Project description:Drosophila Orb, the homologue of vertebrate CPEB is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb has also an essential function during early oogenesis which has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy, by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues. Orb RIP-chip vs mock IP on ovaries from mature 3-5 day old females.
Project description:Drosophila Orb, the homologue of vertebrate CPEB is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb has also an essential function during early oogenesis which has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy, by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues. Orb RIP-chip vs mock IP on ovaries from newly eclosed females.
Project description:The experimental data indicate that during a persistent infection, lymphocytic choriomeningitis virus (LCMV) may both directly or indirectly modulate the regulatory cellular processes and alter the cellular functions that are not critical for the survival, but are needed for the homeostasis in the organism. Two-dimensional differential in-gel electrophoresis (2D-DIGE) and MALDI-TOF MS/MS analyses were used to determine the cellular proteome response of HeLa cell line to persistent LCMV infection. Quantitative analysis revealed 24 differentially abundant proteins, half of which were up-regulated and the rest down-regulated. Functional categorization showed that LCMV-responsive proteins were mainly involved in metabolism, stress and defense responses. Among identified proteins, significant changes were found for peroxiredoxins, family of antioxidant enzymes. Decreased amount of these antioxidant proteins was accompanied with the elevation of ROS content in infected cells.
Project description:This study considers the physiological modulation of liver proteins due to the supplementation with fish oils under two different dietary backgrounds: low- or high- fat and sucrose diets, and the effect of their combination with an antioxidant agent (grape polyphenols) which provides reducing power. For this scope, a quantitative proteomics approach based on the Isobaric Tag for relative and Absolute Quantitation methodology (iTRAQ)-coupled to nano-LC-MS/MS and complemented with 2D-DIGE analysis were used for determining the regulation of liver proteins exerted by the supplementation with fish oils, polyphenols or their combination of Wistar Kyoto rats in the two chosen dietary backgrounds. This experimental design was useful to investigate if the behavior of fish oils changes when the dietary background is modified and the possible synergy between fish oils and polyphenols. Results show that the capacity of fish oils, polyphenols or their combination for down or up-regulating liver proteins depends on the dietary context. In the background of low-fat low-sucrose healthy diets, 10 different proteins were altered by the sum of three supplements, in opposite to the 45 altered proteins found in the high-fat high-sucrose unhealthy diets. In both situations, fish oils seemed to be the main force for regulating liver proteins, although the addition of polyphenols was able to modulate some fish oils effects. Moreover, we provide evidence of the effect of fish oils and their combination with grape polyphenols for improving biochemical parameters and for reducing enzymes of hepatic lipogenesis and glycolysis, for enhancing fatty acid beta oxidation and insulin signaling and for the amelioration of endoplasmic reticule stress and protein oxidation when are included in an unhealthy diet.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin. For determining DamID based UpSET chromatin profile, three biological replicates were used. For evaluating chromatin accessibility, two biological replicates were performed.
Project description:To gain insight into the molecular mechanisms at work during progression through the pre-erythrocytic stages, a comparative microarray based transcriptional study was under taken on radiation attenuated (RAS) and wild type sporozoites (wtSPZ) as well as, and liver stage parasites collected 24 hours (24hrLS) and 48 hours (48hrLS) after wild type sporozoite infection. We were able to identify ~1100 genes significantly differentially expressed during one or more of the pre-erythrocytic stages relative to the mixed blood stages. This study compared the gene expression profiles of radiation attenuated sporozoites (RAS), wild type sporooites (wtSPZ), and infected hepatocytes collected 24hr (24hrLS) and 48hr (48hrLS) after sporozoite infection, using a common reference design. Each sample was hybridized with an equal amount of reference RNA. 48hrLS: 4 biological replicates, with 2 total technical replicates and one dye swap; 24hrLS: 4 biologicla replicates, with 3 total technical replicates; RAS: 2 biological replicates, with 2 technical replicates total, and two tota dye swaps; wtSPZ: 2 biological replicates, with 2 total technical replicates and 2 total dye swaps
Project description:Trypanosoma cruzi, the causative agent of Chagas disease, is a genetically diverse with TcI being the most widespread lineage in the Americas. The protein expression dynamics allows unravel the biological phenomena of pathogens such as disease development and evolution. A large number of studies have shown a direct relationship between genetic variability and the biological characteristics of T. cruzi. However, much remains to be explored regarding the impact of gene expression on the development and evolution of Chagas disease. Two-dimensional (2D) electrophoresis and mass-spectrometry were employed to characterize the global protein expression patterns of epimastigotes from four distinct TcI strains, which display different growth kinetics. Biological differences were identified and confirmed throug metabolic and morphometric assays. Statistical tests (parametric or non-parametric test, as appropriate for the experiment) were used to assess the significance of the observed differences. The Ascending hierarchical classification analysis shows two clusters, that were congruent with their growth kinetics. The proteins expressed in one or the other cluster were identified by mass spectrometry. These proteins were evaluated by independent biological assays, which showed that the parasite’s metabolic activity and morphology is significantly different between the two clusters. We conclude that the protein expression patterns displayed by epimastigotes from fast/slow growth TcI strains are correlated with various biological parameters, including intensity of metabolic activity, metabolic fuel usage and resistance to oxidative stress, as well as morphological characteristics, such as length of the flagellum.
Project description:Pharmaceuticals are pseudo persistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to Acetaminophen: 54.77 ± 34.67; Atenolol: 11.08 ± 7.98 and Carbamazepine: 7.85 ± 0.13 µg•L-1 for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, 7 differently expressed proteins were obtained in comparison with the control, of which 6 could be identified (four up-regulated and 2 down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, 3 features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability due to the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development.
Project description:Collagen type II-induced arthritis (CIA) is an autoimmune disease that is accompanied by a complex host systemic response, which includes inflammatory and autoimmune reactions. Since to develop CIA is required the injection of antigen in complete Freund’s adjuvant (CFA), which is a water-in-mineral-oil emulsion containing killed Mycobacteria, systemic response proteins related with inflammation can confound the detection or diagnosis of arthritis. CIA in CD38 deficient mice (CD38 KO) is milder than that in C57BL/6 (B6 WT) mice. Protein extracts from spleen were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analysis to identify proteins that were differentially expressed in CD38 KO and B6 WT mice with arthritis, with inflammation, or non-immunized. Differential protein expression was validated by ELISA, or Western-blotting.
Project description:Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. The mechanism regulating functional changes of semen composition upon hormone treatment is unknown at present. The aim of the study was to compare proteome of carp spermatozoa and seminal plasma from control (PBS, n=10) and hormonally stimulated males (D-Ala6,Pro9-NEt)-mGnRH+metoclopramide, n=10). Comparative proteomics based on two-dimensional differential in-gel electrophoresis was employed to quantitatively analyze the spermatozoa and seminal plasma proteins changed in abundance due to hormonal stimulation. Changes in phosphorylation and oxidation level of spermatozoa proteins were also analyzed. The proteomic results were validated by western blot, enzymatic assay and mRNA expression analysis.In total we identified 62 spots (38 up and 24 down regulated) corresponding to 48 proteins with differential abundance in spermatozoa and 36 spots (23 up and 13 down regulated) corresponding to 23 proteins differentially abundant in seminal plasma upon hormone treatment (P<0.05; fold change 1.2). The majority of spermatozoa proteins changed in abundance as well as phosphorylation and carbonylation level were involved in cillium assembly and organization, energy metabolism, protein folding, and signal transduction while differentially abundant seminal plasma proteins were associated with immune and stress response, lipid metabolism and tissue remodelling. Our results strongly suggest that hormonal treatment is not only related to hydration of testis but also induces modification of spermatozoa proteins and synthesis of seminal plasma proteins. ROS signaling and cAMP/PKA and calcium/calmodulin pathways are involved in sperm response to hormonal stimulation.