM10 and PM0.5, respectively). PM0.5 concentrated biological and toxic activities linked to organic substances. Transcriptomic analyses showed strong induction of the AhR signalling pathway (a.k.a. dioxin-like activity) for embryos exposed to both rural and urban extracts, correlating with the concentrations of PAHs. Urban extracts, with strong contribution of traffic emissions, specifically de-regulated oxidative stress-related genes, as well as pancreatic and eye-lens specific genes, two organs known to be affected by air pollution in humans. Exposure to rural extracts, with high contribution of wood burning emissions, affected genes implicated in basic cellular functions, in agreement with their strong embryotoxicity. Extracts from rural and urban samples elicited both common and specific transcriptome responses, suggesting different potentially adverse outcomes depending on PM source and composition. The authors thank the financial support of the Spanish Ministry (project TEA-PARTICLE, grant number CGL2011-29621) and the Portuguese Foundation for Science and Technology for the doctoral grant of Sofia R. Mesquita (SFRH/BD/80710/2011) funded by the Program POPH-QREN through the Portuguese Ministry of Education and Science and the European Social Fund, and support through project PEst-C/MAR/LA0015/2013. The PM filter samples used in the present study were PM10 and PM0.5 from the urban site - total of 4 samples from 2 sampling months; and PM0.5 from the rural site - total of 2 samples from 2 sampling months (PM10 samples from the rural site were not tested due to their very low concentration in organic compounds, and insignificant biological activity, previously measured). Sampling occurred during the cold periods of the year 2012/2013. The organic fraction of PM samples was extracted by sonication using a mixture of dichloromethane:methanol. Then, the extracts were filtered, evaporated and re-dissolved in Dimethyl sulfoxide (DMSO). Zebrafish fertilized eggs were exposed to PM organic extracts (0.2% DMSO) from the urban and rural sites from 24 to 120h post-fertilization. Total RNA was isolated from whole embryos (pools of 20 individuals), using Trizol reagent protocol (Invitrogen Life Technologies, Carlsberg, CA). The microarray study was performed using the Agilent Two-colour D. rerio Oligo Microarray v3 platform, following the Agilent Microarray â?? Based Gene Expression Analysis protocol. Three biological replicates were performed for each PM sample and controls. Biological replicates, a technical replicate and a self-to-self, were simultaneously amplified and labelled using Cyanine 3 (Cy3) and Cyanine 5 (Cy5) dye. After cRNA purification (RNeasy Kit, Quiagen GmbH, Hilden, Germany), the cRNA concentration and labelling were quantified in the NanoDrop spectrophotometer, obtaining incorporation rates in the range of 15-20pmol of cyanide dye/Âµl cRNA and yield values > 0.825ug, as recommended. cRNA was fragmented and hybridized in 4x44K arrays, for 17h at 65oC. After the hybridization the array slides were washed, and immediately scanned using Microarray Scanner Agilent G2505C system. Data was extracted using Agilent Feature Extraction Software v10.5.1.1, and the quality of microarray data was evaluated using the Quality Control report provided by Agilent Software. Based on the microarray data analysis, 22 primers were selected and designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Genes were quantified by real-time PCR to validate the microarray data, and a subset of 13 genes were further selected by their robust behaviour in the validation assays, allowing to differentiate the toxic potential of PM from urban and rural sources.

Project description:The fungi in the gastrointestinal tract, the gut mycobiota, are now recognised as a significant part of the gut microbiota, and they may be important to human health. In contrast to the adult gut mycobiota, the establishment of the early gut mycobiota has never been described, and there is little knowledge about the fungal transfer from mother to offspring.In a prospective cohort, we followed 298 pairs of healthy mothers and offspring from 36 weeks of gestation until 2 years of age (1516 samples) and explored the gut mycobiota in maternal and offspring samples. Half of the pregnant mothers were randomised into drinking probiotic milk during and after pregnancy. The probiotic bacteria included Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis Bb-12 and Lactobacillus acidophilus La-5. We quantified the fungal abundance of all the samples using qPCR of the fungal internal transcribed spacer (ITS)1 segment, and we sequenced the 18S rRNA gene ITS1 region of 90 high-quantity samples using the MiSeq platform (Illumina).The gut mycobiota was detected in most of the mothers and the majority of the offspring. The offspring showed increased odds of having detectable faecal fungal DNA if the mother had detectable fungal DNA as well (OR = 1.54, p = 0.04). The fungal alpha diversity in the offspring gut increased from its lowest at 10 days after birth, which was the earliest sampling point. The fungal diversity and fungal species showed a succession towards the maternal mycobiota as the child aged, with Debaryomyces hansenii being the most abundant species during breast-feeding and Saccharomyces cerevisiae as the most abundant after weaning. Probiotic consumption increased the gut mycobiota abundance in pregnant mothers (p = 0.01).This study provides the first insight into the early fungal establishment and the succession of fungal species in the gut mycobiota. The results support the idea that the fungal host phenotype is transferred from mother to offspring.Clinicaltrials.gov NCT00159523.

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The gut mycobiota of rural and urban individuals is shaped by geography.

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The gut mycobiota of rural and urban individuals is shaped by geography.

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