Project description:Anaerobic digestion (AD) is a core technology in management of urban organic wastes, converting a fraction of the organic carbon to methane and the residual digestate, the biorest, have a great potential to become a major organic fertilizer for agricultural soils in the future. At the same time, mitigation of N2O-emissions from the agricultural soils is needed to reduce the climate forcing by food production. Our goal was therefore to enrich for N2O reducing bacteria in AD digestates prior to fertilization, and in this way provide an avenue for large-scale and low-cost cultivation of strongly N2O reducing bacteria which can be directly introduced to agricultural soils in large enough volumes to alter the fate of nitrogen in the soils. Gas kinetics and meta-omics (metagenomics and metaproteomics) analyses of the N2O enriched digestates identified populations of N2O respiring organisms that grew by harvesting fermentation intermediates of the methanogenic consortium.
Project description:The crucial role of nutrition for cerebral health and the impact of dietary habits on brain structure and function have been long far recognized. To date a major health concern is associated with the increased consumption of fructose as added sugar in many types of drinks and processed foods, especially among young people. High-fructose intake has been pointed out as the possible culprit for the raised incidence of chronic diseases, such as obesity, cardiovascular disease, nonalcoholic fatty liver disease, and type 2 diabete. Further, it has been reported that high-fructose intake is associated with the over-activation of its cerebral metabolism, which was proposed to negatively impact on whole brain physiology and cognitive function. Notably, we previously reported that short-term fructose-rich diet induces mitochondrial dysfunction, oxidative stress, and neuroinflammation in hippocampus of young rats, as well as the imbalance of redox homeostasis, autophagic mechanisms and representation of synaptic markers in frontal cortex of both adult and young rats. Animal studies have also revealed the damaging effect of high-fructose diets on hippocampal functions during periods of neurocognitive development, such as childhood and adolescence. Hypothalamus plays a crucial role in maintaining whole body homeostasis. Long-term fructose overfeeding was reported to alter hypothalamic-pituitary-adrenal axis, leading to elevations in glucocorticoids in peri-adolescent rats [22]. Further, fructose overconsumption was associated with impairment of hypothalamic insulin signalling, oxidative stress and inflammation , and it was proposed that fructose-driven perturbations of hypothalamic function may compromise the potential for satiety, thereby increasing the prospect of developing obesity. Data currently available on hypothalamic dysfunctions related to a high-fructose diet essentially refer to the effects of long-term sugar feeding, while information on corresponding alterations associated with a short-term dietary treatment, particularly in the critical period of adolescence, is still lacking. Due to complexity and multiplicity of hypothalamic functions, there is also the need for a holistic characterization aimed at unveiling the general picture of hypothalamic dysfunctions associated with a high-fructose diet. To fill this gap, we investigated adolescent rats fed a fructose-rich or control diet, for 3 weeks. To verify whether the fructose-driven changes are rescued after the switch to a control diet, half of the rats from both animal groups were then fed a control diet for additional 3 weeks until young adulthood phase. Quantitative proteomics on hypothalamic extracts of all animal groups was used to identify molecular alterations triggered by fructose-rich diet and to obtain insights into the relationship between sugar feeding and possible dysfunctions of hypothalamus.
Project description:Exosomes are small RNA and protein containing vesicles that can mediate hetero- and homotypic intercellular communication between normal and malignant cells. Especially, tumor-derived exosomes are believed to mediate reprogramming of the tumor-associated stroma to favor tumor growth and metastasis. In this study we isolated exosomes from three different Ewing’s sarcoma (ES) cell lines by ultracentrifugation. Microarray analysis of ES-derived exosomes and their parental cells was performed to gain insight into the spectrum of transcripts they contain and the functions in which these transcripts might be involved in. In total we analyzed six different samples consisting of three pairs of exosomal and cellular RNA of different Ewing's sarcoma cell lines.
Project description:Human genetics as well as pharmacological intervention reveal that Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by stalling the ribosome near codon 34 of its messenger RNA. Inhibition by PF-06446846 is sensitive to the amino acid sequence of the PCSK9 nascent chain, and not the messenger RNA. PF-06446846 also reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling to examine the proteome-wide effects of PF-06446846, we find that it is exceptionally specific for PCSK9 and has no measurable effect on 99.7% of the translatome at concentrations sufficient for 90% inhibition of PCSK9 expression. Together, PF-06446846 represents the first example of an orally administered small molecule directly targeting PCSK9 that functions by a mechanism inhibiting translation during elongation with a high degree of selectivity. Selective inhibition of translation in human may represent a new approach to target proteins with small molecules.
Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.
Project description:The rat pheochromocytoma cell line PC12 cells were cultured in complete DMEM till 80% confluence, then placed at 5000 cells per squared cm. Cells were then plated in 24-well plates for cell viability assay and in T75 flasks for RNA isolation. Medium was replaced with serum-free fresh medium for 12 hours prior to TMT treatment. Gene expression patterns were then analysed using Rat Expression Array 230A Experiment Overall Design: In this study we analize gene expression patterns in PC12 cells treated with Trimethyltin (TMT). We utilized control cells (untreated) and two different concentration (1 and 5) Experiment Overall Design: We used three biological replicates, for the three concentration tested, according to MIAME guidelines Experiment Overall Design: (total 9 chips were used in this study).
Project description:The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bovine tuberculosis in cattle. We have used the advantages of the murine model to identify promising candidates in the host transcriptome post-infection. RNA from BALB/c splenocytes and lung cells after aerogenic Mycobacterium bovis infection were with high-density microarrays to defining biomarkers of disease progression. In antigen-stimulated splenocytes we found statically significant modulation of 1109 genes early after infection and 1134 at later time-point post-infection. 618 of these genes were modulated at both time points. In the lung 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were granzyme A in spleen, and cxcl9, granzyme B, interleukin-22, interluekin-17A receptor, and ccr6 in lungs. The expression of 20 of the most up-regulated genes identified in the murine studies were evaluated using PBMC from uninfected and naturally infected cattle. We could show that the expression of cxcl9, granzyme A and interleukin-22 following in vitro stimulation with PPD was significantly increased in infected cows compared to naïve animals. Thus, we have demonstrated that murine transcriptome analysis can be used to predict responses in cattle. In addition, we have prioritised CXCL9, GnzA and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis. Two groups of 5 BALB/c mice each were infecetd with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested. One group of 5 mice were used as a control. Their splenocytes and lung cells were stimulated in vitro for 3 days with M7 protein pool. Antigen cell culture stimulations in mice were performed using an equal pool of seven secreted, immunogenic recombinant mycobacterial proteins (Rv1886c, Rv3019c, Rv3763, Rv3804c, Rv0251, Rv0287 and Rv0288) common to M. bovis and BCG, referred here as M7 protein cocktail. Each protein was used at final concentration of 2μg/ml in 3-day culture.
Project description:Background: We have found that extracellular vesicles (EV) secreted by embryonic stem cell-derived cardiovascular progenitor cells (hES-CPg) recapitulate the therapeutic effects of these cells in a model of chronic heart failure (CHF). Objectives: Our goal was to test other cellular sources of EV and to explore their mechanism of action.
Project description:Mutants of the Mnn1 gene are hyper-sensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic. We describe a role for Menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity 2-colour microarray design using 36 microarrays testing shock vs non shock in Drosophila melanogaster. Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNAi are characterized by increased genome instability in response to short, repeated but non-lethal heat shock or hypoxia treatments. The same was true for strains lacking all Hsp70 genes.
Project description:Sertoli cells are highly polarized testicular cells that provide a nurturing environment for germ cell development and maturation during spermatogenesis. The Class III PI3K, PIK3C3/VPS34 plays key roles in endosomal membrane traffic and macroautophagy in various cell types; however, its role in Sertoli cells remains unclear. Here, we generated a mouse line in which Pik3c3 was specifically deleted in Sertoli cells (cKO) and found that after one round of normal spermatogenesis, the cKO mice quickly became infertile and showed disruption of Sertoli cell polarity, loss of germ cells and impaired spermiogenesis. Subsequent proteomics and phosphoproteomics analyses enriched the F-actin cytoskeleton network involved in the disorganized Sertoli-cell structure in cKO testis, and showed significantly increased expression of the F-actin negative regulator SCIN and reduced phosphorylation of the α-tubulin deacetylase HDAC6. Our results further demonstrated that the accumulation of SCIN in cKO Sertoli cells caused the disassembly of the F-actin cytoskeleton, which was related to the failure of SCIN degradation through the autophagy-lysosome pathway. Additionally, we found that the phosphorylation of HDAC6 at site S59 by PIK3C3 was essential for its degradation through the ubiquitin-proteasome pathway. As a result, the HDAC6 that accumulated in cKO Sertoli cells deacetylated SCIN at site K189 and led to a disorganized F-actin cytoskeleton. Taken together, our findings elucidate the indispensable role of PIK3C3 in maintaining Sertoli cell polarity and reveal a new mechanism in which both its protein kinase activity and its regulation of autophagy are required for the stabilization of the actin cytoskeleton.