Project description:Primary dermal fibroblasts from patients with dSSc and healthy controls were treated with TGF beta for up to 24h and the genome-wide patterns of gene expression measured on DNA microarrays. 894 genes were identified as TGF beta-responsive in 4 independent cultures of dermal fibroblasts (2 healthy control and 2 dSSc patients). The 894 genes in the TGF beta-responsive signature are associated with induction of growth factor signaling, collagen synthesis and extracellular matrix deposition.
Project description:Propolis is a resinous honeybee product, rich in polyphenolic compounds and with high economic value. Although extensive studies regarding the chemical composition of different propolis extracts have been carried out, the propolis proteome remained unknown. The present study aimed at characterizing the proteome of two geographically-distinct propolis originating from Belgium and Iran. Propolis extracts were analysed by SDS-PAGE followed by MALDI-TOFand LC/ESI- mass spectrometry (MS). Our in-depth proteomic analysis led to the identification of honeybee venom and royal jelly proteins, and numerous plant-defense proteins in propolis, extending our understanding on the antimicrobial properties of this complex natural substance. The proteome of both propolis was mainly made up of proteins belonging to the poplar tree. The poplar PR-2 (β-1,3-glucanases) and PR-8 (acidic endochitinases) proteins were found to be the major components of both propolis extracts, while bee venom (Api m 1) and royal jelly allergens (MJRP 1and 2) were only identified in the propolis from Belgium. Overall, our proteome analyses revealed that propolis extracts from Belgium and Iran shared a core protein composition originating from the poplar proteome. The majority of identified proteins are involved in the plant defense against pathogens, some belonging to well-known pathogenesis-related families.
Project description:Transforming growth factor-Ã?1 (TGF-Ã?1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-Ã?1â??s activity on hMSCs. Using a proteomics â??Protect and Labelâ?? approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin. Competition assays in an optical biosensor demonstrated that TGF-Ã?1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-Ã?1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14â??24 saccharides (dp14â??24) in length all increased the phosphorylation of SMAD2 after 6 h of stimulation with TGF-Ã?1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-Ã?1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-Ã?1 in hMSCs.
Project description:To determine if aberrant activation of endothelin-1 (Et1) could lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. Mouse dermal fibroblasts were treated with exogenous Et1 peptide for 24 hours. ET1 treatment resulted in significant expression changes - primarily downregulation - of a number of genes. In particular, Tgf-beta-2 and Tgf-beta-3 were among the downregulated genes, which in turn alter the expression status of their many target genes. These data suggest that the stable silencing of Et1 is important for the phenotypic stability of dermal fibroblasts, and perhaps many other cell types as well.<br><br>Three separate biological replicates were derived for both control and treated samples. The primary dermal fibroblasts were derived by explant procedure from the skin of mouse pups aged 0-3 days. By passage 5, cells were split to two separate cultures-- one with 100nM synthetic Et1 peptide added to the medium (treated) and the other with nothing added (control). Cells were exposed to Et1 for 24 hrs, then treated and control populations were harvested for total RNA.
Project description:Driving HUVECs toward mesenchymal fate Comparison of untreated HUVECs, HUVECs treated with TGF-B, HUVECS treated with TGF-B and oxidative stress, and comparator human dermal fibroblasts
Project description:Recent studies in non-human model systems have shown therapeutic potential of modified mRNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the α-galactosidase (GLA) gene in a human cardiac model generated from induced-pluripotent stem cell-derived from two patients with Fabry disease. In line with the clinical phenotype, cardiomyocytes from Fabry patient’s induced pluripotent stem cells show accumulation of the glycosphinolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Further, the patient-specific cardiomyocytes have significant upregulation of lysosomal associated proteins. Upon modRNA treatment, a subset of lysosomal proteins were partially restored to wildtype levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA treated cardiomyocytes demonstrating that α-galactosidase enzymatic activity was restored. Together, our results validate the utility of patient IPSC-derived cardiomyocytes as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
Project description:Filamentous algae (FA) have potential advantages over microalgae for wastewater treatment. However, their implementation at large-scale is hindered by an inability to predict performance. This study compared the cellular responses (photosynthesis and respiration) and composition (pigments and photosystem proteins) of FA Oedogonium acclimatised to average summer and winter conditions (Melbourne, Australia). After 7 days of acclimation the Chl a content of summer acclimated (SA) algae was about half that of the winter acclimated (WA) algae, which can be related to a strategy to reduce photodamage under high light intensities. No statistically significant changes were observed in any identified proteins associated with photosystem PSII and the reaction centre of PSI. Transmission electron microscopy images revealed more prominent lipid bodies within the SA filaments than in WA filaments, but no discernible difference in the abundance of starch granules. Photosynthetic irradiance curves were compared for the SA and WA algae. Consistent with the differences in chlorophyll, the specific gross photosynthetic rate (µP, gross) was generally higher for the WA algae. The relative difference increased from around 2-fold at 15°C to 3-fold at 25°C, and then decreased to less than 1.5-fold at 30 °C and 35 °C. At all the tested temperatures, saturation irradiance levels were in the range of 75 – 500 µmol/m2·s. Photoinhibition was observed at 30 °C (above ~300 µmol/m2·s) and was more severe at 35 °C (above ~500 µmol/m2·s), with WA algae showing greater inhibition. In contrast, the respiration response was similar for the SA and WA algae. The study emphasises the significance of accounting for seasonal variations and their effects on biomass productivity and utilisation. The data obtained will enable the incorporation of acclimation and its effect on biochemistry and photosynthetic response into predictive models of FA performance in outdoor cultures.