Project description:Because of severe abiotic limitations, Antarctic soils represent simplified ecosystems, where microorganisms are the principle drivers of nutrient cycling. This relative simplicity makes these ecosystems particularly vulnerable to perturbations, like global warming, and the Antarctic Peninsula is among the most rapidly warming regions on the planet. However, the consequences of the ongoing warming of Antarctica on microorganisms and the processes they mediate are unknown. Here, using 16S rRNA gene pyrosequencing and qPCR, we report a number of highly consistent changes in microbial community structure and abundance across very disparate sub-Antarctic and Antarctic environments following three years of experimental field warming (+ 0.5-2°C). Specifically, we found significant increases in the abundance of fungi and bacteria and in the Alphaproteobacteria-to-Acidobacteria ratio. These alterations were linked to a significant increase in soil respiration. Furthermore, the shifts toward generalist or opportunistic bacterial communities following warming weakened the linkage between bacterial diversity and functional diversity. Warming also increased the abundance of some organisms related to the N-cycle, detected as an increase in the relative abundance of nitrogenase genes via GeoChip microarray analyses. Our results demonstrate that soil microorganisms across a range of sub-Antarctic and Antarctic environments can respond consistently and rapidly to increasing temperatures, thereby potentially disrupting soil functioning. We conducted in situ warming experiments for three years using open-top chambers (OTCs) at one sub-Antarctic (Falkland Islands, 52ºS) and two Antarctic locations (Signy and Anchorage Islands, 60ºS and 67ºS respectively) (see Supplementary Fig. 1 for a map). OTCs increased annual soil temperature by an average of 0.8°C (at a depth of 5 cm), resulting in 8-43% increase in positive-degree days annually and a decrease in freeze-thaw cycle frequency by an average of 15 cycles per year (8). At each location, we included densely vegetated and bare fell-field soils in the experimental design for a total of six environments. Densely vegetated and bare environments represent two contrasting environments for Antarctic soil microorganisms, with large differences in terms of C and N inputs to soils. Massively parallel pyrosequencing (Roche 454 GS FLX Titanium) of 16S rRNA gene amplicons was used to follow bacterial diversity and community composition [GenBank Accession Numbers: HM641909-HM744649], and functional gene microarrays (GeoChip 2.0)(11) were used to assess changes in functional gene distribution. Bacterial and fungal communities were also quantified using real-time PCR.
Project description:Coordinated migration of B cells within and between secondary lymphoid tissues is required for robust antibody responses to infection or vaccination. Secondary lymphoid tissues normally expose B cells to a low O2 (hypoxic) environment. Recently, we have shown that human B cell migration is modulated by an O2-dependent molecular switch, centrally controlled by the hypoxia-induced (transcription) factor-1a (HIF1A), which can be disrupted by the immunosuppressive calcineurin inhibitor, cyclosporine A (CyA). However, the mechanisms by which low O2 environments attenuate B cell migration remain poorly defined. Proteomics analysis has linked CXCR4 chemokine receptor signaling to cytoskeletal rearrangement. We now hypothesize that the pathways linking the O2 sensing molecular switch to chemokine receptor signaling and cytoskeletal rearrangement would likely contain phosphorylation events, which are typically missed in traditional transcriptomic and/or proteomic analyses. Hence, we have performed a comprehensive phosphoproteomics analysis of human B cells treated with CyA after engagement of the chemokine receptor CXCR4 with CXCL12. Statistical analysis of the separate and synergistic effects of CyA and CXCL12 revealed 116 proteins whose abundance is driven by a synergistic interaction between CyA and CXCL12. Among these 116 proteins, Lymphocyte Specific Protein 1 (LSP1) was most interesting due to its original identification as a protein modulating neutrophil migration. We used our previously described algorithm BONITA to reveal a critical role for LSP1 in cytoskeletal rearrangement. Validating these modeling results, we show experimentally that LSP1 levels in B cells increase with low O2 exposure, and CyA treatment results in decreased LSP1 protein levels. This correlates with the increased chemotactic activity observed after CyA treatment. Lastly, we directly link LSP1 levels to chemotactic capacity, as shRNA knock-down of LSP1 results in significantly increased B cell chemotaxis at low O2 levels. These results directly link the CyA to LSP1-dependent cytoskeletal regulation, demonstrating a previously unrecognized mechanism by which CyA modulates human B cell migration.
Project description:[original title] Gene expression response to the implantation of drug (paclitaxel)-eluting or bare metal stents in denuded human LIMA arteries. Different clinical outcomes have been observed for paclitaxel-eluting and bare metal cardiovascular stents. The aim of this project was to identify genes that might be associated with the observed clinical outcomes.
Project description:[original title] Gene expression response to the implantation of drug (paclitaxel)-eluting or bare metal stents in denuded human LIMA arteries. Different clinical outcomes have been observed for paclitaxel-eluting and bare metal cardiovascular stents. The aim of this project was to identify genes that might be associated with the observed clinical outcomes. Human left internal mammary artery (LIMA) was divided into three segments and and two of the segments were fitted with either a paclitaxel-eluting stent or a bare metal stent. The experiment includes three groups: control, paclitaxel-eluting stent, and bare metal stent, respectively. Each group includes four biological replicates (patients 1, 2, 4 and 5).
Project description:Mycophenolic acid (MPA), an immunosuppressive drug widely used in kidney transplantation, has been suggested to have anti-fibrotic effects. To analyze at a genomic level these effects, we prospectively studied a group of stable kidney transplant recipients (n=35) on cyclosporine (CyA) and azathioprine treatment. Twenty patients were converted from azathioprine to MPA (MPA group) and 15 patients continued on azathioprine (AZA group). RNA was extracted by peripheral blood mononuclear cells at baseline and 3 months thereafter. Genomic analysis, performed on 5 randomly-selected MPA patients, revealed that 17 genes discriminated the transcriptomic profile after conversion. Neutral endopeptidase (NEP), an enzyme degrading angiotensin-II, was the most significant up-regulated gene. NEP expression level was inversely correlated to proteinuria at baseline and after conversion. Immunohistochemistry on graft biopsy of 33 independent patients demonstrated higher glomerular and tubular NEP protein expression in CyA+MPA (n=13) compared to CyA+AZA (n=12) and CyA alone (n=8). Glomerular NEP levels were inversely correlated to proteinuria and glomerulosclerosis. Tubular NEP expression was inversely correlated to interstitial fibrosis. Incubation of proximal tubular cells with MPA led to a dose- and time-dependent increase of NEP gene expression. The direct influence of MPA on NEP expression may suggest a novel therapeutic effect of this drug.
Project description:The faecal indicator bacterium Escherichia coli K12 was used to study the effects of the global regulators RpoS and cAMP at the transcription level using microarray technology during short-term (physiological) adaptation to slow growth under limited nutrient supply. Effects due to the absence of one global regulator were assessed by comparing the mRNA levels isolated from rpoS or cya mutants under glucose-limited continuous culture at a dilution rate of 0.3 h-1 for the rpoS mutant or 0.16 h-1 for the cya mutant with those from wt E. coli grown under the same conditions.
Project description:The spread of antibiotic resistance genes (ARG) into agricultural soils, products, and foods severely limits the use of organic fertilizers in agriculture. In this study, experimental land plots were fertilized, sown, and harvested for two consecutive agricultural cycles using either mineral or three types of organic fertilizers: sewage sludge, pig slurry, or composted organic fraction of municipal solid waste. The analysis of the relative abundances of more than 200,000 ASV (Amplicon Sequence Variants) allowed the identification of a small, but significant (<10%) overlap between soil and fertilizer microbiomes, particularly in soils sampled the same day of the harvest (post-harvest soils). Loads of clinically relevant ARG were significantly higher (up to 100 fold) in fertilized soils relative to the initial soil. The highest increases corresponded to post-harvest soils treated with organic fertilizers, and they correlated with the extend of the contribution of fertilizers to the soil microbiome. Edible products (lettuce and radish) showed low, but measurable loads of ARG (sul1 for lettuces and radish, tetM for lettuces). These loads were minimal in mineral fertilized soils, and strongly dependent on the type of fertilizer. We concluded that at least part of the observed increase on ARG loads in soils and foodstuffs were actual contributions from the fertilizer microbiomes. Thus, we propose that adequate waste management and good pharmacological and veterinarian practices may significantly reduce the potential health risk posed by the presence of ARG in agricultural soils and plant products.